raf kinases: pathway, modulation and modeling · 2013. 12. 10. · signalweges fest; ein e ekt, der...

RAF Kinases Pathway Modulation andModeling

Dissertation zur Erlangung des naturwissenschaftlichenDoktorgrades der Bayerischen Julius-Maximilians-Universitat

Wurzburg

vorgelegt von

Armin Robubi

aus

Teheran Iran

Wurzburg 2007

Eingereicht am

Mitglieder der Promotionskommission

Vorsitzender Prof Dr Martin J MullerGutachter Prof Dr Thomas DandekarGutachter Prof Dr Ulf R Rapp

Tag des Promotionskolloquiums

Doktorurkunde ausgehandigt am


Armin RobubiDepartment of Bioinformatics

University of Wurzburg

December 12 2007

I dedicate this work to my mother

Plain question and plain answer make the shortest road out ofmost perplexities

Mark Twain

Abstract

The RasRAFMEKERK cascade is a central cellular signal transductionpathway involved in cell proliferation differentiation and survival where RAFkinases are pivotal kinases implicated in cancer

The development of specific irreversible kinase inhibitors is a rewardingbut difficult aim CI-1033 was developed to irreversibly inhibit erbB recep-tor tyrosine kinases by reacting to the Cys113 residue (p38α MAP kinasenumbering) of the kinase domain In this study we tried a similar approachto target the RAF oncoproteins which posses a similar cysteine at position108 in the hinge region between the small n-lobe and the large c-lobe of thekinase domain A novel synthetic approach including a lyophilization stepallowed us the synthesis of a diphenyl urea compound with an epoxide moiety(compound 1) Compound 1 possessed inhibitory activity in vitro Howeverour time kinetics experiments and mass spectroscopic studies clearly indicatethat compound 1 does not react covalently with the cysteine residue in thehinge region Moreover in cell culture experiments a strong activation ofthe RAF signaling pathway was observed an effect which is known fromseveral other RAF kinase inhibitors and is here reported for the first timefor a diphenyl urea compound to which the clinically used unspecific kinaseinhibitor BAY 43-9006 (Sorafinib Rcopy Nexavar Rcopy) belongs Although activationwas apparently independent on B- and C-RAF hetero-oligomerization in vitroin vivo experiments support such a mechanism as the activation did not occurin starved knockout cells lacking either B-RAF or C-RAF (Robubi et alChemMedChemndashsubmitted)

Furthermore we developed a mathematical model of the RasRAFMEK-ERK cascade demonstrating how stimuli induce different signal patterns andthereby different cellular responses depending on cell type and the ratiobetween B-RAF and C-RAF Based on biochemical data for activation anddephosphorylation we set up differential equations for a dynamical model ofthe RasRAFMEKERK cascade We find a different signaling pattern andresponse result for B-RAF (strong activation sustained signal) and C-RAF(steep activation transient signal) We further support the significance of such

VI Abstract

differential modulatory signaling by showing different RAF isoform expressionin various cell lines and experimental testing of the predicted kinase activitiesin B-RAF C-RAF as well as mutated versions (Robubi et al 2005)

Additionally the effect of the tumor suppressor DiRas3 (also known asNoey2 or ARHI) on RAF signaling was studied I could show that Di-Ras3 down-regulates the mitogenic pathway by inhibition of MEK (BeckRobubi et al Mol Cellndashsubmitted) a basis for a refined model of theRasRAFMEKERK cascade (Robubi et al in preparation)

Zusammenfassung

Die RasRAFMEKERK Kaskade ist ein zentraler zellularer Signalwegder bei der Regulierung der Proliferation Differenzierung und Uberlebender Zelle eine entscheide Rolle spielt Dabei kommt den RAF Kinasen eineSchlusselrolle bei der Tumorgenese zu

Die Entwicklung von spezifischen irreversiblen Kinasehemmern stellt einenattraktiven jedoch schwierigen Ansatz zur Tumorsupression dar CI-1033wurde erfolgreich mit dem Ziel entwickelt ErbB-Rezeptor-Tyrosinkinasen irre-versibel zu inhibieren indem es kovalent mit dem Cys113 (p38α MAP KinaseNummerierung) in der Kinase-Domane reagiert In dieser Arbeit wird ein ver-gleichbarer Ansatz gegen die RAF-Onkoproteine verfolgt die einen analogenCystein-Rest in der Position 108 aufweisen Dieser ist in der Hinge-Regionzwischen dem kleinen n-lobe und dem groszligen c-lobe der Kinase-Domane loka-lisiert Ein neuer synthetischer Ansatz der einen Lyophilisierungsschritt miteinschloss erlaubte hierfur die Synthese einer Diphenylharnstoff-Verbindungmit einer Epoxidgruppe (Verbindung 1)

Verbindung 1 zeigt in vitro tatsachlich eine inhibitorische Aktivitat gegenRAF-Kinasen Jedoch zeigen unsere zeitkinetischen Experimente sowie un-sere massenspektrometrischen Analysen dass Verbindung 1 keine kovalenteBindung mit dem Cystein-Rest in der Hinge-Region bildet Auszligerdem stelltenwir in Zellkulturexperimenten eine starke Aktivierung des RAF-induziertenSignalweges fest ein Effekt der bereits fur andere RAF-Kinase-Inhibitorenbeschrieben wurde jedoch hier erstmalig auch fur eine Diphenylharnstoff-Verbindung zu der auch BAY 43-9006 (Sarafinib Rcopy Nexavar Rcopy) gehort BAY 43-9006 ist ein unspezifischer fur die Behandlung von Krebs zugelassener KinaseInhibitor Obwohl die Aktivierung in vitro scheinbar unabhangig von einerHeterooligomerisierung von B-RAF und C-RAF war unterstutzen in vivoExperimente einen solchen Mechanismus da in gehungerten knockout Zellenin denen B-RAF oder C-RAF fehlte keine Aktivierung beobachtet werdenkonnte (Robubi et al ChemMedChemndasheingereicht)

Des Weiteren zeigten wir in einem mathematischen Modell wie abhangigvom B-RAFC-RAF-Verhaltnis verschiedene Zellantworten durch unterschied-

VIII Zusammenfassung

liche Stimuli induzierbar werden Basierend auf biochemischen Daten uberAktivierung und Dephosphorylierung sowie auf den Differentialgleichungen un-seres Rechenmodells fanden wir eine unterschiedliche Signalkinetik fur B-RAF(starke Aktivierung anhaltendes Signal) und C-RAF (schwache Aktivierungtransientes Signal) Die Bedeutung dieser differenzierten Signalmodifikationwurde auch durch unterschiedliche Expression der RAF Isoformen in verschie-denen Zelllinien und durch die experimentelle Messung der Kinaseaktivitatvon B- und C-RAF sowie mutierte Formen uberpruft (Robubi et al 2005)

Zusatzlich wurde der Effekt des Tumorsupressorproteins DiRas3 (auchbekannt als Noey2 oder ARHI) auf den RAF-Signalweg untersucht Wirkonnten zeigen dass DiRas3 den mitogenen Signalweges durch Inhibierungder mitogen-aktivierten Proteinkinase Kinase (MEK) negativ reguliert (BeckRobubi et al Mol Cellndasheingereicht) eine Basis fur ein verfeinertes Modellder RasRAFMEKERK Kaskade (Robubi et al in Vorbereitung)

Contents

Abstract V

Zusammenfassung VII

1 Introduction 19

11 History and nomenclature of RAF kinases 19

12 RAF kinase signaling 22

13 Mouse knockout models 23

14 RAF kinases in cancer 23

15 Architecture of Raf kinases 25

151 Kinase domain 25

16 Development of a novel RAF kinase inhibitor 28

17 Dynamic pathway modeling 29

18 DiRas3 30

2 Materials and Methods 33

21 Compound characterization 33

22 Cell culture 33

221 Conditions for inhibitor studies 33

222 Conditions used for modeling studies 33

23 Immuno blot analysis 34

24 Kinase assay (immuno blot) 34

25 Kinase assay (ELISA) 35

26 Kinase assay (DiRas3) 36

27 Biosensor measurements 36

28 Mass spectrometry measurements 36

29 Gel filtration 37

210 Bioinformatics 37

2101 Molecular modeling 37


X Contents

3 Results 3931 Development of a novel RAF kinase inhibitor 39

311 Homology modeling 39312 Activity of compound 1 in vitro 43313 Activation in cell culture 44314 Other compounds 47

32 Dynamic modeling 4933 DiRas3 58

331 DiRas3 interacts in vitro efficiently with active C-RAFand MEK 58

332 Inhibition of MEK activity by DiRas3 in vitro 59

4 Discussion 6341 Developing a novel RAF kinase inhibitor 6342 Dynamic modeling 6643 DiRas3 68

Bibliography 71

Acknowledgments 85

Curriculum vitae 87

List of publications 89Poster Abstracts 90Oral presentations 91

A Supplementary material 93

Erklarung 95

List of Figures

11 Some important steps in RAF research 2012 Scheme of the mitogenic signaling pathway 2413 Multiple alignment of A- B- and C-RAF 2614 3D crystal structure of the kinase domain B-RAF 2715 RAF kinase inhibitors 28

31 Development of a new lead compound 4032 Model of compound 1 in complex with B-RAF 4133 Synthesis of compound 1 4234 Inhibition of RAF kinases in an in vitro kinase assay 4435 Time kinetics experiment 4536 Mass spectrometry data 4637 Elevated levels of pERK after treatment with compound 1 4738 No activation by compound 1 in starved RAF knockout cells 4839 Hetero-oligomerization of B-RAF and C-RAF in vitro 48310 Synthesis of compounds 10 and 16 50311 Model of the Ras-ERK signaling pathway depiction of the

parameters 52312 Response curve for the Ras-ERK pathway under standard

conditions 53313 Simulation showing the qualitative differences between B-RAF

and C-RAF 54314 Gel showing different expression levels of RAF kinases in dif-

ferent tissues 55315 Kinase assays showing the activity of different preparations of

RAF kinases 56316 DiRas3 interaction with C-RAF and MEKmdashBIAcore 59317 DiRas3 interaction with C-RAF and MEKmdashsummary 60

41 Reaction mechanism between a cysteine and an epoxide 6342 Model of the mitogenic signaling pathway 65

XII List of Figures

43 DiRas3 binds to RAF as well as to MEK and blocks MEKfrom phosphorylating ERK 69

A1 Scanning different parameter values 94

List of Tables

11 Nomenclature of RAF kinases 2112 Cellular signals and responses 30

31 Inhibition of C-RAF and MEK by different inhibitors in vitro 5132 Parameter values 55

List of Abbreviations

SI-Units are not listed

Rcopy registered3D three dimensionalA adenineadenosineAA amio acidabs absoluteAML acute myeloid leukemiaANP atrial natriuretic peptide receptorATP adenosine 5rsquo-triphosphateATR attenuated total reflectanceBLK block residue as defined in the Modeller packageBoc2O di-tert-butyl dicarbonatebp base pairBSA bovine serum albuminC cytosine cysteinec-lobe C-terminal lobeCFC cardio-facio-cutaneousCR1 2 3 conserved region 1 2 3CRD cysteine rich domainCys cysteineD aspartateDa daltonDFG aspartatendashphenylalaninendashglycineDMAP 4-dimethylaminopyridineDMEM dulbeccorsquos modified eagle mediumDMSO dimethyl sulfoxideDNA deoxyribonucleic acidDTT dithiothreitolE glutamate

XVI List of Tables

ECL enhanced chemoluminiscenceE coli Escherichia coliEDTA ethylenediamine tetraacetic acideg for example Lat exempli gratiaEGF epidermal growth factorEGFR epidermal growth factor receptorELISA enzyme-linked immunosorbent assayERK extracellular signal-regulated kinaseEt2O diethyl etherEt3N triethyl amineEtOH ethanolFCS fetal calf serumFDA food and drug administrationFGF fibroblast growth factorFRS2 fibroblast growth factor receptor substrate 2FT-IR fourier transform infraredG-loop glycine rich loopGAP GTPase activating protein GTPase accelerating proteinGDP guanosine diphosphateGEF guanine nucleotide exchange factorGrb2 growth factor receptor-bound protein 2GS glutathione sepharoseGST glutathione S-transferaseGTP guanosine triphosphateHEK293 human embryonic kidney cellsHepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acidHS horse serumJNK c-Jun N-terminal kinaseKSR kinase suppressor of RasLck eukocyte-specific protein tyrosine kinaseMA MassachusettsMAPK mitogen-activated protein kinaseMAPKK mitogen-activated protein kinase kinaseMAPKKK mitogen-activated protein kinase kinase kinaseMDR multi drug resistanceMEK mitogen-activated protein kinase kinaseMeOH methanolMg-loop magnesium positioning loopMH2 Mil-Hill No 2MSV mouse sarcoma virusMTP micro titer plate

List of Tables XVII

n-lobe N-terminal lobeNGF nerve growth factorNi-NTA nickel-nitrilotriacetic acidNP40 nonidet 40NR n-regionNSCLC non-small-cell lung cancerODE ordinary differential equationOMIM online mendelian inheritance in manOPD o-phenylenediamine hydrochloridePAGE sodium dodecyl sulfate polyacrylamide gel electrophoresisPBS phosphate buffered salinePC12 rat pheochromocytomaPDB protein data bankPDGFR platelet-derived growth factor receptorPMA phorbol 12-myristate 13-acetatePNS post-nuclear supernatantRAF rapidly growing fibrosarcomaRAF-ER RAF-estrogen receptorRBD Ras binding domainRET rearranged during transfectionRNA ribonucleic acidRPMI roswell park memorial institute medium 1640rt room temperatureRTK receptor tyrosine kinaseRU response unitsSDS sodium dodecyl sulfateSer serineSHC Src homology 2 domain (or SH2 domain)si-oligos small interfering oligonucleotidessiRNA small interfering RNASOS son of sevenlessSpeg striated muscle-specific serinethreonine protein kinaseT threonineTBST tris-buffered saline Tween-20TCA trichloroacetic acidTFA trifluoroacetic acidTHF tetrahydrofuranThr threonineTPA tetradecanoylphorbol acetateTyr tyrosineVEGFR vascular endothelial growth factor receptor

XVIII List of Tables

wt wild typeY tyrosine

Chapter 1

Introduction

RAF kinases are an important group of proto-oncoporteins They play a keyrole in the mitogenic signaling pathway (RasRAFMEKERK) a highlyconserved signaling pathway which controls proliferation differentiation andsurvival The mitogenic signaling pathway was found to be hyper-regulated inabout 30 of solid tumors (Hoshino et al 1999) Ras a small GTP bindingprotein is a common proto-oncoprotein that binds directly to RAF kinasesand initiates a highly complex process of activation RAF kinases are the beststudied effectors of Ras The fact that B-RAF mutations were also found inhuman cancers underlines their prominent role in oncogenesis (Davies et al2002)

My project covers a wide range of issues around RAF kinases (i) It startedfrom structural analysis of the kinase domain of B-RAF followed by an effortto develop a novel irreversible RAF kinase inhibitor Three novel compoundswere synthesized and their behavior against RAF kinases were studied (ii) Amathematical model was developed to study dynamic properties of the Ras-RAFMEKERK signaling pathway (iii) Additionally the interaction of RAFkinases with the tumor suppressor DiRas3 (also known as Noey2ARHI) wasstudied I could show that DiRas3 downregulates the mitogenic pathway byinhibition of the mitogen-activated protein kinase kinase (MEK) which is themajor substrate of RAF kinases This provides a basis for a refined model ofthe cascade signaling

11 History and nomenclature of RAF kinases

Figure 11 winds up important milestones in the research of RAF kinases

The oncogene of the acutely transforming replication-defective mouse typeC virus 3611-MSV was characterized in 1983 (Rapp et al 1983) Since 3611-

20 Chapter 1 Introduction

MSV induces rapidly growing fibrosarcomas the transforming viral oncogenewas called v-raf Its cellular homologs in mouse and in human were thereforecalled c-raf-1 and c-raf-2 respectively

In the same year the avian acute leukemia retrovirus Mil-Hill No 2 (MH2)was found to carry a second oncogene in addition to v-myc which was termedv-mil after the virus Its cellular homolog was termed c-mil (Jansen et al1983) c-mil turned out to be the avian homolog of the mamalian c-raf(Jansen et al 1984) c-raf-2 later turned out to be a pseudogene (Bonneret al 1985) The product of the c-raf-1 gene became c-Raf-1 (eg c-raf-1craf1 C-Raf-1) or just Raf-1

In 1986 a new paralog of c-Raf-1 was found and termed A-Raf accordingto the nomenclature of that time Two A-Raf genes were found in humansand mice and termed A-Raf-1 and A-Raf-2 (Huebner et al 1986) A-Raf-1 isa functional gene located on chromosome X whereas A-Raf-2 is a pseudogene

Finally in 1988 a second paralog of c-Raf was identified as a homologof transforming gene in a human Ewing sarcoma (Ikawa et al 1988) Alsoin 1988 the avian homolog of c-mil was identified and found to transformneuroretinal cells in chicken It was termed c-Rmil to point out its retinalorigin and its homology with c-mil (the chicken has no ortholog of A-Raf )As with the other Raf isoforms two B-Raf genes were found in human One(B1) being functional the other (B2) being a pseudogene (Sithanandam et al1992)

Table 11 summarizes the different nomenclatures of RAF kinases Re-cently Wellbrock et al (2004) suggested a nomenclature using A-RAF B-RAFand C-RAF for the functional proteins and A-RAF B-RAF C-RAF for thecorresponding genes in human and A-Raf B-Raf and C-Raf (and A-Raf

1983 2007

Time

1993RAF as effector of Ras [4]

1985A-RAF discovered [2]

2006RAF germline

mutations discovered[8]

2002B-RAF mutations in human

cancers discovered[6]

1983C-RAF discovered [1]

1988B-RAF discovered [3]

2000BAY 43-9006 [5]

2005BAY 43-9006

appoved for treatment [7]

Figure 11 Some important steps in RAF research The time barshows starting from the discovery of C-RAF the major discoveries in RAFresearch with references 1 Rapp et al (1983) 2 Huebner et al (1986) Ikawaet al (1988) 4 Zhang et al (1993) 5 Lowinger et al (2002) 6 Davies et al(2002) 7 Strumberg et al (2007) 8 Duesbery and Woude (2006)


In this work Alternative names Hugo names

A-RAF A-Raf A-Raf-1 araf arafB-RAF B-Raf braf BRAF BRAF1 B-Raf-1 c-Rmil

p94 v-Raf murine sarcoma viral oncogenehomolog B1

braf

C-RAF Raf-1 craf craf1 cRaf-1 c-Raf-1 c-mil v-raf-1 murine leukemia viral oncogene homolog1 v-raf murine sarcoma 3611 viral oncogenehomolog

craf1

Table 11 Nomenclature of RAF kinases

B-Raf C-Raf ) for the corresponding murine proteins (and genes) HereafterI will use the former spelling throughout the entire thesis

RAF kinases were found to bind directly to the small GTP binding proteinRas (Koide et al 1993 Zhang et al 1993) Ras proteins were well establishedproto-oncoproteins and Ras mutations were already found in many humancancers (Malumbres and Barbacid 2003) Thus RAF kinasesmdashparticularlyC-RAFmdashwere intensively studied in the following years

BAY 43-9006 (Sorafenib Rcopy Nexavar Rcopy) is a C-RAF targeted small moleculekinase inhibitor developed by the pharmaceutical company Bayer Rcopy (Lowingeret al 2002) It entered clinical trials in 2002 (Richly et al 2003 Gollob et al2005 Strumberg et al 2007) The drug received FDA approval in December2005 for the treatment of patients with advanced renal cell carcinoma and morerecentlymdashin November 2007mdashfor the treatment of advanced hepatocellularcarcinoma BAY 43-9006 is not a specific C-RAF inhibitor but shows activityagainst a wide range of protein kinases including other RAF kinase isoformsas well as a number of tyrosine kinases such as platelet-derived growthfactor receptor β (PDGFR-β) vascular endothelial growth factor receptors(VEGFR-1 and VEGFR-2) Flt-3 and c-Kit (Wilhelm et al 2004) as well asrearranged during transfection (RET) (Carlomagno et al 2006)

Davies et al (2002) demonstrated that mutations of the B-RAF geneoccur in a high number of human tumors moving the attention somewhatfrom C-RAF

Mouse models demonstrated that the mitogenic signaling pathway wasof crucial importance for cellular function and for development Germ linemutations with strong impact in one or the other way were considered tobe lethal during embryonal development It was thus a real surprise for thescientific community when gain-of-function mutations in key components ofthat pathway were reported to cause mendelian disorders in human (Duesbery


and Woude 2006) Cardio-facio-cutaneous (CFC) syndrome (OMIM115150)is caused by activating mutations of either the K-Ras B-RAF MEK-1 orMEK-2 gene Interestingly the gain-of-function mutations in B-RAF aredistinct from the ones observed in cancer (Rodriguez-Viciana et al 2006) TheCFC syndrom overlaps clinically with Costello syndrome (OMIM218040)which is caused by gain-of-function mutations of the H-Ras gene (Aoki et al2005) Germ line mutations of the C-RAF gene have also been reported incausing acute myeloid leukemia (AML) (Zebisch et al 2006)

12 RAF kinase signaling

The mitogenic signaling pathway is shown in Figure 12 Receptor tyrosinekinases (RTKs) bind growth factors in the extracellular space This bindingleads to their oligomerization and trans-phosphorylation The growth factorreceptor-bound protein 2 (Grb2) binds to the phosphorylated RTKs throughits SHC domain The signal is passed over to the G-protein exchange factor(GEF) son of sevenless (SOS) SOS facilitates the nucleotide exchange of Rasreplacing its GDP by GTP Ras-GTP recruited RAF kinases as well as anumber of other effectors RAF kinases bind to Ras-GTP (Koide et al 1993Zhang et al 1993) after which a complex ensemble of kinases phosphatasesscaffold proteins and lipids is required for their activation For all RAFisoforms the exact mechanism of activation has not been fully elucidatedThe activation of C-RAF has been studied most intensively but is probablyalso the least understood

Expression of constitutively active RAF kinase activates the extracellularsignal-regulated kinasemdashERK (Dent et al 1992 Howe et al 1992) ERKis not directly phosphorylated by RAF kinases but through the mitogen-activated protein kinase kinase (MEK) MEK is a dual specificity proteinkinase which activates ERK by phosphorylating a tyrosine and a threonineresidue in its activation segment Thus RAF kinases act as MAP kinasekinase kinases (MAPKKKs) activating MEK which in turn activates theMAP kinase ERK (Kyriakis et al 1992)

Two isoforms of ERK are found in mammals ERK-1 and ERK-2 Theformer has a molecular mass of 44 kDa the latter 42 kDa They share about43 sequence identity and are expressed in varying extends in all tissuesDeletion of ERK-2 leads to early embyonic lethality (Saba-El-Leil et al2003) whereas deletion ERK-1 does not (Pages et al 1999) indicating thatthey have distinct functions Two residues of the conserved TEY motivein the activation segmentmdashT183 and Y185mdashneed to be phosphorylated tofully activate ERK (Payne et al 1991 Robbins et al 1993) Mutation of


those amino acids to acidic residues is however not sufficient to generate aconstitutively active kinase (Canagarajah et al 1997)

MEK-1 and MEK-2 are the only protein kinases known so far to phos-phorylate ERK-1 and ERK-2 In fact ERK-12 are also the only MEK-12substrates known so far MEK is a dual specificity kinase which phosphory-lates both required residues in the activation segment of ERK MEK on theother hand needs to be phosphorylated on two serine residues (S218 S222) ofits activation segmentmdashLIDSMANSmdashby RAF kinases to be active Mutationof the two residues to acidic amino acids gives rise to a constitutively activekinase (S218E S222E LIDEMANE) MEK-1 knock out mice die at an earlyembryonic stage (Giroux et al 1999) whereas deletion of MEK-2 gives nosignificant phenotype (Belanger et al 2003)

13 Mouse knockout models

The distinct functions of RAF kinase isoforms can be studied using mouseknockout models Knockout mice for all three RAF isoforms have beengenerated Surprisingly all three RAF isoforms appear to be very importantAll RAF knockout mice display distinct but severy phenotypes with A-RAFknockouts showing the mildest phenotype (Pritchard et al 1996 Mikulaet al 2001 Wojnowski et al 1997)

This is rather surprising since other proteins in the signaling moduleexhibit redundancy to a much higher extent Even in the case of Ras onlythe K-Ras gene seems to be essential (Malumbres and Barbacid 2003)

14 RAF kinases in cancer

In one study the mitogenic signaling pathway was shown to be upregulatedin 50 of 138 human tumor cell lines (Hoshino et al 1999) Upregulation canbe caused by gain-of-function mutations or by overexpression of a number ofproteins Particularly Ras mutations are commonly found in tumors Howevera considerable number of tumors were shown to carry mutations in the B-RAF gene (Davies et al 2002) in particular malignant melanoma (27ndash70)papillary thyroid cancer (36ndash53) colorectal cancer (5ndash22) and serousovarian cancer (sim30) Virtually all B-RAF mutations found in humancancers are located either in the N-terminal region of the activation segmentor in the glycine rich loop (Figure 13) More than 40 different mutationsof the B-RAF gene have been observed in human cancers However a singlethymine to adenine transversion accounts for about 90 of the cases This


Grb2

RTK

Sos

RasGDP

SHC

RasGTPGAPs

RAFRAF

Plasma membrane

Sig

na

l

MEK

ERK

MEK

ERK

PP

PP

Figure 12 Scheme of the mitogenic signaling pathway Growth factorsbind to receptor tyrosine kinases (RTKs) inducing oligomerization and trans-phosphorylation Growth factor receptor-bound protein 2 (Grb2) binds tothe phosphorylated RTKs through its SHC domain Son of sevenless (SOS)is activated by Grb2 and facilitates the nucleotide substitution of GDP byGTP bound by Ras proteins Ras-GTP binds directly to RAF kinasesinducing a complex process of activation Active RAF kinases activate themitogen activated protein kinase kinase (MEK) by phosphorylation of tworesidues in the activation segment Activated MEK in turn phosphorylates theextracellular signal-regulated kinase (ERK) Phosphorylated ERK has a largenumber of substrates and is involved in many cellular processes (Campbellet al 1998) Negative feedback regulation by active ERK is described atthe level of SOS and RAF kinases (Chen et al 1996 Brummer et al 2003Dougherty et al 2005 Hekman et al 2005)


mutation converts a valine residue in the N-terminal region of the activationsegment into a glutamate (B-RAF-V600E) and gives rise to a constitutivelyactive kinase (Garnett and Marais 2004)

15 Architecture of Raf kinases

RAF kinases are multi domain proteins Most vertebrates possess three RAFisoforms referred to A-RAF B-RAF and C-RAF The overall architectureof A-RAF B-RAF and C-RAF resemble each other All three possess threehighly conserved regions CR1 at the N-terminus CR3 at the C-terminusand CR2 in between CR3 encodes the kinase domain the most conservedregion (Figure 13)

RAF kinases are subject to complex regulation which is also reflected bythe high number of phosphorylation sites which are distributed throughout thewhole protein While some phosphorylation sites are conserved throughoutthe whole protein family others are not indicating that different isoformsmay be subject to distinct modes of regulation

CR3 constitutes the catalytic kinase domain of the protein (Figure 14)Its sequence is highly conserved between different RAF isoformes and showsa higher sequence homology to tyrosine kinases than other serinethreoninekinases Nevertheless RAF kinases appear to act as serinethreonine kinasesexclusively

CR2 contains the S256 (C-RAF numbering) residue which binds 14-3-3 proteins upon phosphorylation and is a major negative regulatory site(Hekman et al 2004)

CR1 contains the Ras binding domain (RBD) and the cysteine rich domain(CRD) and is important for Ras-GTP and membrane association

151 Kinase domain

The kinase domain is highly conserved between RAF paralogs and orthologsThe catalytic function of RAF kinases that is the transfer of an ortho-phosphate from ATP to a protein is solely depended on that domain Itis also the target of all current RAF kinase inhibitors including the onedeveloped in this work

Tyrosine and serinethreonine kinases are structurally closely relatedThere are the largest family of proteins encoded by the human genome Dueto their high importance a large number of crystal structures of kinase domainswere solved Parts of the kinase domain of B-RAF could be solved with aresolution of 295 A (Wan et al 2004) The kinase domain has a structure


$

amp

(

)+-

0120134567863097+-

0+014+-lt13764=6713

Figure 13 Multiple alignment of A- B- and C-RAF Residue numbering(human sequences) is indicated on the left Dots indicate every tenth B-RAF residueStrictly conserved residues are shown as red blocks with white lettering Similar positionsare highlighted in red and boxed Dots in the sequences indicate gaps Indicated are thefollowing motifs CR1 CR2 and CR3 The N-terminal CR1 contains the Ras bindingdomain (RBD) and the cysteine rich domain (CRD) CR2 is situated in the middle ofthe protein and bears a conserved 14-3-3 binding site CR3 contains the kinase domainwith several important motives the N-region (NR) the glycine rich loop (G-loop) thehinge region the catalytic loop the magnesium positioning loop (Mg-loop) the activationsegment and the C-terminal 14-3-3 binding site


Figure 14 3D crystal structure of the kinase domain B-RAF Imageis based on the PDB entry 1UWH (Wan et al 2004) All β-strands are shownin blue α-helices in red The kinase domain of RAF kinases resembles thegeneral architecture of all serinethereoninetyrosine kinases It consists of asmall N-terminal section (n-lobe left) which is build up by three β-strandsan α-helix and two further β-strands and a large C-terminal lobe (c-loberight) which is predominantly built up by α-helices The ATP molecule canbe bound by the narrow cleft between the lobes The stretch of the activationsegment is quite flexible and is therefore not detectable in the electron densitymap of many crystal structures this is also the case for the B-RAF crystalstructure

known from other serinethreoninetyrosine protein kinases as shown in thecrystal structure in Figure 14 It consists of a small n-lobe which in turnis built up by five β-sheets and one α-helix and a larger c-lobe which ispredominantly built up by α-helices The ATP molecule is bound by thenarrow cleft between the lobes See also Figure 13 for the crucial residues inthe kinase domain (CR3)


16 Development of a novel RAF kinase in-

hibitor

A number of compounds which were developed as RAF kinase inhibitorsdo inhibit RAF kinases in vitro yet paradoxically activate RAF kinases incell culture independent of substance classes (Figure 15) These compoundsinclude ZM 336372 (Hall-Jackson et al 1999a) GW 5074 (Lackey et al2000 Chin et al 2004) and SB 203580 (Hall-Jackson et al 1999b) Sofar only BAY 43-9006 (Sorafinib Rcopy Nexavar Rcopy) a diphenyl urea compoundpassed clinical trials for cancer treatment BAY 43-9006 acts like most kinaseinhibitors in a reversible manner

As here a bundle of different methods had to be combined the completestrategy is briefly summarized here (i) generate models of the kinase domainof B-RAF in complex with diphenyl urea ligands (ii) synthesis of a noveldiphenyl urea lead compound with an epoxide moiety (iii) in depth biochem-ical characterization of the lead in vitro as well as in cell culture We firstgenerated a homology model of the kinase domain of B-RAF in complex withBAY 43-9006 Therein we observed a close proximity between the pyridinemoiety of the inhibitor and a cysteine residue in the hinge region (Figure 13)of the kinase domain Since few protein kinases possess a cysteine at this

O

OH

NH

HN

N

CH3

H3C

CH3

O

S

F

O

NHN

N

H3C

I

Br

Br

O

HO

NH

H

ZM 336372 SB 203580 GW 5074

Figure 15 RAF kinase inhibitors ZM 336372 (Hall-Jackson et al 1999a)SB 203580 (Hall-Jackson et al 1999b) and GW 5074 (Lackey et al 2000Chin et al 2004)


position we argued that it may be an attractive nucleophile to covalentlylink inhibitor molecules with mildly electrophilic groups to the kinase domainand thus irreversibly and specifically diminish the moleculersquos kinase activityThis led to the successful synthesis of a diphenyl urea lead compound withan epoxide moiety We did not detect covalent binding to the targeted cys-teine residue which may be explained by sterical problems although we didachieve inhibition of RAF kinase (B C) at an IC50 of 1 and 100 microM which isabout three orders of magnitude higher than for BAY 43-9006 In contrast toBAY 43-9006 compound 1 strongly elevated the content of phosphorylatedERK in RAF transformed NIH 3T3 cells This is the first report of a diphenylurea compound activating RAF kinase in vivo The underlying mechanismhas not been definitively delineated Although there was no evidence forthe exact mechanism in vitro in vivo data provide suggestive evidence forheterooligomer formation because no activation could be observed in starvedknockout cells lacking B-RAF or C-RAF

17 Dynamic pathway modeling

The high complexity of RAF kinase regulation offers more options for reg-ulation than any other step of the pathway The intensity and duration ofkinase signals are important determinants (Table 12) for cellular responses(Marshall 1995 Kerkhoff and Rapp 1998) In PC12 rat pheochromocytomacells nerve growth factor (NGF) induces sustained activation of Ras (Quiand Green 1992) The activity of the B-RAF isoform essentially follows Ras-GTP whereas the C-RAF isoform after strong initial activation is quicklyinactivated (Wixler et al 1996) The prolonged activation of B-RAF causessustained activation of the mitogenic signaling pathway which inhibits cellgrowth and induces differentiation On the other hand epidermal growthfactor (EGF) induces short activation of Ras (B-RAF and C-RAF) The re-sulting transient ERK activation stimulates cell growth (Tombes et al 1998)In rat hepatocytes both NGF and EGF induce phasic activation of C-RAFand sustained activation of B-RAF However with both growth factors phasicactivation of the mitogenic signaling pathway is observed leading to increasedcell growth Sustained activation of ERK using a RAF-ER construct blockscell growth as in PC12 cells (Tombes et al 1998)

We start from a mathematical formalism suggested by Heinrich et al(2002) Conceptually our model includes the following advances (i) Weconsider the central RAF-MEK-ERK signaling pathway To obtain accurateparameter estimations the model presented here was carefully constructedexploiting available experimental data on the RAF-kinase cascade (eg Ras-


GTP half-life) (ii) This new model considers the effect of kinase isoformson signaling cascades specifically B-RAF and C-RAF (iii) Furthermore westudied their differential inactivation by phosphatases (iv) Including all thesefeatures we can then theoretically model and experimentally show that dif-ferential expression and ratios of different RAF isoforms can partially explaindifferent mitogenic signaling behavior in different cell types This includesdirect tests on the predicted kinase activities and differential phosphataseinactivation on wild-type and mutated RAF isoforms

18 DiRas3

The mechanisms and components influencing RAF activation and RAF activ-ity are widely studied however still not fully understood Also very littleis known about the regulation of MEK the only physiologically validatedsubstrate of RAF kinases and best candidate to specifically regulate ERKactivity One candidate that may negatively regulate RAF-MEK-ERK signal-ing is the Ras-like GTP binding protein DiRas3 (also called ARHI or Noey2)It was found to inhibit epidermal growth factor (EGF) but not phorbol12-myristate 13-acetate (PMA) mediated phosphorylation of ERK (Luo et al2003) and could therefore be involved in the RAF signaling pathway DiRas3is encoded by a maternally imprinted tumor suppressor gene and expressedin human ovarian and breast tissue (Hisatomi et al 2002 Lu et al 2006Rosen et al 2004 Wang et al 2003 Yu et al 2005 1999) In cells DiRas3is predominantly GTP-bound

Expression of DiRas3 reduces cell proliferation which is accompanied by

Signal Response

Transientintensive Proliferation (Marshall 1995 Wixler et al 1996Tombes et al 1998)

Sustainedintensive Cell cycle arrest differentiation (Marshall 1995Wixler et al 1996 Sewing et al 1997 Woodset al 1997 Kerkhoff and Rapp 1998 Tombeset al 1998)

Transientlow SurvivalSustainedlow Transformation (Kerkhoff and Rapp 1997 1998)

Table 12 Cellular signals and responses The mitogenic signaling path-way can induce different cellular responses depending on its intensity andduration

18 DiRas3 31

the downregulation of the cyclin D1 promoter (Luo et al 2003 Yu et al 1999)This function is barely nucleotide dependent supporting the assumption thatits expression is regulated like in the Rnd group of permanent GTP boundproteins (Chardin 2003) The N-terminal 34 amino acids do not exhibitsignificant sequence homology to any other proteins and are required forthe anti-proliferative effect of DiRas3 (Luo et al 2003) The molecularmechanisms by which DiRas3 exerts its functions are not known yet

In this thesis we provide a molecular explanation of how DiRas3 actsas tumor suppressor We demonstrate that Di-Ras3 is tethered via N- andC-terminal residues to the plasma membrane At the plasma membraneit binds to activated C-RAF Ras binding to C-RAF is cooperative withDiRas3 but not vice versa (Beck Robubi et alndashsubmitted) Unexpectedlybinding of DiRas3 to C-RAF does not affect its kinase activity HoweverDiRas3 binds and inhibits MEK Thus DiRas3 represents the first Ras-likeGTP binding protein directly inhibiting MEK and therefore suppressingERK phosphorylation DiRas3 expression has been shown to be controlledtranscriptionally via DNA methylation and histon deacetylase complexesas well as posttranscriptionally (Feng et al 2007 Lu et al 2006) Ourdata now suggest that C-RAF functions as an ldquoandrdquo gate integrating atleast two GTPase signaling inputs leading to a block of the RAF signalingcascade at the level of MEK As the nucleotide binding state of DiRas3 didnot influence its binding to C-RAF we propose that the regulation of theRas-RAF-MEK-ERK cascade might occur at the level of DiRas3 expression

Chapter 2

Materials and Methods

21 Compound characterization

IR spectra recorded as ATR were obtained by using a Biorad PharmalyzIRFT-IR spectrometer 400-MHz 1H and 100-MHz 13C-NMR spectra weredetermined on a Bruker AV-400 spectrometer

22 Cell culture

221 Conditions for inhibitor studies

NIH 3T3 cells were transformed with constitutively active Gag-v-RAF usingthe EHneo plasmid (Rennefahrt et al 2002 Heidecker et al 1992) The cellswere cultured in Dulbeccorsquos modified Eaglersquos medium (DMEM) supplementedwith 10 heat-inactivated fetal calf serum (FCS) and with 2 mM L-glutamineand 100 unitsml penicillinstreptomycin Cells were cultured at 37 C inhumidified air containing 5 CO2 The C-RAFminusminus and B-RAFminusminus cells(Zhong et al 2001) were cultured using the same protocol After inhibitortreatment the cells were washed once in phosphate-buffered saline (PBS) andthen lysed for 10 min on ice in RIPA buffer (25 mM Tris-HCl pH 76 150 mMNaCl 1 NP-40 1 sodium deoxycholate 01 SDS and common proteaseinhibitors) Subsequently cells were centrifuged for 10 min at 20000timesg and4 C The supernatant was subjected to immono blot analysis

222 Conditions used for modeling studies

HEK293 and HepG2 cells were cultured in Dulbeccorsquos modified Eagle medium(DMEM) supplemented with 10 heat-inactivated fetal calf serum (FCS)

34 Chapter 2 Materials and Methods

PC12 cells were also grown in DMEM but supplemented with 5 FCS and10 heat-inactivated horse serum (HS) HeLa cells and the human melanomacell line IF6 were maintained in RPMI 1640 medium with 10 FCS In addi-tion all media were supplemented with 2 mM L-glutamine and 100 unitsmlpenicillinstreptomycin Cells were cultured at 37 C in humidified air con-taining 5 CO2 Cells were washed once in ice-cold phosphate-buffered saline(PBS) and then lysed for 5 min on ice in 50 mM Hepes (pH 78) 032 M su-crose 06 Nonidet P-40 100 mM KCl 20 mM NaCl 20 mM iodoacetamideand common protease inhibitors Subsequently cells were centrifuged for5 min at 1000timesg and 4 C The post-nuclear supernatant (PNS) was collectedand analyzed by immuno blotting

23 Immuno blot analysis

Protein concentration was assessed using the Pierce BCA-Kit and equalamounts of protein (25 microg) were separated by SDS-PAGE and transferred tonitrocellulose The blots were blocked for one hour in TBST (Tris-BufferedSaline with Tween-20) supplemented with 5 non-fat milk They weresubsequently incubated over night at 4 C in primary antibody namely anti-penta-His (Quiagen) anti-phospho-MEK (CellSignalling) anti-phospho-ERK(9106 New England Biolabs) and anti-ERK (K23 Santa Cruz Biotech-nology) After washing blots were incubated with secondary antibodies andthen detected using the enhanced chemi-luminescence (ECL) detection system(Amersham)

24 Kinase assay (immuno blot)

For the production of recombinant RAF kinases Sf9 cells were infected withbaculoviruses at a multiplicity of infection of 5 and incubated for 48 h at30 C The cells were then washed with PBS and pelleted at 230timesg TheSf9 cell pellets (2times 108 cells) were lysed in 10 ml of Nonidet P-40 lysis buffercontaining 25 mM Tris-HCl pH 76 150 mM NaCl 10 mM Na-pyrophosphate25 mM β-glycerophosphate 25 mM NaF 10 glycerol 075 Nonidet P-40and common proteinase inhibitors for 45 min with gentle rotation at 4 CThe lysate was centrifuged at 27000timesg for 30 min at 4 C The supernatants(10 ml) containing GST-tagged RAF kinases were incubated with 05 ml ofGS beads (Amersham) for 2 h at 4 C with rotation After incubation theGS beads were washed three times with Nonidet P-40 buffer with the thirdwash containing only 02 Nonidet P-40 instead of 075 The RAF kinases


bound to the beads were eluted three times with 05 ml of 25 mM Tris-HClpH 76 150 mM NaCl 25 mM β-glycerophosphate 25 mM NaF 10 glycerol01 Nonidet P-40 and 20 mM glutathione The purification procedurefor His-tagged RAF kinases was similar to that described above with theexception that the Sf9 cell lysates (10 ml) were incubated with 05 ml ofNi-NTA-agarose The bound proteins were then eluted with imidazole usinga step gradient The purity of the RAF kinase preparations was documentedby SDS-polyacrylamide gel electrophoresis (10 gels) and staining withCoomassie blue (gels not shown) Kinase assays with RAF proteins wereperformed using recombinant MEK-1 and ERK-2 as substrates in 25 mMHepes pH 76 150 mM NaCl 25 mM β-glycerophosphate 10 mM MgCl21 mM dithiothreitol and 1 mM sodium ortho vanadate buffer (50 ml finalvolume) Following additions of purified RAF kinases (5ndash10 ml) and ATP(500 mM) the samples were incubated for 30 min at 26 C The incubation wasterminated by the addition of Lammli sample buffer and the proteins wereseparated by 10 SDS-PAGE and transferred to nitrocellulose membranesThe extent of ERK phosphorylation was determined by anti-phospho-ERKantibodies (9106 New England Biolabs) and detected using an enhancedchemiluminescence (ECL) detection system (Amersham)

25 Kinase assay (ELISA)

The inhibitors were dissolved in DMSO The mitogenic signaling pathway wasreconstructed using GST-C-RAF-Y340DY341D or His-B-RAF expressed inSf9 insect cells GST-MEK-1 expressed in E coli and His-ERK-2 expressedin E coli The reaction mixture (1 mM ATP 10 mM MgCl2 150 mM NaCl25 mM β-glycerophosphate 25 mM Hepes pH 75 and 20ndash150 ng MEK ERKand RAF respectively) was pre-incubated with the inhibitors for 30 minutesat ambient temperature The kinase reaction was started by uniting thepre-incubated kinases (50 microl final volume) and stirring at 26 C for 30 minutesThe reaction was terminated by addition of SDS (2 final concentration)and heating (50 C 10 min) 96 well micro titer plates (MTPs) coated withanti-ERK antibodies (K-23 Santa Cruz Biotechnology) were incubated withthe reaction mixture (60 min) and subsequently washed three times withTBST (25 mM Tris 140 mM NaCl 3 mM KCl 005 Tween-20 pH 74)The MTPs were incubated with anti-phospho-ERK antibody (9106 NewEngland Biolabs 1500 1 BSA TBST) at 4 C over night and washed threetimes with TBST Subsequently the MTPs were incubated with IgGPOD

conjugated secondary mouse antibody (NA931 Pharmacia 12500 1 h)and washed three times with TBST The phospho-ERK levels were mea-


sured colorimetrically in an ELISA reader at 492 nm after incubation witho-phenylenediamine hydrochloride (OPD) buffer (37 C 30 min 50 microl)

26 Kinase assay (DiRas3)

Kinase activity assays were performed as described in Kinase assay (immunoblot) using purified MEK-1-His6 and His6-ERK as substrates in 25 mM HepespH 80 150 mM NaCl 25 mM β-glycerophosphate 10 mM MgCl2 and 1 mMsodium vanadate buffer (50 microl final volume) Following additions of purifiedGST-C-RAF-Y340DY341D kinase (05 microg) and increasing amounts of pu-rified His6-DiRas3 or His6-∆N-DiRas3 and ATP (1 mM) the mixtures wereincubated at 30 C for 20 min The incubation was terminated by additionof SDS loading buffer and boiling at 96 C for 5 min The samples wereapplied to SDS-PAGE blotted and stained against pMEK and pERK Toobtain an active MEK preparation purified GST-MEK-1 isolated from E coliwas incubated with purified His-B-RAF for 50 min at 27 C in 25 mM HepespH 80 150 mM NaCl 25 mM β-glycerophosphate 10 mM MgCl2 buffer and1 mM ATP The phosphorylated and active GST-MEK-1 was subsequentlyseparated from B-RAF using GSH-Sepharose

27 Biosensor measurements

The biosensor measurements were carried out either on a BIAcore-J system(Biacore AB Uppsala Sweden) at 25 C To measure DiRas3-RAF interactionsthe biosensor chip CM5 was loaded with anti-GST antibody using covalentderivatization according to the manufacturerrsquos instructions The GST-taggedC-RAF and C-RAF mutants were expressed in Sf9 insect cells and purifiedas described in Hekman et al (2002) These C-RAF preparations wereimmobilized in biosensor buffer (10 mM Hepes pH 74 150 mM NaCl and001 NP-40) at a flow rate of 10 mlmin which resulted in a depositionof approximately 800ndash1200 response units (RU) Next purified DiRas3-GDPwas injected The unspecific binding was measured in the reference cell andsubtracted

28 Mass spectrometry measurements

GST-tagged C-RAF-Y340DY341D was expressed in Sf9 cells partially puri-fied (Robubi et al 2005) treated with compound 1 (100 microM 60 min 30 C)and applied to SDS-PAGE (5 pmol) Proteins were visualized by subsequent


Coomassie Blue applying the method described in Neuhoff et al (1988) In-gelreduction acetamidation and tryptic digestion were done according to Wilmet al (1996) After elution of the peptides solutions were desalted usingMillipore C18 ZipTip according to the manufacturers instructions ESI-MSwas performed on a Bruker APEX II FT-ICR mass spectrometer (BrukerDaltonic GmbH Bremen)

29 Gel filtration

His-tagged C-RAF and His- tagged B-RAF were coexpressed in Sf9 insect cellsThe cells were treated with inhibitors for 30 minutes and subsequently lysedfor 30 minutes at 4 C in lysis buffer (25 mM Tris 150 mM NaCl 15 glycerol1 Chaps 25 mM NaF 25 mM β-glycerophosphate 01 β-mercaptoethanoland common protease inhibitors pH 74) The lysate was directly subjected togel filtration chromatography (Akta Explorer 100 Superdex 200 25 mM Tris150 mM NaCl 15 glycerol pH 74) after centrifugation (20000timesg 30 min4 C) Runs with thyroglobulin (670 kD) and aldolase (158 kD) were used asstandards The proteins were collected in fractions of equal volume (1 ml)and precipitated with trichloroacetic acid (TCA) The precipitated proteinswere solved in Lammli buffer and subjected to immuno blot analysis

210 Bioinformatics

2101 Molecular modeling

The crystal structures of active Lck (Zhu et al 1999) and p38 MAP kinasein complex with diphenyl urea inhibitors (Pargellis et al 2002) were usedto model the structure of the kinase domain of B-RAF in complex withBAY 43-9006 The coordinates of the activation segment were taken from thekinase domain of the insulin receptor with the DFG amino acid motif in theldquoDFG-outrdquo conformation as in the p38 MAP kinase structure (Hubbard et al1994) The PDB coordinate file of Lck required some editing in a standardtext browser in order to be accepted by the Modeller package In particularthe phosphorylated tyrosines and serines are unknown to Modeller andhad to be replaced by unphosphorylated versions of these amino acids Thesequences were aligned manually using Seaview Alignments were adjusted ina standard text editor The model was generated with Modeller (Sali andBlundell 1993) based on the alignment using the standard parameter settingof the Modeller package The inhibitor molecules were included as blockresidues (BLK)



For calculation of the kinase-phosphatase cascade the formalism given inEquation 31 on page 51 was applied Several assumptions for simplifiedmodeling were used in particular first-order rate constants allowed theconcise formula given to be obtained The Matlab software library wasobtained from MathWorks Inc A custom written program with differentsubroutines first solved the set of four ordinary differential equations (ODEs)summarized in Figure 311 and next plotted different parameter settingsand values as described in the Results Calculations took between secondsand several minutes on a PC with a Pentium 4 processor depending onthe time frame calculated and the convergence of the ODE set accordingto the parameter set used Concentrations of Ras RAF MEK and ERKin Figure 312 were set at 1 10 20 and 30 respectively Concentrationsfor Figure 313 were according to experimental data and set at CRas = 33CRAF = 17 CMEK = 1300 and CERK = 1250 (concentrations according toFerrell (1996))

For the activity values in Figure 312 all cascade members were modeledto be active with αRAF = 01 αMEK = 1 αERK = 5 βRAF = 05 βMEK =05 βERK = 1 CRas = 1 CRAF = 10 CMEK = 20 CERK = 30 andλ = 1 In Figure 313 (page 54) we systematically varied activation anddephosphorylation parameters and then solved the set of differential equationsFigure A1 (page 94) shows plots for the following RAF-specific settings B-RAF α1 = 1 80 1000 50000 β1 = 8 C-RAF α1 = 1 80 1000 50000 β1 = 70For the other proteins (Ras ERK and MEK) activation and phosphorylationwas set at λ = 0069 (Ras receptor-module activation halflife of 600 s) for theRas-GTP receptor module decay and activation of αMEK = 600 αERK = 600with dephosphorylation at βMEK = 170 and βERK = 170 Parameter settingsfor time were systematically varied between 30 s and 3 h

Chapter 3

Results


hibitor

311 Homology modeling

The crystal structures of active Lck (Zhu et al 1999) and p38 MAP kinasein complex with diphenyl urea inhibitors (Pargellis et al 2002) were usedto model the structure of the kinase domain of B-RAF in complex withBAY 43-9006 The coordinates of the activation segment were taken fromthe kinase domain of the insulin receptor (Hubbard et al 1994) with theDFG amino acid motif in the ldquoDFG-outrdquo conformation as in the p38 MAPkinase structure The sequences were aligned manually and the model wasgenerated with Modeller (Sali and Blundell 1993) The crystal structurepublished later (Wan et al 2004) was strikingly similar to our model buthas an unresolved activation segment A look at the PDB entry (1UWH)showed that the crystal structures of Lck and p38 MAP kinase were usedfor the refinement Our homology model revealed the close proximity of thepyridine moiety of the bound BAY 43-9006 molecule and the cysteine 532residue (Cys109 in p38α) This residue is situated at the hinge region of thekinase domain between the small n-lobe and the large c-lobe (Figure 31A)

We designed the structure of compound 1 (Figure 31B) The diphenylmoiety was used to mimic BAY 43-9006 and the epoxy moiety was introducedin order to provide a mild electrophilic group for the nucleophilic sulfur atomof the Cys532 residue to react with (Figure 32) The molecular modelingcoordinate file of compound 1 in complex with the kinase domain of B-RAF is deposited in Appendix A The nucleophilic attack would open thetight ring system of the epoxide and thus irreversibly link 1 to the protein

40 Chapter 3 Results

HN

HN

OCl

CF3

O

BAY 43-9006

N

HN

CH3

O

HN

HN

OCl

CF3

NH

O

O

1

Figure 31 Development of a new lead compound A Homologymodel of BAY 43-9006 (stick model) in complex with B-RAF (ribbons) Allβ-strands are shown in blue α-helices are shown in orange The pyridineresidue of the BAY 43-9006 molecule forms a hydrogen bond with a conservedcysteine residue in the hinge region of the RAF kinase Our model fits wellto crystallographic data Wan et al (2004) B Structure of BAY 43-9006and compound 1 The diphenyl urea moiety (right) is preserved while thepyridine moiety is replaced by an epoxide group (left) C Alignment of thehinge region of different kinases The leucine residue 537 is strictly conservedand shown in a red box with white lettering Similar residues are boxed andwith red lettering The dots show gaps The cysteine 532 residue is presentin all mammalian RAF kinases but not in most other protein kinase families


Figure 32 Model of compound 1 in complex with B-RAF Homologymodel of compound 1 (stick model) in complex with B-RAF (ribbons) Allβ-strands are shown in blue α-helices are shown in orange The epoxideresidue of the compound 1 molecule forms a hydrogen bond with a conservedcysteine residue in the hinge region of the RAF kinase The orientation of theepoxide moiety relative to the nucleophilic sulfur atom of the Cys532 residueis crucial the sulfur atom needs to attack the epoxide group from the backof the beta carbon

(Figure 41) Previously CI-1033 an irreversible pan-erbB inhibitor wasdeveloped successfully in the same manner Apart from inhibiting the receptortyrosine kinase activity the covalent modification also proved to increase thedegradation of the protein (Fry 2003) Additionally irreversible inhibitors aregenerally better suited to provide prolonged suppression of signaling pathways(Allen et al 2002) and are in principle less sensitive to multi drug resistance(MDR) However it should be noted that the cysteine residue in the hingeregion of the RAF kinases has a different position compared to the cysteineresidue in the erbB receptor tyrosine kinases to which CI-1033 is targetedWe were therefore dealing with a truly novel system

Cys532 is conserved throughout all mammalian RAF kinase isoforms (A-B- and C-RAF) but is found in few other protein kinases (Figure 31C) Thuswe were expecting compound 1 to possess high specificity and due to thefact that an epoxide is an relatively mild electrophile low toxicity

Synthesis of N-(2-4-[([4-chloro-3-(trifluoromethyl)phenyl]amino-carbonyl)amino]phenylethyl)oxirane-2-carboxamide (1) All stepsare depicted in Figure 33

(a) KBr (20 g) DL-serine (2) (525 g 50 mmol) and HBr (62) (137 g)were dissolved in H2O (50 ml) A solution of NaNO2 (38 g 55 mmol) in


COOHHO

NH2

HOCOOH

Br

COOK

O

COOH

O

NH2

NH

O

O

HN

HN

OCl

CF3

NH

O

O

1

2 3 4

5 6

a b

c d

e

4

6

Figure 33 Synthesis of compound 1 The synthesis route isbriefly sketched The reaction conditions were (a) H2O KBr HBrNaNO2 minus15 C (b) MeOH KOH (2 eq) minus50 C (c) Ion exchange (Dowex50Wx2) and lyophilization (d) THF 4-methyl-morpholine isobutyl chlo-roformate 2-(4-aminophenyl)ethylamine minus15 C (e) CH2Cl2 4-chloro-3-(trifluoromethyl)phenyl isocyanate 0 C Details are given in the text

H2O (20 ml) was added dropwise (1 h) at minus15 C with stirring The reactionmixture was further stirred over night and then extracted seven times withEt2O (50 ml) The combined organic extracts were dried over Na2SO4 filteredand the solvent was removed in vacuo Yield 94 7975 g RS-2-Bromo-3-hydroxy-propionic-acid (3) as an yellow oil (Grosjean et al 1994) 1H NMR(CDCl3) δ (ppm) = 435 (dd J = 53 73 Hz 1H) 40 (ddd J = 63 121173 Hz 2H) 13C NMR (CDCl3) δ (ppm) = 1718 639 446

(b) RS-2-Bromo-3-hydroxy-propionic-acid (3) (763 g 45 mmol) was dis-solved in MeOH (60 ml) and cooled to minus50 C KOH (512 g 90 mmol 2 eq)dissolved in MeOH (35 ml) was added dropwise (45 min) The pH of thesolution became neutral after stirring for three hours at room temperatureThen the solvent was removed in vacuo and the potassium salts were precipi-tated by adding Et2O (150 ml) and stirring for one hour The salt cake wasremoved by suction and heated with EtOH (100 ml) under reflux After hot


filtration the potassium-oxiranyl carboxylate precipitated when the solutionwas cooled to room temperature and further cooling over night at minus30 CYield 33 g potassium-oxiranyl carboxylate (4) Grosjean et al (1994) 1HNMR (D2O) δ (ppm) = 34 (dd J = 28 47 Hz 1H) 29 (m 2H) 13C NMR(D2O) δ (ppm) = 1718 64 446 1767 495 460 Anal (C3H3O3K) Ccalcd 2857 found 2621 H calcd 238 found 288

(c) Potassium-oxiranyl carboxylate (33 g 26 mmol) was dissolved in H2O(20 ml) and subjected to ion exchange chromatography on an acidic stationaryphase (Dowex 50Wx2) The free acid (5) was obtained by subsequent freezedrying over night as an yellow oil (193 g 183 mmol 70 yield) 13C NMR(D2O) δ (ppm) = 1736 477 466

(d) Compound 4 (212 mg 2 mmol) was solved in abs THF (10 ml) andcooled to minus15 C Then 4-methyl-morpholine (202 mg 2 mmol) isobutylchloroformate (273 mg 2 mmol) and 2-(4-aminophenyl)ethylamine (272 mg2 mmol) were added and the mixture was stirred for thirty minutes at minus15 CThe mixture was allowed to warm to rt and the precipitate was removed byfiltration and washed with THF The THF was partially removed in vacuoand CH2Cl2 (25 ml) was added and the solution was extracted two times with15 ml phosphate buffer (pH 7) The organic phase was dried over Na2SO4filtered and used directly for the next step

(e) 4-Chloro-3-(trifluoromethyl)phenyl isocyanate (200 mg 1 mmol) wasadded to the organic phase with stirring (0 C 30 min) The product (50 mg11 yield) readily precipitated from the solution and was removed by suctionand dried 1H NMR (DMSO-d6) δ (ppm) = 91 (s 1H) 87 (s 1H) 811 (s1H) 809 (m 1H) 76 (m 2H) 74 (d J = 84 Hz 2H) 71 (d J = 84 Hz2H) 33 (m 3H) 28 (m 4H) 13C NMR (DMSO-d6) δ (ppm) = 1676 15241394 1373 1331 1319 1289 1267 (CF3) 1229 1187 485 456 399342 IR ν cmminus1 1655 1596 1542 1515 1484 1416 1310 1258 1227 11751129 1032 888 828 685 662 Anal (C19H17N3O3ClF3) H C calcd 5334found 5132 N calcd 982 found 920

312 Activity of compound 1 in vitro

Compound 1 showed inhibitory activity toward B-RAF and C-RAF in vitrothat was lower compared to BAY 43-9006 as shown in Figure 34 and Table 31In addition we did not detect that 1 was an irreversible inhibitor in timekinetics experiments suggesting a very slow reaction rate (see Figure 35) Todetect very low levels of modified protein we performed mass spectrometricmeasurements of the RAF protein pre-incubated with 1Mass spectrometry GST-C-RAF-Y340DY341D was digested with trypsinafter respectively without pretreatment with compound 1 (100 microM 50 min


30 C) The resulting peptides were used for mass spectrometric analysis Theratio of the signal intensities corresponding to peptides containing cysteine657 (corresponding to Cys532 in B-RAF) and other peptides was comparablein both samples (Figure 36) indicating that at least the major amountof protein was not covalently modified at cysteine 657 Furthermore nosignal corresponding to compound 1 linked by a thioether bonding with apeptide containing cysteine 657 could be detected Our results indicate thatno significant covalent binding occurs between 1 and the protein

313 Activation in cell culture

A striking observation was made in cell culture experiments NIH 3T3 fibrob-lasts transformed by constitutively active RAF were treated with compound 1The transformed phenotype was not reversed (data not shown) Examinationof pERK levels revealed that the mitogenic signaling pathway was actually$ampamp

($)

+++- ++ ++- + +- - + - +++

+

0+

+

1+

++

+230-45++24678

230-45++94678

9amplt=gt24678

9amplt=gt94678

$amp(amp)+

-$amp))amp0

Figure 34 Inhibition of RAF kinases in an in vitro kinase assay Theplot depicts inhibition of B-RAF and C-RAF by compound 1 and BAY 43-9006 in vitro using a coupled RafMEKERK ELISA assay The kinaseactivity relative to the DMSO control is plotted against the concentration ofthe inhibitors BAY 43-9006 posses higher potency than 1 However the IC50

values we measured were considerable higher than described in the literatureThe assay was repeated three time and gave highly reproducible results

31 Development of a novel RAF kinase inhibitor 45$

amp()+

+ + + -+ + + ++

+

+

+

0+

++

+

+

+

++)12

-3)12

+)12

-3)12

)12

+3-)12

+3)12

+3+-)12

+3+)12

+3++-)12

$amp()$+-$

amp$$01

Figure 35 Time kinetics experiment Performed to distinguish reversiblefrom irreversible inhibition C-RAF was pre-incubated with compound 1 atroom temperature for a variable amount of time (x-axis) Subsequently ATPwas added and the kinase activity was detected as described in Materials andMethods An irreversible inhibitor would show enhanced inhibition (y-axis)when it is given more time to react to the protein Compound 1 did notshow any increased activity at any concentration (different graphs) when thepre-incubation time was increased

up-regulated after treatment with the inhibitor The activation was profound(Figure 37A) and rapid (Figure 37B) The counter-intuitive activation of theRAF signaling pathway in cell culture was already described for other RAFkinase inhibitors such as ZM 336372 (Hall-Jackson et al 1999a) GW 5074(Lackey et al 2000 Chin et al 2004) and SB 203580 (Hall-Jackson et al1999b) (Figure 15) but the mechanism is unknown

Dimerization of RAF kinases leads to activation in a Ras-dependentmanner (Farrar et al 1996 Luo et al 1996 Rushworth et al 2006) Manyoncogenic mutants of B-RAF have impaired kinase activity but neverthelessactivate the mitogenic signaling pathway by dimerizing with C-RAF (Wanet al 2004) Most of these mutations are detected in the glycine rich loopand the activation segment and thus overlap with the inhibitor binding siteDiphenyl urea inhibitors bind to protein kinases in the DFG-out conformation(Pargellis et al 2002 Wan et al 2004) The fact that the kinase domainof B-RAF could only be crystallized in the presence of BAY 43-9006 (Wan


Figure 36 Mass spectrometry data This diagram shows the mz range1850ndash2050 obtained from peptide samples generated by tryptic in-gel digestionof GST-C-RAF-Y340DY341D after (upper part) respectively without (lowerpart) pretreatment with compound 1 (100 microM 60 min 30 C) Peptides withthe mz 185787 correspond to AA861ndash877 peptides with the mz 204499correspond to AA490ndash508 Peptides with the mz 198394 correspond to thepeptide containing the cysteine corresponding to cysteine 532 (AA648ndash664)

et al 2004) is another indication that RAF-RAF interactions are effectedby binding to these ligands We assumed that compound 1 may activateRAF kinases by induction of hetero-dimerization of B- and C-RAF Totest this hypothesis we treated starved mouse embryonal fibroblasts fromC-RAFminusminus and B-RAFminusminus mouse embryos with compound 1 BAY 43-9006and ZM 336372 We did not detect a significant activation by compound 1and BAY 43-9006 indicating that compound 1 indeed activates through theformation of heterodimers (Rushworth et al 2006) In contrast activation wasobserved in the presence of ZM 336372 (Figure 38) This may indicate thatthere are multiple mechanism for paradoxical activation or that ZM 336372recruits A-RAF for hetero-oligomerization

To test for the formation of heterooligomers we co-expressed His-taggedB-RAF and C-RAF in Sf9 insect cells The cells were treated with inhibitorfor thirty minutes prior to lysis The lysates were subjected to size exclusion


Figure 37 Elevated levels of pERK after treatment with compound1 A Lanes 1 and 7 DMSO controls lanes 2ndash6 decreasing concentrations ofBAY 43-9006 (10 8 6 4 and 2 microM) lanes 8ndash12 decreasing concentrationsof compound 1 (24 22 20 18 16 microM) B pERK levels after different timepoints Lane 1 DMSO control lanes 2ndash12 10 20 30 40 50 60 70 80 90100 110 min treatment with compound 1 (20 microM)

gel filtration chromatography The fractions were subjected to SDS-PAGEimmuno blotting using an anti-penta-His antibody to detect RAF proteinsWe could clearly show that C-RAF elutes only in high mass and the lowmass fractions indicating that it is in a partially oligomerized state Howeveraddition of inhibitors did not alter the profile (Figure 39)

Thus the alternative hypothesis should also be considered activation byinhibition of an inhibitory kinase in a pathway not active under starvationIn fact several such kinases would be potential targets for this (Appendix A)

314 Other compounds

Compound 1 is was the last of several compound which were synthesized andtested in in vitro kinase assays

Synthesis of N-(24-[([4chloro3(trifluoromethyl)phenyl]amino-carbonyl)amino]phenylethyl)acrylamide (7)


Figure 38 No activation by compound 1 in starved RAF knockoutcells Mouse embryonal fibroblasts (MEFs) from C-RAFminusminus and B-RAFminusminus

knockout mouse embryos were starved for 42 hours in 005 serum priorto treatment with different kinase inhibitors for one hour Lanes 1 and 7DMSO controls lane 2 3 and 8 stimulation with 20 FCS lane 6 and 12ZM 336372 inhibitor (10 microM) lane 5 and 11 compound 1 (20 microM) lane 4 9and 10 BAY 43-9006 (800 nM)

Figure 39 Hetero-oligomerization of B-RAF and C-RAF in vitroHis tagged B- and C-RAF were coexpressed in Sf9 cells and subjected to gelfiltration chromatography as described in Experimental Section The proteinstandards thyroglobulin (670 kDa) and aldolase (158 kDa) have elusion peaksas indicated by the arrows

(a) 4-Dimethylaminopyridine (DMAP 9 mg) di-tert-butyl dicarbonate(Boc2O 151 mg) triethylamine (111 microl) and acrylic acid (48 microl) were dissolvedin CH2Cl2 and stirred (30 min) at room temperature 2-(4-Aminophenyl)ethyl-amine (9) was added and the solution was stirred (3 h) at room temperatureThe solution was extracted three times with 10 ml phosphate buffer (pH 7)The organic phase was dried over Na2SO4 filtered and the solvent was removedin vacuo gaining 8

32 Dynamic modeling 49

(b) 8 was dissolved in abs THF and 4-chloro-3-(trifluoromethyl)phenylisocyanate (94 mg 042 mmol) was added with stirring (0 C 30 min) Theproduct (7) readily precipitated from the solution and was separated bysuction and dried

Synthesis of 4-[([4-Chloro-3-(trifluoromethyl)phenyl]aminocar-bonyl)amino]phenyl acrylate (10)

(c) p-Aminophenol (11 5 g 46 mmol) and di-tert-butyl dicarbonate (Boc2O10 g 46 mmol) were stirred in THF (18 h) at room temperature The THFwas removed in vacuo (8)

(d) The potassium salt of 8 (5 mmol) was dissolved in THF and acrylicacid chloride (045 g 5 mmol) was added (minus10 C 18 h) The solution wasfiltered and the THF was removed in vacuo 13 was crystalized in isopropanol

(e) 13 (01 g 038 mmol) was was dissolved in CH2Cl2 and trifluoroaceticacid (TFA 12 eq) was added After stirring at room temperature (4 d) 10 mlof a saturated Na2CO3 solution was added and the two phases were separatedThe aqueous solution was extracted two times with CHCl3 the organic phaseswere united dried over NaSO4 and the solvent was removed in vacuo (14)

(f) 14 (0054 g 0331 mmol) and 4-chloro-3-(trifluoromethyl)phenyl iso-cyanate were stirred in Et2O (1 h) at room temperature 10 precipitatedreadily from the reaction mixture It and was separated by suction and dried

Inhibition of C-RAF and MEK by 1 7 10 and commercial RAF kinaseinhibitors is presented in Table 31

32 Dynamic modeling

As Table 12 on page 30 shows there are many different cellular responsesmediated by RAF in the RAF-MEK-ERK cascade such as proliferationcell cycle arrest or differentiation survival and transformation From a cellbiology point of view these various responses depend on the cellular contextTo examine how far these different effects can be mediated by the type ofRAF-molecule present we first did mathematical modeling of the RAF-ERKcascade as shown in Figure 12 on page 24 The signaling module consists ofseveral steps (Ras-GTP RAF MEK ERK) Furthermore there is an intensiveinterplay between kinases (with specific activities αi) and phosphatases (withspecific activities βi) as depicted in Figure 311 To model these activitieswe use a formalism introduced by Heinrich et al (2002) Applied on themitogenic signaling pathway this leads to a set of differential equations for theactivities of each kinase or phosphatase implicated in the pathway accordingto the summary Equation 31


NH2

NH

O

HN

HN

OCl

CF3

NH

O

7

9 8

a

b8

NH2

H2N

NH2

HO

NHBoc

HO

NH2

O

O

HN

O

O

HN

OCl

CF3

NHBoc

O

O

11

10

12

13 14

13c d

e

f14

Figure 310 Synthesis of compounds 10 and 16 The synthesis routeis briefly sketched The reaction conditions were (a) DMAP Boc2OEt3N acrylic acid CH2Cl2 rt (b) THF 4-chloro-3-(trifluoromethyl)phenylisocyanate 0 C (c) p-Aminophenol Boc2O THF rt (d) Acrylic acidchloride THF minus10 C (e) CF3COOH CH2Cl2 rt (f) 4-Chloro-3-(trifluoromethyl)phenyl isocyanate CH2Cl2 rt Details are given in thetext


Conc 1 7 10 BAY ZM GW 1 7 10

3 nM 97 100 119 140 107 88 111 104 8610 nM 95 105 117 137 99 83 100 96 9333 nM 79 91 99 81 74 58 108 90 91

100 nM 81 96 104 4 51 44 87 91 95333 nM 63 86 117 4 17 33 87 93 97

1 microM 56 70 111 4 8 14 106 73 9833 microM 40 63 100 3 4 3 95 77 9910 microM 20 53 76 4 3 2 97 79 9833 microM 11 50 49 3 3 3 78 70 82

100 microM 6 19 14 4 3 2 25 48 23

Table 31 Inhibition of C-RAF and MEK by different inhibitorsin vitro Compounds 1 7 and 10 inhibit ERK phosphorylation in the acoupled C-RAF-MEK-ERK ELISA assay The inhibitory activity of 7 and 10is very low compared to the commercial RAF kinase inhibitors BAY 43-9006(BAY ) ZM 336372 (ZM ) and GW 5074 (GW ) (Figure 15 p 28)and also compared to 1 (Figure 31 p 40) The same activity is detected inan MEK-ERK kinase assay () indicating that 7 and 10 do not show anysignificant binding to RAF kinases However 1 clearly shows inhibitionmdashandthus bindingmdashto C-RAF

dXi

dt= αiXiminus1

(1minus Xi

Ci

)minus βiXi (31)

Although there are a number of components involved modeling usingMatlab (MathWorks Inc Natick MA) to solve the set of differential equationssummarized by the formula in Equation 31 shows for standard parametersthat the basic function of the cascade is signal amplification an input signalactivates the Ras receptor module (assumed to be one module for simplicity)with an exponential decay Subsequent peaks of RAF MEK and ERK followeach other with some time delay (time in arbitrary units) and the signal peakis augmented throughout the cascade (Figure 312)

However this standard behavior does not yet explain the complex differ-ential responses known from cell biology studies (Table 12) We reasonedthat the different isoforms of RAF in particular B-RAF and C-RAF arecentral for the differential responses mediated by the cascade In particularthere are indications for differences in their dephosphorylation and activationin the RAF-ERK cascade For example data (Lew 2003) for ERK showthat for each phosphorylation step the activity increases in a specific manner


Grb2

RTK

Sos

RasGDP

SHC

RasGTPGAPs

RAFRAF

Plasma membrane

1

1

Sig

na

l

MEK

ERK

MEK

ERK

PP

2

PP3

2

3

Figure 311 Model of the Ras-ERK signaling pathway Mapping theparameters of the ordinary differential equation (Equation 31) by Heinrichet al (2002) to the components of the RasRAFMEKERK cascade Symbolsused Xi denotes the concentration of active kinase i αi is the second-orderrate constant for the phosphorylation of kinase i by kinase (i minus 1) βi is afirst-order rate constant for the dephosphorylation of kinase i and Ci is thetotal concentration of kinase i

(for the first phosphorylation an increase of either 80 or 1000 fold activityfor a combined phosphorylation a 50000 fold increase) Since RAF is thecentral part in this cascade we next investigated how far known differences inactivation and dephosphorylation could influence the output obtained fromthe cascade Data indicate that B-RAF can be stronger activated whereasC-RAF is weaker activated

The total concentrations of the respective kinases were set to values typicalfor eukaryotic cell lines reported by Ferrell (1996) (CRas = 33 CRAF = 17CMEK = 1300 CERK = 1250) Values for α were estimated to be 600 forMEK and ERK Based on kinetic data reported by Lew (2003) (αMEK = 600αERK = 600) Ras-GTP was assumed to have a half life of 10 minutesaccording to data from Qui and Green (1992) (setting of λ = 0069 half lifedecay measured in seconds) Unfortunately we could not apply experimentallyproven β values for the respective phosphatases however we were able to


0 10 20 30 40 50 60 70 80 90 100minus02

0

02

04

06

08

1

12Model of the RAF minus Cascade

Inte

nsity

X(t)

Time t

RASRAFMEKERK

Figure 312 Response curve for the Ras-ERK pathway under stan-dard conditions An input signal activates the Ras receptor module withan exponential decay Subsequent peaks of RAF MEK and ERK followeach other with some time delay (time in arbitrary units) and the signalis amplified throughout the cascade Parameters used for generating thegraphs were αRAF = 01 αMEK = 1 αERK = 5 βRAF = 05 βMEK = 05βERK = 1 CRas = 1 CRAF = 10 CMEK = 20 CERK = 30 λ = 1

set sensible values for the phosphatases by applying kinetic parameters fromthe Brenda database (Schomburg et al 2004) Since usually more than onephosphatase is involved in inactivation of the pathway our estimations are onlyapproximate (βMEK = 170 βERK = 170) The situation is even more complexwith RAF which is both positively and negatively regulated by phosphatasesThus we could only estimate the β values taking into account the highersensitivity for C-RAF kinase activity towards phosphatases C-RAF requiresphosphorylation on residues S338 and Y341 for complete activation In B-RAF S445 (equivalent to S338 in C-RAF) is constitutively phosphorylated


0 5 10 15 20 25 300

20

40

60

80

100

120

140

160BminusRAF

Activ

atio

n X(

t)

Time t (minutes)

RASRAFMEKERK

A

0 5 10 15 20 25 300

05

1

15CminusRAF

Activ

atio

n X(

t)Time t (minutes)

RASRAFMEKERK

B

0 05 1 15 20

50

100

150

BminusRAF

Activ

atio

n X(

t)

Time t (minutes)

RASRAFMEKERK

C

0 02 04 06 08 1 12 14 16 18 20

02

04

06

08

1

12

14

CminusRAF

Activ

atio

n X(

t)

Time t (minutes)

RASRAFMEKERK

D

Figure 313 Simulation showing the qualitative differences betweenB-RAF and C-RAF C-RAF shows a short-lived intense peak decreasingquickly with time whereas B-RAF shows a sustained strong activity A rapidresponse of the cascade with almost no delay in the output signal mediated byERK is observed for both RAF isoforms We obtained different qualities forthe signal peak mediated by B-RAF (A and C same simulation but differentscaling of the x-axis) compared to the peak mediated by C-RAF (B and Dsame simulation but different scaling of the x-axis) The parameters used areshown in Table 32


Parameter Value Reference

Protein concCRas 33 Ferrell (1996) Robubi et al (2005)CRAF 17 Ferrell (1996) Robubi et al (2005)CMEK 1300 Ferrell (1996) Robubi et al (2005)CERK 1250 Ferrell (1996) Robubi et al (2005)

Kinetic constantsλ 0069 Qui and Green (1992) Robubi et al (2005)αRAF 1000 10 Robubi et al (2005)αMEK 600 Lew (2003) Robubi et al (2005)αERK 600 Lew (2003) Robubi et al (2005)βRAF 8 80 Robubi et al (2005)βMEK 170 Robubi et al (2005)βERK 170 Robubi et al (2005)

Table 32 Parameter values for the simulations of the RasRAFMEK-ERK cascade (Figure 311) The simulation results are presented in Fig-ure 313 based on the set of ordinary differential equations (Equation 31) byHeinrich et al (2002)

$amp

$(

)

+

-+

(

0+1

2(

3$

45657$845-57$8

Figure 314 Gel showing different expression levels of RAF kinasesin different tissues The total kinase concentration can have profoundeffects on signal intensity but only a slight effect on signal duration whichin our model depend primarily on the kinetic parameters The proteinconcentrations for several cell types are shown Equal amounts of totalprotein (25 microg) were loaded


His

-B-R

afH

is-C

-Raf

-wt

GS

T-C

-Raf

-RL

GS

T-C

-Raf

-375

WG

ST-

C-R

af-3

403

41D

DG

ST-

C-R

af-w

t

P-ERK

Figure 315 Kinase assays showing the activity of different prepara-tions of RAF kinases purified from Sf9 cells The protein purification theassay conditions and the immuno blotting are described in Materials andMethods B-RAF shows far higher kinase activity than C-RAF irrespectiveof the tag However C-RAF-Y340DY341D shows high kinase activity asdoes C-RAF-RL for which C-RAF was coexpressed with oncogenic RasV12and Lck C-RAF-K375W shows no kinase activity (negative control) Therange between 25 and 50 kDa is shown P-ERK has a mass of approximately42 kDa No further bands were detected

and the Y341 residue is replaced by aspartate Thus B-RAF is primed foractivation and more resistant against inactivation (Garnett and Marais 2004)

Furthermore we tested a range of parameter values additional data andplots are shown in Figure A1 in Appendix A (the wide range of parametersinvestigated leads to different scales on the y-axis of the plots) The plotsin Figure 313 are close to the real situation using the above available dataand estimates and combining them with our model formalism we obtaineddifferent qualities for the signal peak mediated by B-RAF compared to thepeak mediated by C-RAF (Figure 313) The specific parameters estimatedfor B-RAF (αBminusRAF = 1000 βBminusRAF = 8) lead to a broad concave peak(Figure 313A) and with an almost constant behavior for the output signalin the early time steps (Figure 313C) Note furthermore that parameterswe estimate to be present in the tissue change the behavior of the cascadefrom that in Figure 312 into a rapid response of the cascade with almost nodelay in the output signal mediated by ERK (Figure 313C within secondsthe cascade is also at top activation for ERK) This rapid signal mediation isalso observed for C-RAF (Figure 313D αCminusRAF =10 βCminusRAF =80) Howeverthe response curve is qualitatively different of convex shape and leads to


an intensive short peak which is rapidly declining Of course it is only asimplified model however we took for most parameters available biochemicaldata which allow us to demonstrate that indeed B-RAF behaves qualitativelydifferently from C-RAF

The tissue-specific graphs in Figure 313 model the qualitative differenceswhich exists between B-RAF (sustained high level on state) and C-RAF(short high peak then going down quickly with time) in the cellular cascadethe phosphatases react fast leading to quick inactivation C-RAF is quicklyactive (steep and strong signal amplitude) but also quickly deactivated B-RAF is somewhat slower active but very slowly inactivated its activity curvealso in the model follows somewhat Ras-GTP

Furthermore if we now take Table 12 into account we realize that theparameters for activated B-RAF are indeed well suited to mediate functionsknown for B-RAF ie cell cycle arrest and differentiation whereas thetransient intensive peak predicted for C-RAF should explain why for C-RAFoften a proliferation response is observed The overall signal in a cell whichhas both RAF isoformes would of course be a combination of both effects

According to this model we would expect and predict that in variouscell types the distribution of B-RAF and C-RAF is in fact different to allowmediation of different cellular responses in a variety of tissues To furthersupport this we investigated the respective amount of B-RAF and C-RAF invarious cell lines (Figure 314) The protein concentrations for several celltypes are shown Equal amounts (determined by direct colorimetric assaysee Materials and Methods) of total protein (25 microg) were loaded to allowcomparisons between different cell lines As the immuno blot data indicateB-RAF is present in high amounts in melanoma cells HepG2 cells and PC12cells whereas C-RAF is the more dominating RAF in HEK293 cells andHeLa cells We can thus indeed demonstrate a strong variation of B-RAFand C-RAF in these different cell lines Note that the levels of B-RAF as wellas of C-RAF isoforms change in specific tissues Our immuno blots providea good estimate of the relative changes regarding one isoform in differenttissues and show that levels for one isoform do vary in different tissues Incontrast the exact ratio between B-RAF and C-RAF is only approximatedby the band intensities as different antibodies were used for each isoform

To test our model predictions in respect of kinase activity differences weexpressed tagged RAF kinases in Sf9 cells and performed a coupled kinaseassay on the purified proteins (Figure 315)

We can show that B-RAF performs a high kinase activity without anyspecific intervention whereas C-RAF kinase activity is comparatively lowQuantitatively the difference in activities is about two orders of magnitudeThe model prediction for comparison is an 180 fold difference of RAF kinase


activity resulting in a 100 fold difference for ERK kinase activity at theend of the cascade (Figure 313) To obtain highly active C-RAF the modelprediction suggests that the effect of the phosphatase is a critical aspect Oneway to explore this experimentally would be to treat cells with phosphataseinhibitors prior to stimulation and assay immunoprecipitates of RAF kinasesfor activity However dephosphorylation is important also for RAF kinaseactivation and treatment of cells with unspecific phosphatase inhibitors suchas Okadaic acid was in fact shown to block activation of C-RAF (Kubiceket al 2002) The effect of the phosphatase can be tested more specifically bygenetic experiments with mutations The important phosphorylation sitesfor activation in C-RAF are Y340 and Y341 as has been shown by previousinvestigations (Mason et al 1999) We mutated these to aspartate residuesto mimic constant phosphorylation In fact the resulting mutations at thephosphorylation sites Y340Y341 to aspartates (equivalent to D447D448 inB-RAF) lead to a greatly increased kinase activity in C-RAF (Figure 315) Inan additional test we show that Lck a tyrosine kinase able to phosphorylateC-RAF at 340341 achieves the same effect if it is coexpressed together withRasV12 (Figure 315 lane GST-C-RAF-RL) Thus also the experimentaldata support that the two RAF isoforms differ mainly in their sensitivitytowards phosphatases

33 DiRas3

331 DiRas3 interacts in vitro efficiently with activeC-RAF and MEK

To test in vitro the in vivo binding data regarding C-RAF association withDiRas3 we used BIAcore technology For that purpose purified GST-taggedC-RAF or MEK were immobilized to a CM5 chip coated with anti-GSTantibody Next the association and dissociation with purified DiRas3 weremonitored (Figure 316 Figure 317A) In accordance with our in vivo resultsDiRas3 bound with high affinity to C-RAF activated with RasV12 and Lck(C-RAF-RL) compared to non-activated C-RAF While the Ras bindingdomain (RBD) of C-RAF did not bind DiRas3 the catalytic domain of C-RAF (C-RAF-BXB-Y340DY341D designated as C-RAF-CT-DD) exhibitedhigh binding affinity Surprisingly the most efficient binding to DiRas3 wasrecorded with purified MEK Thus DiRas3 interacts in vitro with the catalyticdomain of C-RAF and even better with MEK The apparent affinity constants(KD values) revealed that MEK binding was about four fold higher thanbinding of DiRas3 to active C-RAF (018 microM and 080 microM respectively) In

33 DiRas3 59

comparison the binding of DiRas3 to MEK was even 25 times stronger thanthe interaction between H-Ras-GTP and C-RAF (018 microM versus 046 microM)

332 Inhibition of MEK activity by DiRas3 in vitro

To investigate the influence of His-DiRas3 and His-∆N-DiRas3 on kinaseactivities of the RAF-MEK-ERK signalling cascade we performed coupledkinase assays using an active mutant of C-RAF GST-C-RAF-Y340DY341D(designated as C-RAF-DD) purified MEK-1 and ERK-2 and increasing con-centrations of His-DiRas3 or His-∆N-DiRas3 Surprisingly DiRas3 inhibitedERK phosphorylation by MEK but not MEK phosphorylation by C-RAF(Figure 317B) The N-terminally truncated DiRas3 inhibited MEK activityto a much lower degree (compare lane 4ndash6 with 7ndash9) But again no effect onC-RAF activity was detected

Figure 316 DiRas3 interaction with C-RAF and MEKmdashBIAcoreThe biosensor chip CM5 was loaded with anti-GST antibody using covalentderivatization GST-tagged proteins were immobilized on the biosensor whichresulted in a deposition of approximately 800ndash1200 response units (RU) Nextpurified DiRas3-GDP was injected The unspecific binding was measuredin the reference cell and subtracted DiRas3 binds efficiently to MEK andactive C-RAF preparations (C-RAF C-RAF-BXB-DD) but reveals nosignificant binding to inactive C-RAF wild type (wt) The association ratesdiffer between the probes whereas the dissociation rate is similar and very lowfor all the probes tested GST is used as a negative control GST∆N-DiRas3shows no significant binding to MEK nor any other protein tested (data notshown)


Figure 317 DiRas3 interaction with C-RAF and MEK A Biosensoranalysis A CM5 sensor chip was loaded with anti-GST antibody using covalent derivatiza-tion Purified GST-tagged MEK full-length C-RAF C-RAF-RBD and a constitutivelyactive C-terminal part of C-RAF (C-RAF-BXB-DD) were immobilized considering theirmolecular size Following DiRas3-GDP injection (400 nM) association-dissociation curveswere monitored The bar represents the maximal association degrees BndashC) DiRas3inhibits MEK but not C-RAF in in vitro kinase assays B The effect of DiRas3 on MEKand ERK phosphorylation were monitored by use of an in vitro kinase assay with equalconcentrations of purified MEK-1 ERK-2 and GST-C-RAF-Y340DY341D (C-RAF-DD)The assay conditions were as described in Materials and Methods Lane1 no DiRas3 lane2kinase dead GST-C-RAF-K375W was used as a negative control lanes 3ndash6 01 microg 05 microg1 microg and 15 microg DiRas3-GDP lanes 7ndash9 1 microg 3 microg 5 microg ∆N-DiRas3-GDP C ERK-2 wasphosphorylated by active MEK-1 in presence of DiRas3 (15 microg) or ∆N-DiRas3 (15 microg)alone in presence of GST-C-RAF-DD (05 microg) or His-B-RAF (05 microg) D This experimentwas performed by Beck et al MCF10A cells treated with si-oligos targeting DiRas3 werestarved stimulated with serum after indicated time points lysed and analysed by immunoblot detecting total MEK and pMEK levels

33 DiRas3 61

To investigate whether the inhibition of MEK by DiRas3 is C-RAF de-pendent a MEK-ERK assay was performed omitting C-RAF Active MEKwas obtained by in vitro phosphorylation of purified MEK-1 using B-RAFand subsequent removal of B-RAF as described in Materials and MethodsThe MEK preparation obtained was highly active but not quantitativelyphosphorylated DiRas3 inhibited the kinase activity of MEK irrespective ofthe addition of active C-RAF or B-RAF (Figure 317C) On the other handno suppression of kinase activity was detected in the presence of ∆N-DiRas3In fact in the presence of active RAF kinases ERK phosphorylation waselevated compared to the MEK probe (lane 1) presumably because the MEKpreparation was not completely phosphorylated and the presence of activeRAF kinases led to elevation of pERK Based on these results we concludethat DiRas3 is a specific MEK inhibitor and that RAF kinases are not requiredfor this effect

Consequently Beck et al studied the effect of DiRas3 on MEK phosphory-lation in vivo In DiRas3 downregulated MCF10A cells no difference in MEKphosphorylation compared to control cells was detectable (Figure 317D)However as ERK phosphorylation was modified in comparable experiments(Beck et alndashsubmitted) we conclude that DiRas3 did not inhibit or alterMEK phosphorylation but reduced MEK activity to phosphorylate ERKThus we provide here in vitro and in vivo evidence that DiRas3 is a MEKinhibitor

Chapter 4

Discussion

41 Developing a novel RAF kinase inhibitor

In search for a novel irreversible RAF kinase inhibitor we were stimulated bythe unique cysteine 532 residue (B-RAF numbering GI50403720) The highreactivity of the epoxide moiety was a challenging task for the synthesis Theapproach to provide oxiranylcarboxylic acid (5) described in the literature(Grosjean et al 1994) did not work in our hands Eventually a novel syntheticapproach including a lyophilization step let ultimately to 5 We started thesynthesis with racemic serine and therefore obtained 4 as a racemate Howeverour synthetic strategy is well suited for the synthesis of enantiopure 4 andtherefore also compound 1

Compound 1 showed a clear and direct RAF kinase inhibition in vitroalbeit weaker than BAY 43-9006 (Figure 34 and Table 31) indicating thatit is delivered to the targeted site in the kinase domain However kineticand mass spectroscopic experiments strongly argue that the inhibitor wasprobably not covalently bound to the specific cysteine residue The homology

Figure 41 Reaction mechanism between a cysteine and an epoxideThe nucleophilic sulfur atom of the cysteine can only attack and covalentlybind to the epoxide moiety if it the back of the beta carbon atom is exposedto it See also Figure 32 on page 41 and Appendix A for a three dimensionalview

64 Chapter 4 Discussion

model and the in vitro data indicate that compound 1 can successfully bindto the targeted site however the orientation of the epoxide moiety relativeto the nucleophilic sulfur atom is crucial the sulfur atom needs to attackthe epoxide group from the back of the beta carbon (Figure 41) Thenatural ligand at this site is the planar purine ring system of ATP Theepoxide moiety is probably ill suited to mimic this electron-rich π-ring systemThe corresponding acrylamide derivative did also not show an irreversibleinhibition of RAF kinases in our time kinetics analysis (data not shown)

Compound 1 did not inhibit RAF kinases in cell culture In fact in RAFtransformed cell lines the compound even strongly activated the mitogenicsignaling pathway RAF activation through ZM 336372 or SB 203580 wasusually explained by feedback regulation of RAF (Figure 42A) Inhibition ofRAF also leads to inhibition of negative feedback regulation and therefore toactivation (in the absence of the inhibitor) Negative feedback regulation isdescribed at the level of SOS (Chen et al 1996) as well as RAF (Brummeret al 2003 Dougherty et al 2005 Hekman et al 2005) However thefact that inhibitors of MEK do not cause this activation argues against thathypothesis It appears more likely that a different target X is affected byRAF inhibitors (Figure 42B) This different target may be RAF itself ifthe activation in vivo relies on oligomerization of RAF kinases (Rushworthet al 2006) Such an oligomerization has already been hypothesized forSB 203580 but was not experimentally shown (Hall-Jackson et al 1999b)Although no evidence for oligomerization was obtained in vitro experimentswith RAF knockout cells were consistent with this hypothesis An alternativeexplanation might be the inhibition of an inhibitory kinase All proteinkinases with Thr106 (p38α numbering) could be candidates for such a role(Hall-Jackson et al 1999b)(see Appendix A) However this criterion is notthat strict for diphenyl urea compounds as some non-Thr106 kinases (such asVEGFRs) are also potently targeted by such inhibitors

Compound 1 is the first diphenyl urea compound for which an activationof RAF kinases in cell culture was described The implications this may havefor BAY 43-9006 and other compounds are not clear BAY 43-9006 is a rathernon specific inhibitor with activity against a wide range of important targetsIt blocks the mitogenic signaling pathway in many tumor cell lines but notin human non-small-cell lung cancer (NSCLC) carcinoma lines This may bedue to a RAF independent activation of MEK in this cell line (Wilhelm et al2004) However BAY 43-9006 also failed to impair ERK phosphorylationand reduce tumor size in a transgenic mouse tumor model with C-RAFdriven lung adenomas arguing against a RAF independent mechanism ofMEK activation in lung cancer In the same mouse model treatment withCI-1040 (PD 184352) a potent MEK inhibitor lead to a significant reduction

41 Developing a novel RAF kinase inhibitor 65

Grb2

RTK

Sos

RasGDP

SHC

RasGTPGAPs

RAFRAF

Plasma membrane

Sig

na

l

MEK

ERK

MEK

ERK

PP

PP

Inhibitor

X

Grb2

RTK

Sos

RasGDP

SHC

RasGTPGAPs

RAFRAF

Plasma membrane

Sig

na

l

MEK

ERK

MEK

ERK

PP

PP

B

A

Figure 42 Model of the mitogenic signaling pathway A Negativefeedback regulation has been reported at the level of SOS (Chen et al 1996)and RAF (Brummer et al 2003 Dougherty et al 2005 Hekman et al 2005)B RAF kinase inhibitors may however activate by binding to a protein Xwhich may facilitate its activation This protein might in fact be RAF itself(Hall-Jackson et al 1999b) However we were not able to detect increasedRAF association upon treatment with inhibitor The differences observedin different cell type (Hall-Jackson et al 1999b Wilhelm et al 2004) alsosuggest that protein X is a different protein kinase


of ERK phosphorylation and adenoma size (Kramer et al 2004) Thereforea conditional RAF activation by BAY 43-9006 can at least not be ruled out

We have set up the entire route for the development of a kinase inhibitortargeted against an Thr106 and Cys109 (p38α numbering) protein kinaseRAF including molecular modeling the synthesis in vitro assay cell cultureand mass spectrometry Given that there are only 14 genes in the humangenome coding for with a Thr106 and Cys109 kinase domain (Speg PDGFRαPDGFRβ Kit Fms KSR ANP-A ANP-B RETGC-1 RETGC-2 NEK11and A- B- and C-RAF) a specific irreversible RAF kinase inhibitor on thisbasis is a real perspective andmdashin the opinion of the authormdashneeds to bepursued further

42 Dynamic modeling

We show here how tissue specific variation in RAF-response can be explainedin terms of different distribution of B-RAF and C-RAF and their differentresponse to activation by kinases and inactivation by phosphatases Inparticular we could adopt a standard model of response by introducing moreaccurate parameters known from experimental data and show that this leadsto qualitatively different behavior in B-RAF and C-RAF signaling Thedifferences in peak shape and length accord with their different effects oncells To further support our hypothesis of differential effects in tissues bydifferential behavior of B-RAF and C-RAF we experimentally confirm thattheir quantitative distribution varies strongly in different cell lines Certainlyour analysis is based on a simplistic model but nevertheless it demonstratesthat core signaling molecules existing in different isoforms can in fact mediatedifferent tissue specific signals (Table 12 p 30) for the concrete system ofthe RAF-MEK-ERK cascade

Our mathematical model which was based on a formalism for a linearsignaling cascade described by Heinrich et al (2002) but now takes differentisoforms and their ratios into account strongly simplifies a number of furtherfactors that have been described in literature In this study we did not takeinto account possible crosstalk with other signaling modules like Rap1 nordid we include negative feedback regulation (Dougherty et al 2005 Hekmanet al 2005) A-RAF was not considered since it possesses the lowest kinaseactivity of the RAF kinases and is mostly expressed in urogenital tissue (Stormet al 1990) We further simplified the complex regulation of RAF kinases byusing single rate constants to calculate their activation and inactivation inour model

More complex models include different terms producing more complex


results Thus negative feedback regulation is an important factor in MAPKcascades For example this was predicted to lead to quantitative differencesin the EGF and NGF signaling in PC12 cells In this model which usesonly one type of RAF this factor was found to be an important ingredientin determining cascade activation (Brightman and Fell 2000) HoweverYamada et al (2004) did not find this effect in their simulations includingfeedback regulation In contrast they investigated the effect and found asignificant role for fibroblast growth factor receptor substrate 2 (FRS2) inthe NGFFGF pathway regarding sustained MAPK activation In this casethe authors used a detailed model of the receptor activation including Grb2-SOS and FRS2 Thus differential effects of feedback regulation do have animportant modulatory effect on the mitotic signaling pathway and durationof activation Moreover the feedback regulation of C-RAF (Dougherty et al2005) might be rather different from the partly ERK-mediated feedback inB-RAF (Brummer et al 2003) however there is no quantitative data onB-RAF feedback regulation Furthermore these are in addition and separatefrom the effect of the different RAF isoforms the focus of this study andmodeled here in the simplified cascade shown in Figure 311 on page 52 basedon parameters shown in Table 32 on page 55

A clear limitation arises from our Ras term It assumes that Ras-GTP ispresent at high concentrations at time point 0 and declines in a first orderreaction This is an approximation that doesnrsquot hold true for most realsystems Note also that our model results are in line with a detailed model ofRasRAFMEKERK activation presented in a recent article by Sasagawaet al (2005) focusing on the interplay between Ras and Rap1 For thismodel PC12 cells was considered and clearly distinct dynamics of transientand sustained ERK activation resulted by the rapid increase of epidermalgrowth factor and nerve growth factor but not on their final concentrationThis was validated by measurements of ERK phosphorylation Peyker et al(2005) experimentally observed clear effector differences between differentRas isoforms In the context of our model different receptor tyrosine kinasespossess different rates of deactivation Slower rates of deactivation for receptortyrosine kinases (and Ras) will lead to prolonged signals as seen in manycancer cells whereas high expression levels cause higher signal intensities Thehuge number of receptor tyrosine kinases suggests a high degree of regulationalready at this step (Offterdinger et al 2004) For simplicity we did notconsider complex effects of scaffolds and other factors further modifying andchanging kinase activity in B-RAF and C-RAF These complicating factorswill be included in later studies However our model despite of its simplicitysuggests different cellular responses (Table 12 p 30) mediated by the differentisoforms


Before time series experiments provide detailed kinetic data on the com-plete cascade we can only conclude that our model is supported by all thekinetic data reported on the cascade so far and by the experimental datashown here on isoform specific different expression levels in different tissuesand differential behavior of the RAF kinase isoforms against phosphatases

The regulation of RAF kinases and B-RAF in particular is also a focusfor cancer research Mutations of B-RAF are detected in a number of tumorsMost mutations generate a B-RAF with elevated and constitutive kinaseactivity however some B-RAF mutants possess impaired but neverthelessconstitutive kinase activity These rare mutations may coincide with Rasmutations which are not detected in tumors with highly activating B-RAFmutations These data indicate that tumours depend on a prolonged buttightly modulated B-RAF signaling (Garnett and Marais 2004)

Another point is that the concentration of RAF kinases might be ratherdynamical for a given cell type (Cleveland et al 1994) Higher proteinexpression levels of the kinases will elevate signal intensity but have almost noeffect on signal duration which is primarily determined by kinetic propertiesand the expression levels of the phosphatases

It might be assumed from comparison of both RAF kinases that C-RAFis not predominant However in situations for which activation by B-RAFalone is not sufficient the additional activation by C-RAF may becomecritical This depends on the quantitative ratio between B-RAF and C-RAFwhich was not yet accurately modeled here considering further modifyingfactors and scaffolds Indeed Trakul et al (2005) showed in siRNA depletionexperiments both C-RAF and B-RAF are important as the total RAF activityis reduced by 60 versus 90 respectively if one or the other RAF isoformis inactivated In fact both are required as predicted but further technicalimprovements will be necessary to get exact quantitative data B- and C-RAFare almost equally important for the initial signal intensity but it is mostlyB-RAF which is responsible for signal duration

43 DiRas3

Results presented in this contribution demonstrate that DiRas3 a Ras-likeGTPase interacts with activated C-RAF and is a direct negative regulatorof MEK activity The interaction of DiRas3 with C-RAF is in several waysunusual First DiRas3 bound directly to the catalytic half of C-RAF (Fig-ure 316 p 59) It represents the first GTPase with such an affinity Yet wedid not detect an inhibition of C-RAF kinase activity in an in vitro kinaseassay where already activated C-RAF was used (Figure 317B p 60) Also

43 DiRas3 69

DiRas3 downregulation did not affect MEK phosphorylation (Figure 317DBeck et alndashsubmitted) Though we did not detect a functional consequenceof this interaction the function of other proteins binding to RAF like 14-3-3paxillin or KSR might be modified (McKay and Morrison 2007) SecondDiRas3 and Ras-GTP can bind simultaneously to C-RAF Additionally Di-Ras3 increases the amount of Ras-GTP bound to the DiRas3-C-RAF-complex(Beck et alndashsubmitted)

This cooperativity might be caused by a DiRas3 mediated stabilization of aparticular RAF conformation which engages the CRD Therefore by blockingthe RAF-kinase cascade DiRas3 may trap Ras-GTP in signalling dead endRAF-complexes C-RAF seems to act similarly to WASP (Wiskott-Aldrichsyndrome protein) as an ldquoandrdquo gate whereby integrating two distinct GTPasesignals (Prehoda et al 2000) The first signal leading to the activation ofRas and the second one leading to the association of DiRas3 to RAF seemto be both necessary for MEK inhibition at the plasma membrane Thesecond signal is likely to be triggered by steroid hormones as DiRas3 ismainly expressed in ovarian and breast tissue (Yu et al 1999) which undergomonthly cycles of proliferation and apoptosis This scenario is supported byup to four different principles of expression regulation of DiRas3 (Yu et al2005) It remains to be established whether DiRas3 interacts also with A-

Grb2

RTK

Sos

RasGDP

SHC

RasGTPGAPs

RAFRAF

Plasma membrane

Sig

na

l

MEK

ERK

MEK

ERK

PP

PP

DiRas3

Figure 43 DiRas3 binds to RAF as well as to MEK and blocksMEK from phosphorylating ERK A detailed description of the signalingcascade is given in Figure 12 on page 24 Our data show clearly that DiRas3does not inhibit RAF kinases despite binding to C-RAF


and B-RAFBased on our data we propose the following model for the regulation of

the mitogenic signalling cascade by DiRas3 signal induced Ras-GTP recruitsC-RAF within the plasma membrane to initiate RAF activation ThereafterDiRas3 can bind to the open conformation of C-RAF Beck et al identifiedAA150ndash331 of C-RAF encompassing the CRD and CR2 and the catalyticCR3 domain in C-RAF as potential binding interfaces between DiRas3 andC-RAF (Beck et alndashsubmitted) Not all of these domains may be boundsimultaneously to DiRas3 Remarkably 14-3-3 proteins bind to CR2 and CR3and upregulate RAF kinase activity as shown in several model organisms(Wilker and Yaffe 2004) Thus DiRas3 may displace in vivo 14-3-3 from RAFto downregulate the RAF activity Consistently DiRas3 did not interfere withRAF activity in the in vitro assays where already activated RAF kinase wasused The binding of DiRas3 to the CR3 region of C-RAF may also result inreduced access of activating kinases or in impaired binding of the substrates ofthe RAF kinase Thus DiRas3 might negatively influence the complete RAFactivation within the plasma membrane In addition to its ability to bindactive C-RAF in the plasma membrane we demonstrate here that DiRas3 canefficiently associate with MEK and inhibit its kinase activity Interestinglyin vitro and in vivo experiments demonstrate that phosphorylation of MEKby C-RAF is not influenced by DiRas3 The signal transduction from MEKto ERK is however nearly abolished in the presence of DiRas3 (Figure 317p 60) Thus DiRas3 might not only influence the activation of C-RAF but itmight also need active C-RAF to inhibit MEK suggesting a scaffold function(Figure 43)

Efficient activation of RAF needs the redistribution of Ras-GTP from raftmicro domains into non-raft regions of the plasma membrane (Prior et al2001) Therefore a similar relocation of the RAF-DiRas3-complex at theplasma membrane may release the binding domain of DiRas3 that is neededfor MEK association We suggest that a change of lipid micro-environmentmay induce association of DiRas3 with MEK terminating RAF signaling

Bibliography

L F Allen P F Lenehan I A Eiseman W L Elliott and D W FryPotential benefits of the irreversible pan-erbB inhibitor CI-1033 in thetreatment of breast cancer Semin Oncol 29(3 Suppl 11)11ndash21 Jun 200241

Y Aoki T Niihori H Kawame K Kurosawa H Ohashi Y TanakaM Filocamo K Kato Y Suzuki S Kure and Y Matsubara Germlinemutations in HRAS proto-oncogene cause Costello syndrome Nat Genet 37(10)1038ndash1040 Oct 2005 doi 101038ng1641 URL httpdxdoiorg

101038ng1641 22

T I Bonner S B Kerby P Sutrave M A Gunnell G Mark andU R Rapp Structure and biological activity of human homologsof the rafmil oncogene Mol Cell Biol 5(6)1400ndash1407 Jun 1985URL httpwwwpubmedcentralnihgovarticlerenderfcgitool=

pubmedamppubmedid=2993863 20

F A Brightman and D A Fell Differential feedback regulation ofthe MAPK cascade underlies the quantitative differences in EGF andNGF signalling in PC12 cells FEBS Lett 482(3)169ndash174 Oct 2000doi 101016S0014-5793(00)02037-8 URL httpdxdoiorg101016

S0014-5793(00)02037-8 67

T Brummer H Naegele M Reth and Y Misawa Identification of novelERK-mediated feedback phosphorylation sites at the C-terminus of B-RafOncogene 22(55)8823ndash8834 Dec 2003 doi 101038sjonc1207185 URLhttpdxdoiorg101038sjonc1207185 24 64 65 67

L-F Belanger S Roy M Tremblay B Brott A-M Steff W MouradP Hugo R Erikson and J Charron Mek2 is dispensable for mousegrowth and development Mol Cell Biol 23(14)4778ndash4787 Jul 2003 doi101128MCB23144778-47872003 URL httpdxdoiorg101128

MCB23144778-47872003 23

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org101038sjonc1202174 24

B J Canagarajah A Khokhlatchev M H Cobb and E J GoldsmithActivation mechanism of the MAP kinase ERK2 by dual phosphorylationCell 90(5)859ndash869 Sep 1997 doi 101016S0092-8674(00)80351-7 URLhttpdxdoiorg101016S0092-8674(00)80351-7 23

F Carlomagno S Anaganti T Guida G Salvatore G Troncone S MWilhelm and M Santoro BAY 43-9006 inhibition of oncogenic RET mutantsJ Natl Cancer Inst 98(5)326ndash334 Mar 2006 doi 101093jncidjj069 URLhttpdxdoiorg101093jncidjj069 21

P Chardin GTPase regulation getting aRnd Rock and Rho inhibitionCurr Biol 13(18)R702ndashR704 Sep 2003 doi 101016jcub200308042URL httpdxdoiorg101016jcub200308042 31

D Chen S B Waters K H Holt and J E Pessin SOS phosphorylationand disassociation of the Grb2-SOS complex by the ERK and JNK signalingpathways J Biol Chem 271(11)6328ndash6332 Mar 1996 doi 101074jbc271116328 URL httpdxdoiorg101074jbc271116328 24 64 65

P C Chin L Liu B E Morrison A Siddiq R R Ratan T Bottiglieriand S R DrsquoMello The c-Raf inhibitor GW5074 provides neuroprotection invitro and in an animal model of neurodegeneration through a MEK-ERK andAkt-independent mechanism J Neurochem 90(3)595ndash608 Aug 2004 doi101111j1471-4159200402530x URL httpdxdoiorg101111j

1471-4159200402530x 28 45

J L Cleveland J Troppmair G Packham D S Askew P LloydM Gonzalez-Garcia G Nunez J N Ihle and U R Rapp v-raf sup-presses apoptosis and promotes growth of interleukin-3-dependent myeloidcells Oncogene 9(8)2217ndash2226 Aug 1994 68

H Davies G R Bignell C Cox P Stephens S Edkins S Clegg J TeagueH Woffendin M J Garnett W Bottomley N Davis E Dicks R EwingY Floyd K Gray S Hall R Hawes J Hughes V Kosmidou A MenziesC Mould A Parker C Stevens S Watt S Hooper R Wilson H Jayati-lake B A Gusterson C Cooper J Shipley D Hargrave K Pritchard-JonesN Maitland G Chenevix-Trench G J Riggins D D Bigner G PalmieriA Cossu A Flanagan A Nicholson J W C Ho S Y Leung S T Yuen

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P Dent W Haser T A Haystead L A Vincent T M Roberts andT W Sturgill Activation of mitogen-activated protein kinase kinase byv-Raf in NIH 3T3 cells and in vitro Science 257(5075)1404ndash1407 Sep1992 doi 101126science1326789 URL httpdxdoiorg101126

science1326789 22

M K Dougherty J Muller D A Ritt M Zhou X Z Zhou T DCopeland T P Conrads T D Veenstra K P Lu and D K MorrisonRegulation of Raf-1 by direct feedback phosphorylation Mol Cell 17(2)215ndash224 Jan 2005 doi 101016jmolcel200411055 URL http

dxdoiorg101016jmolcel200411055 24 64 65 66 67

N Duesbery and G V Woude BRAF and MEK mutations make a late en-trance Sci STKE 2006(328)pe15 Mar 2006 doi 101126stke3282006pe15URL httpdxdoiorg101126stke3282006pe15 20 21

M A Farrar Alberol-Ila and R M Perlmutter Activation of the Raf-1kinase cascade by coumermycin-induced dimerization Nature 383(6596)178ndash181 Sep 1996 doi 101038383178a0 URL httpdxdoiorg10

1038383178a0 45

W Feng Z Lu R Z Luo X Zhang E Seto W S-L Liao and Y YuMultiple histone deacetylases repress tumor suppressor gene ARHI in breastcancer Int J Cancer 120(8)1664ndash1668 Apr 2007 doi 101002ijc22474URL httpdxdoiorg101002ijc22474 31

J E Ferrell Tripping the switch fantastic how a protein kinase cascadecan convert graded inputs into switch-like outputs Trends Biochem Sci21(12)460ndash466 Dec 1996 doi 101016S0968-0004(96)20026-X URLhttpdxdoiorg101016S0968-0004(96)20026-X 38 52 55

D W Fry Mechanism of action of erbB tyrosine kinase inhibitors Exp CellRes 284(1)131ndash139 Mar 2003 doi 101016S0014-4827(02)00095-2 URLhttpdxdoiorg101016S0014-4827(02)00095-2 41

M J Garnett and R Marais Guilty as charged B-RAF is a human oncogeneCancer Cell 6(4)313ndash319 Oct 2004 doi 101016jccr200409022 URLhttpdxdoiorg101016jccr200409022 25 56 68

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S Giroux M Tremblay D Bernard J F Cardin-Girard S AubryL Larouche S Rousseau J Huot J Landry L Jeannotte and J Char-ron Embryonic death of Mek1-deficient mice reveals a role for this ki-nase in angiogenesis in the labyrinthine region of the placenta CurrBiol 9(7)369ndash372 Apr 1999 doi 101016S0960-9822(99)80164-X URLhttpdxdoiorg101016S0960-9822(99)80164-X 23

J A Gollob K Moran T Richmond J M Jones T E BaellW K Rathmell and B L Peterson Phase II trial of sorafenib(BAY 43-9006) in combination with interferon alpha 2b in patients withmetastatic renal cell carcinoma Ejc Supplements 3(2)226ndash227 Oct 2005doi 101016S1359-6349(05)81088-2 URL httpdxdoiorg101016

S1359-6349(05)81088-2 21

F Grosjean M Huche M Larcheveque J J Legendre and Y Petit Etudepar la modelisation moleculaire de la regioselectivite de lrsquoOuverture desacides glycidiques par les amines aliphatiques Tetrahedron 50(31)9325ndash9334 1994 URL httpwwwsciencedirectcomsciencearticle

B6THR-42GDSWV-6J248240e503ac7ac6f5f492a3befd39450 42 43 63

C A Hall-Jackson P A Eyers P Cohen M Goedert F T Boyle N He-witt H Plant and P Hedge Paradoxical activation of Raf by a novel Rafinhibitor Chem Biol 6(8)559ndash568 Aug 1999a 28 45

C A Hall-Jackson M Goedert P Hedge and P Cohen Effect of SB203580 on the activity of c-Raf in vitro and in vivo Oncogene 18(12)2047ndash2054 Mar 1999b doi 101038sjonc1202603 URL httpdxdoi

org101038sjonc1202603 28 45 64 65

G Heidecker W Kolch D K Morrison and U R Rapp The role of Raf-1phosphorylation in signal transduction Adv Cancer Res 5853ndash73 1992 33

R Heinrich B G Neel and T A Rapoport Mathematical models ofprotein kinase signal transduction Mol Cell 9(5)957ndash970 May 2002doi 101016S1097-2765(02)00528-2 URL httpdxdoiorg101016

S1097-2765(02)00528-2 29 49 52 55 66

M Hekman H Hamm A V Villar B Bader J Kuhlmann J Nickel andU R Rapp Associations of B- and C-Raf with cholesterol phosphatidylser-ine and lipid second messengers preferential binding of Raf to artificiallipid rafts J Biol Chem 277(27)24090ndash24102 Jul 2002 doi 101074jbcM200576200 URL httpdxdoiorg101074jbcM200576200 36

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M Hekman S Wiese R Metz S Albert J Troppmair J NickelM Sendtner and U R Rapp Dynamic changes in C-Raf phosphory-lation and 14-3-3 protein binding in response to growth factor stimu-lation differential roles of 14-3-3 protein binding sites J Biol Chem279(14)14074ndash14086 Apr 2004 doi 101074jbcM309620200 URLhttpdxdoiorg101074jbcM309620200 25

M Hekman A Fischer L P Wennogle Y K Wang S L Campbelland U R Rapp Novel C-Raf phosphorylation sites serine 296 and 301participate in Raf regulation FEBS Lett 579(2)464ndash468 Jan 2005 doi 101016jfebslet200411105 URL httpdxdoiorg101016jfebslet

200411105 24 64 65 66

H Hisatomi K Nagao K Wakita and N Kohno ARHINOEY2 inac-tivation may be important in breast tumor pathogenesis Oncology 62(2)136ndash140 2002 doi 101159000048259 URL httpdxdoiorg10

1159000048259 30

R Hoshino Y Chatani T Yamori T Tsuruo H Oka O Yoshida Y Shi-mada S Ari-i H Wada J Fujimoto and M Kohno Constitutive activationof the 41-43-kDa mitogen-activated protein kinase signaling pathway inhuman tumors Oncogene 18(3)813ndash822 Jan 1999 doi 101038sjonc1202367 URL httpdxdoiorg101038sjonc1202367 19 23

L R Howe S J Leevers N Gomez S Nakielny P Cohen and C JMarshall Activation of the MAP kinase pathway by the protein kinase rafCell 71(2)335ndash342 Oct 1992 doi 1010160092-8674(92)90361-F URLhttpdxdoiorg1010160092-8674(92)90361-F 22

S R Hubbard L Wei L Ellis and W A Hendrickson Crystal structureof the tyrosine kinase domain of the human insulin receptor Nature 372(6508)746ndash754 1994 doi 101038372746a0 URL httpdxdoiorg

101038372746a0 37 39

K Huebner A ar Rushdi C A Griffin M Isobe C Kozak B S EmanuelL Nagarajan J L Cleveland T I Bonner and M D Goldsborough Ac-tively transcribed genes in the raf oncogene group located on the X chromo-some in mouse and human Proc Natl Acad Sci U S A 83(11)3934ndash3938 Jun1986 URL httpwwwpubmedcentralnihgovarticlerenderfcgi

tool=pubmedamppubmedid=3520560 20

S Ikawa M Fukui Y Ueyama N Tamaoki T Yamamoto andK Toyoshima B-raf a new member of the raf family is activated

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by DNA rearrangement Mol Cell Biol 8(6)2651ndash2654 Jun 1988URL httpwwwpubmedcentralnihgovarticlerenderfcgitool=


H W Jansen B Ruckert R Lurz and K Bister Two unrelated cell-derivedsequences in the genome of avian leukemia and carcinoma inducing retrovirusMH2 EMBO J 2(11)1969ndash1975 1983 URL httpwwwpubmedcentral

nihgovarticlerenderfcgitool=pubmedamppubmedid=6315409 20

H W Jansen R Lurz K Bister T I Bonner G E Mark and U RRapp Homologous cell-derived oncogenes in avian carcinoma virus MH2and murine sarcoma virus 3611 Nature 307(5948)281ndash284 1984 doi101038307281a0 URL httpdxdoiorg101038307281a0 20

E Kerkhoff and U R Rapp Induction of cell proliferation in quiescentNIH 3T3 cells by oncogenic c-Raf-1 Mol Cell Biol 17(5)2576ndash2586 May1997 URL httpwwwpubmedcentralnihgovarticlerenderfcgi


E Kerkhoff and U R Rapp High-intensity Raf signals convert mitoticcell cycling into cellular growth Cancer Res 58(8)1636ndash1640 Apr 1998URL httpcancerresaacrjournalsorgcgicontentabstract58

81636 29 30

H Koide T Satoh M Nakafuku and Y Kaziro GTP-dependent associationof Raf-1 with Ha-Ras identification of Raf as a target downstream of Rasin mammalian cells Proc Natl Acad Sci U S A 90(18)8683ndash8686 Sep1993 URL httpwwwpubmedcentralnihgovarticlerenderfcgi

tool=pubmedamppubmedid=8378348 21 22

B W Kramer R Gotz and U R Rapp Use of mitogenic cascade blockersfor treatment of C-Raf induced lung adenoma in vivo CI-1040 stronglyreduces growth and improves lung structure BMC Cancer 424 Jun2004 doi 1011861471-2407-4-24 URL httpdxdoiorg101186

1471-2407-4-24 66

M Kubicek M Pacher D Abraham K Podar M Eulitz and M BaccariniDephosphorylation of Ser-259 regulates Raf-1 membrane association J BiolChem 277(10)7913ndash7919 Mar 2002 doi 101074jbcM108733200 URLhttpdxdoiorg101074jbcM108733200 58

J M Kyriakis H App X F Zhang P Banerjee D L Brautigan U RRapp and J Avruch Raf-1 activates MAP kinase-kinase Nature 358(6385)

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417ndash421 Jul 1992 doi 101038358417a0 URL httpdxdoiorg10

1038358417a0 22

K Lackey M Cory R Davis S V Frye P A Harris R N Hunter D KJung O B McDonald R W McNutt M R Peel R D Rutkowske J MVeal and E R Wood The discovery of potent cRaf1 kinase inhibitors BioorgMed Chem Lett 10(3)223ndash226 Feb 2000 doi 101016S0960-894X(99)00668-X URL httpdxdoiorg101016S0960-894X(99)00668-X28 45

J Lew MAP kinases and CDKs kinetic basis for catalytic activationBiochemistry 42(4)849ndash856 Feb 2003 doi 101021bi0269761 URLhttpdxdoiorg101021bi0269761 51 52 55

T B Lowinger B Riedl J Dumas and R A Smith Design and discoveryof small molecules targeting raf-1 kinase Curr Pharm Des 8(25)2269ndash22782002 doi 1021741381612023393125 URL httpdxdoiorg102174

1381612023393125 20 21

Z Lu R Z Luo H Peng D G Rosen E N Atkinson C WarnekeM Huang A Nishmoto J Liu W S-L Liao Y Yu and R C BastTranscriptional and posttranscriptional down-regulation of the imprintedtumor suppressor gene ARHI (DRAS3) in ovarian cancer Clin Cancer Res12(8)2404ndash2413 Apr 2006 doi 1011581078-0432CCR-05-1036 URLhttpdxdoiorg1011581078-0432CCR-05-1036 30 31

R Z Luo X Fang R Marquez S-Y Liu G B Mills W S-L LiaoY Yu and R C Bast ARHI is a Ras-related small G-protein with a novelN-terminal extension that inhibits growth of ovarian and breast cancersOncogene 22(19)2897ndash2909 May 2003 doi 101038sjonc1206380 URLhttpdxdoiorg101038sjonc1206380 30 31

Z Luo G Tzivion P J Belshaw D Vavvas M Marshall and J AvruchOligomerization activates c-Raf-1 through a Ras-dependent mechanismNature 383(6596)181ndash185 Sep 1996 doi 101038383181a0 URL http

dxdoiorg101038383181a0 45

M Malumbres and M Barbacid RAS oncogenes the first 30 years NatRev Cancer 3(6)459ndash465 Jun 2003 doi 101038nrc1097 URL http

dxdoiorg101038nrc1097 21 23

C J Marshall Specificity of receptor tyrosine kinase signaling transientversus sustained extracellular signal-regulated kinase activation Cell 80

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(2)179ndash185 Jan 1995 URL httpwwwcellcomcontentarticle

abstractuid=PII0092867495904018 29 30

C S Mason C J Springer R G Cooper G Superti-Furga C J Marshalland R Marais Serine and tyrosine phosphorylations cooperate in Raf-1 butnot B-Raf activation EMBO J 18(8)2137ndash2148 Apr 1999 doi 101093emboj1882137 URL httpdxdoiorg101093emboj188213758

M M McKay and D K Morrison Integrating signals from RTKs toERKMAPK Oncogene 26(22)3113ndash3121 May 2007 doi 101038sjonc1210394 URL httpdxdoiorg101038sjonc1210394 69

M Mikula M Schreiber Z Husak L Kucerova J Ruth R WieserK Zatloukal H Beug E F Wagner and M Baccarini Embryonic lethalityand fetal liver apoptosis in mice lacking the c-raf-1 gene EMBO J 20(8)1952ndash1962 Apr 2001 doi 101093emboj2081952 URL httpdxdoi

org101093emboj2081952 23

V Neuhoff N Arold D Taube and W Ehrhardt Improved staining ofproteins in polyacrylamide gels including isoelectric focusing gels with clearbackground at nanogram sensitivity using Coomassie Brilliant Blue G-250and R-250 Electrophoresis 9(6)255ndash262 Jun 1988 doi 101002elps1150090603 URL httpdxdoiorg101002elps1150090603 37

M Offterdinger V Georget A Girod and P I H Bastiaens Imagingphosphorylation dynamics of the epidermal growth factor receptor J BiolChem 279(35)36972ndash36981 Aug 2004 doi 101074jbcM405830200 URLhttpdxdoiorg101074jbcM405830200 67

G Pages S Guerin D Grall F Bonino A Smith F Anjuere P Aubergerand J Pouyssegur Defective thymocyte maturation in p44 MAP kinase(Erk 1) knockout mice Science 286(5443)1374ndash1377 Nov 1999 doi 101126science28654431374 URL httpdxdoiorg101126science

28654431374 22

C Pargellis L Tong L Churchill P F Cirillo T Gilmore A G GrahamP M Grob E R Hickey N Moss S Pav and J Regan Inhibition of p38MAP kinase by utilizing a novel allosteric binding site Nat Struct Biol 9(4)268ndash272 Apr 2002 doi 101038nsb770 URL httpdxdoiorg10

1038nsb770 37 39 45

D M Payne A J Rossomando P Martino A K Erickson J HHer J Shabanowitz D F Hunt M J Weber and T W Sturgill

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Identification of the regulatory phosphorylation sites in pp42mitogen-activated protein kinase (MAP kinase) EMBO J 10(4)885ndash892 Apr1991 URL httpwwwpubmedcentralnihgovarticlerenderfcgi


A Peyker O Rocks and P I H Bastiaens Imaging activation of twoRas isoforms simultaneously in a single cell Chembiochem 6(1)78ndash85 Jan2005 doi 101002cbic200400280 URL httpdxdoiorg101002

cbic200400280 67

K E Prehoda J A Scott R D Mullins and W A Lim Integrationof multiple signals through cooperative regulation of the N-WASP-Arp23complex Science 290(5492)801ndash806 Oct 2000 doi 101126science2905492801 URL httpdxdoiorg101126science2905492801 69

I A Prior A Harding J Yan J Sluimer R G Parton and J F HancockGTP-dependent segregation of H-ras from lipid rafts is required for biologicalactivity Nat Cell Biol 3(4)368ndash375 Apr 2001 doi 10103835070050URL httpdxdoiorg10103835070050 70

C A Pritchard L Bolin R Slattery R Murray and M McMahon Post-natal lethality and neurological and gastrointestinal defects in mice withtargeted disruption of the A-Raf protein kinase gene Curr Biol 6(5)614ndash617 May 1996 doi 101016S0960-9822(02)00548-1 URL http

dxdoiorg101016S0960-9822(02)00548-1 23

M S Qui and S H Green PC12 cell neuronal differentiation is associatedwith prolonged p21ras activity and consequent prolonged ERK activityNeuron 9(4)705ndash717 Oct 1992 URL httpwwwneuronorgcontent

articleabstractuid=PII089662739290033A 29 52 55

U R Rapp M D Goldsborough G E Mark T I Bonner J GroffenF H Reynolds and J R Stephenson Structure and biological activity ofv-raf a unique oncogene transduced by a retrovirus Proc Natl Acad Sci US A 80(14)4218ndash4222 Jul 1983 URL httpwwwpubmedcentralnih

govarticlerenderfcgitool=pubmedamppubmedid=6308607 19 20

U E E Rennefahrt B Illert E Kerkhoff J Troppmair and U R RappConstitutive JNK activation in NIH 3T3 fibroblasts induces a partiallytransformed phenotype J Biol Chem 277(33)29510ndash29518 Aug 2002doi 101074jbcM203010200 URL httpdxdoiorg101074jbc

M203010200 33

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H Richly P Kupsch K Passage M Grubert R A Hilger S KredtkeD Voliotis M E Scheulen S Seeber and D Strumberg A phase Iclinical and pharmacokinetic study of the Raf kinase inhibitor (RKI) BAY43-9006 administered in combination with doxorubicin in patients withsolid tumors Int J Clin Pharmacol Ther 41(12)620ndash621 Dec 2003 URLhttpwwwdustricomzecpsamplecopycp12620pdf 21

D J Robbins E Zhen H Owaki C A Vanderbilt D Ebert T D Geppertand M H Cobb Regulation and properties of extracellular signal-regulatedprotein kinases 1 and 2 in vitro J Biol Chem 268(7)5097ndash5106 Mar 1993URL httpwwwjbcorgcgicontentabstract26875097 22

A Robubi T Mueller J Fueller M Hekman U R Rapp and T DandekarB-Raf and C-Raf signaling investigated in a simplified model of the mitogenickinase cascade Biol Chem 386(11)1165ndash1171 Nov 2005 doi 101515BC2005133 URL httpdxdoiorg101515BC2005133 VI VIII 3655

P Rodriguez-Viciana O Tetsu W E Tidyman A L Estep B A CongerM S Cruz F McCormick and K A Rauen Germline mutations ingenes within the MAPK pathway cause cardio-facio-cutaneous syndromeScience 311(5765)1287ndash1290 Mar 2006 doi 101126science1124642 URLhttpdxdoiorg101126science1124642 22

D G Rosen L Wang A N Jain K H Lu R Z Luo Y Yu J Liu andR C Bast Expression of the tumor suppressor gene ARHI in epithelialovarian cancer is associated with increased expression of p21WAF1CIP1and prolonged progression-free survival Clin Cancer Res 10(19)6559ndash6566Oct 2004 doi 1011581078-0432CCR-04-0698 URL httpdxdoi

org1011581078-0432CCR-04-0698 30

L K Rushworth A D Hindley E OrsquoNeill and W Kolch Regulation androle of Raf-1B-Raf heterodimerization Mol Cell Biol 26(6)2262ndash2272Mar 2006 doi 101128MCB2662262-22722006 URL httpdxdoi

org101128MCB2662262-22722006 45 46 64

M K Saba-El-Leil F D J Vella B Vernay L Voisin L ChenN Labrecque S-L Ang and S Meloche An essential function of themitogen-activated protein kinase Erk2 in mouse trophoblast developmentEMBO Rep 4(10)964ndash968 Oct 2003 doi 101038sjemborembor939 URLhttpdxdoiorg101038sjemborembor939 22

Bibliography 81

A Sali and T L Blundell Comparative protein modelling by satisfaction ofspatial restraints J Mol Biol 234(3)779ndash815 Dec 1993 doi 101006jmbi19931626 URL httpdxdoiorg101006jmbi19931626 37 39

S Sasagawa Y ichi Ozaki K Fujita and S Kuroda Prediction andvalidation of the distinct dynamics of transient and sustained ERK activationNat Cell Biol 7(4)365ndash373 Apr 2005 doi 101038ncb1233 URL http

dxdoiorg101038ncb1233 67

I Schomburg A Chang C Ebeling M Gremse C Heldt G Huhn andD Schomburg BRENDA the enzyme database updates and major newdevelopments Nucleic Acids Res 32(Database issue)D431ndashD433 Jan 2004doi 101093nargkh081 URL httpdxdoiorg101093nargkh08153

A Sewing B Wiseman A C Lloyd and H Land High-intensityRaf signal causes cell cycle arrest mediated by p21Cip1 Mol Cell Biol17(9)5588ndash5597 Sep 1997 URL httpwwwpubmedcentralnihgov

articlerenderfcgitool=pubmedamppubmedid=9271434 30

G Sithanandam T Druck L A Cannizzaro G Leuzzi K Huebner andU R Rapp B-raf and a B-raf pseudogene are located on 7q in manOncogene 7(4)795ndash799 Apr 1992 20

S M Storm J L Cleveland and U R Rapp Expression of raf familyproto-oncogenes in normal mouse tissues Oncogene 5(3)345ndash351 Mar 199066

D Strumberg J W Clark A Awada M J Moore H Richly A HendliszH W Hirte J P Eder H-J Lenz and B Schwartz Safety pharmacoki-netics and preliminary antitumor activity of sorafenib a review of fourphase I trials in patients with advanced refractory solid tumors Oncolo-gist 12(4)426ndash437 Apr 2007 doi 101634theoncologist12-4-426 URLhttpdxdoiorg101634theoncologist12-4-426 20 21

R M Tombes K L Auer R Mikkelsen K Valerie M P WymannC J Marshall M McMahon and P Dent The mitogen-activated protein(MAP) kinase cascade can either stimulate or inhibit DNA synthesis inprimary cultures of rat hepatocytes depending upon whether its activa-tion is acutephasic or chronic Biochem J 330 ( Pt 3)1451ndash1460 Mar1998 URL httpwwwpubmedcentralnihgovarticlerenderfcgi


82 Bibliography

N Trakul R E Menard G R Schade Z Qian and M R Rosner Rafkinase inhibitory protein regulates Raf-1 but not B-Raf kinase activation JBiol Chem 280(26)24931ndash24940 Jul 2005 doi 101074jbcM413929200URL httpdxdoiorg101074jbcM413929200 68

P T C Wan M J Garnett S M Roe S Lee D Niculescu-Duvaz V MGood C M Jones C J Marshall C J Springer D Barford R Maraisand C G Project Mechanism of activation of the RAF-ERK signalingpathway by oncogenic mutations of B-RAF Cell 116(6)855ndash867 Mar 2004doi 101016S0092-8674(04)00215-6 URL httpdxdoiorg101016

S0092-8674(04)00215-6 25 27 39 40 45

L Wang A Hoque R Z Luo J Yuan Z Lu A Nishimoto J Liu A ASahin S M Lippman R C Bast and Y Yu Loss of the expression ofthe tumor suppressor gene ARHI is associated with progression of breastcancer Clin Cancer Res 9(10 Pt 1)3660ndash3666 Sep 2003 URL http

clincancerresaacrjournalsorgcgicontentfull9103660 30

C Wellbrock M Karasarides and R Marais The RAF proteins take centrestage Nat Rev Mol Cell Biol 5(11)875ndash885 Nov 2004 doi 101038nrm1498 URL httpdxdoiorg101038nrm1498 20

S M Wilhelm C Carter L Tang D Wilkie A McNabola H RongC Chen X Zhang P Vincent M McHugh Y Cao J Shujath S GawlakD Eveleigh B Rowley L Liu L Adnane M Lynch D Auclair I TaylorR Gedrich A Voznesensky B Riedl L E Post G Bollag and P A TrailBAY 43-9006 exhibits broad spectrum oral antitumor activity and targetsthe RAFMEKERK pathway and receptor tyrosine kinases involved intumor progression and angiogenesis Cancer Res 64(19)7099ndash7109 Oct2004 doi 1011580008-5472CAN-04-1443 URL httpdxdoiorg10

11580008-5472CAN-04-1443 21 64 65

E Wilker and M B Yaffe 14-3-3 Proteinsndasha focus on cancer and humandisease J Mol Cell Cardiol 37(3)633ndash642 Sep 2004 doi 101016jyjmcc200404015 URL httpdxdoiorg101016jyjmcc20040401570

M Wilm A Shevchenko T Houthaeve S Breit L Schweigerer T Fotsisand M Mann Femtomole sequencing of proteins from polyacrylamide gels bynano-electrospray mass spectrometry Nature 379(6564)466ndash469 Feb 1996doi 101038379466a0 URL httpdxdoiorg101038379466a0 37

Bibliography 83

V Wixler U Smola M Schuler and U Rapp Differential regula-tion of Raf isozymes by growth versus differentiation inducing factors inPC12 pheochromocytoma cells FEBS Lett 385(3)131ndash137 May 1996doi 1010160014-5793(96)00363-8 URL httpdxdoiorg101016

0014-5793(96)00363-8 29 30

L Wojnowski A M Zimmer T W Beck H Hahn R Bernal U RRapp and A Zimmer Endothelial apoptosis in Braf-deficient mice NatGenet 16(3)293ndash297 Jul 1997 doi 101038ng0797-293 URL http

dxdoiorg101038ng0797-293 23

D Woods D Parry H Cherwinski E Bosch E Lees and M McMa-hon Raf-induced proliferation or cell cycle arrest is determined by thelevel of Raf activity with arrest mediated by p21Cip1 Mol Cell Biol17(9)5598ndash5611 Sep 1997 URL httpwwwpubmedcentralnihgov


S Yamada T Taketomi and A Yoshimura Model analysis of differencebetween EGF pathway and FGF pathway Biochem Biophys Res Commun314(4)1113ndash1120 Feb 2004 doi 101016jbbrc200401009 URL http

dxdoiorg101016jbbrc200401009 67

Y Yu F Xu H Peng X Fang S Zhao Y Li B Cuevas W L Kuo J WGray M Siciliano G B Mills and R C Bast NOEY2 (ARHI) an imprintedputative tumor suppressor gene in ovarian and breast carcinomas Proc NatlAcad Sci U S A 96(1)214ndash219 Jan 1999 URL httpwwwpubmedcentral

nihgovarticlerenderfcgitool=pubmedamppubmedid=9874798 30 3169

Y Yu R Luo Z Lu W W Feng D Badgwell J-P Issa D GRosen J Liu and R C Bast Biochemistry and Biology of ARHI (DI-RAS3) an Imprinted Tumor Suppressor Gene Whose Expression Is Lostin Ovarian and Breast Cancers Methods Enzymol 407455ndash468 2005doi 101016S0076-6879(05)07037-0 URL httpdxdoiorg101016

S0076-6879(05)07037-0 30 69

A Zebisch P B Staber A Delavar C Bodner K Hiden K FischerederM Janakiraman W Linkesch H W Auner W Emberger C Wind-passinger M G Schimek G Hoefler J Troppmair and H Sill Twotransforming C-RAF germ-line mutations identified in patients with therapy-related acute myeloid leukemia Cancer Res 66(7)3401ndash3408 Apr 2006doi 1011580008-5472CAN-05-0115 URL httpdxdoiorg101158

0008-5472CAN-05-0115 22

84 Bibliography

X F Zhang J Settleman J M Kyriakis E Takeuchi-Suzuki S J ElledgeM S Marshall J T Bruder U R Rapp and J Avruch Normal andoncogenic p21ras proteins bind to the amino-terminal regulatory domain ofc-Raf-1 Nature 364(6435)308ndash313 Jul 1993 doi 101038364308a0 URLhttpdxdoiorg101038364308a0 20 21 22

J Zhong J Troppmair and U R Rapp Independent control of cell survivalby Raf-1 and Bcl-2 at the mitochondria Oncogene 20(35)4807ndash4816 Aug2001 doi 101038sjonc1204614 URL httpdxdoiorg101038sj

onc1204614 33

X Zhu J L Kim J R Newcomb P E Rose D R Stover L MToledo H Zhao and K A Morgenstern Structural analysis of thelymphocyte-specific kinase Lck in complex with non-selective and Srcfamily selective kinase inhibitors Structure 7(6)651ndash661 Jun 1999doi 101016S0969-2126(99)80086-0 URL httpdxdoiorg101016

S0969-2126(99)80086-0 37 39

Acknowledgments

I want to thank my thesis board Prof Dr Thomas Dandkar (supervisor)Prof Dr Ulf R Rapp (advisor) and Prof Dr Shamil Sunyaev (externaladvisor)

I also want to thank my co-authors Prof Dr Claus Herdeis Mirko HekmanJochen Fuller Tobias Muller Marcus Dittrich Ruth Kroschewski MirkoKlingauf Matthias Beck Stephan Heinzer Yagmur Turgayand and WernerSchmitz

and many other people for their support Ulrike Rennefahrt AndreasFischer Birgit Pils Elena Nekhoroshkova Stefan Albert Barbara BauerLudmilla Wixler Tina Schull Maureen Menning Renate Metz and FrankForster

I gratefully acknowledge funding by the Bavarian state (BIGSS elitenetwork IZKF grant B-36) and the BMBF (Systems Biology grant 03 13074D)

Curriculum vitae

Name RobubiGiven Name ArminPrevious name RobobiPostal address Lange Bogen 2 D-97074 Wurzburg GermanyPhone +49-931-2969754Email mearmineuorg

Personal data

Date of birth Sep 22nd 1977Place of birth Tehran IranMarital status unmarriedChildren noneNationality Austrian

88 Curriculum vitae

Education

Lower education1984ndash1988 Primary school Vienna Austria1988ndash1996 Grammar school Vienna Austria1996 School leaving examination (Matura)

Higher Education1996ndash2000 Chemical Engineering Technical University of

Vienna (TU-Wien) Vienna Austria2000ndash2002 Masters Program of Bioinformatics Chalmers

Goteborg SwedenAugust 2002 Masters of Science in Bioinformatics Supervi-

sor Dr Per-Georg Nyholm Structural chemistrygroup Department of Medical Chemistry Univer-sity of Goteborg (GU) Goteborg Sweden

Sep 2002ndashSep 2004 Research project Supervisor Prof Dr Ulf RRapp Protein group Institute for Medical Radia-tion and Cell Research University of WurzburgWurzburg Germany

October 2004ndashpresent Thesis project ndash BioMedTec Franken Interna-tional Graduate School (BIGSS)mdashElite Networkof Bavaria Supervisor Prof Dr Thomas Dan-dekar Department of Bioinformatics Universityof Wurzburg Wurzburg Germany

List of publications

bull Beck M Robubi A Klingauf M Hekman M Heinzer S Turgay YRapp U R Kroschewski R lsquoThe Ras-like tumor suppressor DiRas3(Noey2 ARHI) downregulates specifically the RAF-MEK-ERK signalingpathway by inhibiting MEKrsquo Molecular Cellndashsubmitted

bull Robubi A Schmitz W Herdeis C Rapp U R Dandekar T lsquoA proof-of-concept study with a novel diphenyl urea lead compound to irreversiblyinhibit the Cys109 protein kinase and oncoprotein RAF design synthe-sis activity in vitro and in vivorsquo ChemMedChemndashsubmitted

bull Robubi A Mueller T Fueller J Hekman M Rapp U R Dandekar TlsquoB-Raf and C-Raf signaling investigated in a simplified model of themitogenic kinase cascadersquo Biological Chemistry 386 (2005) 1165ndash1171DOI 101515BC2005133

bull Rosen J Robubi A Nyholm PG lsquoThe conformations of the O-specificpolysaccharides of Shigella dysenteriae type 4 and Escherichia coli O159studied with molecular mechanics (MM3) filtered systematic searchrsquoCarbohydrate Research 339 (2004) 961ndash966DOI 101016jcarres200311018

bull Rosen J Robubi A Nyholm PG lsquoConformation of the branched O-specific polysaccharide of Shigella dysenteriae type 2rsquo CarbohydrateResearch 337 (2002) 1633ndash1640DOI 101016S0008-6215(02)00089-7

bull Conformational studies on the O-antigens of some gram negative bacteria(Master thesis)httpwwwmathchalmersseStatBioinfoMasterTheses20027pdf

90 List of publications

Poster Abstracts

bull Robubi A Schmitz W Herdeis C Rapp UR and Dandekar TlsquoA novel diphenyl urea lead compound to irreversibly inhibit the Cys109protein kinase and oncoprotein RAF design synthesis activity in vitroand in vivorsquo Cancer Genomics and Epigenomics (Keystone) Feb 19ndash242008 Taos New Mexico USA

bull Walter D Kreutz C Neubert K McNelly S von Weizsacker FTimmer J Robubi A lsquoComplex regulation of caspase-3 processingand activity in FasCD95L-treated primary mouse hepatocytesrsquo Con-ference on Systems Biology of Mammalian Cells (SBMC) Jul 12ndash142006 Heidelberg Germany

bull Robubi A lsquoStructural basis of Raf Kinase Mutations and Raf Kinaseinhibitorsrsquo HUGOrsquos 10th Annual Genome Meeting HGM2005 Apr18ndash21 2005 Kyoto Japan

bull Robubi A Dandekar T Herdeis C Rapp UR lsquoStructural basisof Raf Kinase Mutations and Raf Kinase Inhibitorsrsquo Ras-dependentpathways in human cancer Nov 28ndashDec 1 2004 Kloster Banz Germany

bull Robubi A Hekman M Fischer A Albert S Rapp UR Wiese SlsquoRegulation of Raf protein kinases by 14-3-3 proteins and KSRrsquo ELSO2004 Sep 4ndash8 2004 Nice France

bull Robubi A Rapp UR Dandekar T lsquoMitogen activated proteinkinase pathways Improving bioinformatical strategies for inhibitorscreeningrsquo Deutsche Pharmazeutischen Gesellschaft (DPhG) Oct 8ndash112003 Wurzburg Germany

bull Rosen J Robobi A Nyholm PG lsquoConformation of the branchedO-specific polysaccharide of Shigella dysenteriae type 2rsquo Eurocarb Sep2ndash11 2001 Lisbon Portugal

List of publications 91

Oral presentations

bull lsquoModeling of the Raf signaling pathwayrsquo Bioinformatics SymposiumJul 27 Wurzburg Germany

bull lsquoStructural basis of Raf Kinase Mutations and Raf Kinase InhibitorsrsquoRabensteiner Kolleg May 26ndash28 Pottenstein Germany

Wurzburg December 12 2007

(Armin Robubi)

Appendix A

Supplementary material

This section also includes supplements which are too large to be printed inthe final thesis They can only be accessed in the electronic PDF version ofthe doctoral thesis using a PDF viewer that supports file attachment such asAdobe Reader 40 or higher

modelpdbgz this file contains the homology model of the kinasedomain of B-RAF in complex with compound 1 (PDB format compressedwith GZIP)

scriptszip contains a set of Matlab files These scriptsndashand theparameters thereinndashwere used for our bioinformatics models of the RAFsignaling pathway

thr106aln list of all protein kinases in the human genome with athreoninemdashor any other amino acid with small side chainmdashat position 106(p38α MAP kinase numbering)

supplmodelpdb

ATOM 1 N SER 1 7438 20972 92429 100 5764 13ATOM 2 CA SER 1 6099 20369 92256 100 5764 13ATOM 3 C SER 1 5661 20461 90834 100 5764 13ATOM 4 O SER 1 6258 19857 89944 100 5764 13ATOM 5 CB SER 1 5064 21108 93120 100 5764 13ATOM 6 OG SER 1 5387 20976 94497 100 5764 13ATOM 7 N SER 2 4570 21213 90600 100 8459 13ATOM 8 CA SER 2 4034 21395 89286 100 8459 13ATOM 9 C SER 2 4978 22246 88502 100 8459 13ATOM 10 O SER 2 5070 22120 87282 100 8459 13ATOM 11 CB SER 2 2670 22110 89290 100 8459 13ATOM 12 OG SER 2 1711 21328 89987 100 8459 13ATOM 13 N ASP 3 5724 23127 89195 100 070 13ATOM 14 CA ASP 3 6586 24051 88518 100 070 13ATOM 15 C ASP 3 7534 23293 87643 100 070 13ATOM 16 O ASP 3 8317 22466 88108 100 070 13ATOM 17 CB ASP 3 7426 24911 89478 100 070 13ATOM 18 CG ASP 3 8035 26049 88676 100 070 13ATOM 19 OD1 ASP 3 7390 26483 87686 100 070 13ATOM 20 OD2 ASP 3 9157 26494 89036 100 070 13ATOM 21 N ASP 4 7477 23579 86328 100 6884 13ATOM 22 CA ASP 4 8306 22934 85349 100 6884 13ATOM 23 C ASP 4 9735 23346 85539 100 6884 13ATOM 24 O ASP 4 10652 22554 85329 100 6884 13ATOM 25 CB ASP 4 7916 23293 83906 100 6884 13ATOM 26 CG ASP 4 6550 22682 83631 100 6884 13ATOM 27 OD1 ASP 4 5939 22150 84596 100 6884 13ATOM 28 OD2 ASP 4 6099 22741 82456 100 6884 13ATOM 29 N TRP 5 9953 24613 85929 100 8796 13ATOM 30 CA TRP 5 11269 25174 86054 100 8796 13ATOM 31 C TRP 5 12073 24569 87167 100 8796 13ATOM 32 O TRP 5 13297 24515 87069 100 8796 13ATOM 33 CB TRP 5 11284 26705 86202 100 8796 13ATOM 34 CG TRP 5 10994 27410 84898 100 8796 13ATOM 35 CD1 TRP 5 11867 27764 83912 100 8796 13ATOM 36 CD2 TRP 5 9696 27845 84466 100 8796 13ATOM 37 NE1 TRP 5 11195 28387 82889 100 8796 13ATOM 38 CE2 TRP 5 9857 28446 83217 100 8796 13ATOM 39 CE3 TRP 5 8470 27754 85061 100 8796 13ATOM 40 CZ2 TRP 5 8792 28966 82541 100 8796 13ATOM 41 CZ3 TRP 5 7397 28279 84375 100 8796 13ATOM 42 CH2 TRP 5 7555 28872 83140 100 8796 13ATOM 43 N GLU 6 11439 24129 88271 100 8522 13ATOM 44 CA GLU 6 12213 23619 89374 100 8522 13ATOM 45 C GLU 6 13005 22421 88931 100 8522 13ATOM 46 O GLU 6 12520 21606 88146 100 8522 13ATOM 47 CB GLU 6 11345 23210 90583 100 8522 13ATOM 48 CG GLU 6 12138 22757 91813 100 8522 13ATOM 49 CD GLU 6 12657 23988 92547 100 8522 13ATOM 50 OE1 GLU 6 12536 25112 91992 100 8522 13ATOM 51 OE2 GLU 6 13183 23820 93679 100 8522 13ATOM 52 N ILE 7 14269 22302 89414 100 015 13ATOM 53 CA ILE 7 15102 21182 89050 100 015 13ATOM 54 C ILE 7 15741 20728 90322 100 015 13ATOM 55 O ILE 7 15858 21496 91276 100 015 13ATOM 56 CB ILE 7 16337 21504 88268 100 015 13ATOM 57 CG1 ILE 7 16177 22811 87509 100 015 13ATOM 58 CG2 ILE 7 16671 20289 87385 100 015 13ATOM 59 CD1 ILE 7 16327 23992 88472 100 015 13ATOM 60 N PRO 8 16181 19503 90362 100 044 13ATOM 61 CA PRO 8 16896 19050 91515 100 044 13ATOM 62 C PRO 8 18229 19726 91505 100 044 13ATOM 63 O PRO 8 18766 19971 90426 100 044 13ATOM 64 CB PRO 8 16940 17521 91418 100 044 13ATOM 65 CG PRO 8 16403 17199 90008 100 044 13ATOM 66 CD PRO 8 15524 18412 89664 100 044 13ATOM 67 N ASP 9 18778 20030 92693 100 8572 13ATOM 68 CA ASP 9 20025 20730 92811 100 8572 13ATOM 69 C ASP 9 21126 19878 92255 100 8572 13ATOM 70 O ASP 9 22110 20388 91721 100 8572 13ATOM 71 CB ASP 9 20389 21065 94268 100 8572 13ATOM 72 CG ASP 9 21489 22116 94244 100 8572 13ATOM 73 OD1 ASP 9 21774 22644 93135 100 8572 13ATOM 74 OD2 ASP 9 22059 22406 95330 100 8572 13ATOM 75 N GLY 10 20969 18546 92342 100 4158 13ATOM 76 CA GLY 10 21988 17609 91954 100 4158 13ATOM 77 C GLY 10 22364 17799 90518 100 4158 13ATOM 78 O GLY 10 23507 17555 90134 100 4158 13ATOM 79 N GLN 11 21408 18216 89674 100 060 13ATOM 80 CA GLN 11 21671 18335 88268 100 060 13ATOM 81 C GLN 11 22715 19379 88004 100 060 13ATOM 82 O GLN 11 23371 19344 86962 100 060 13ATOM 83 CB GLN 11 20422 18617 87417 100 060 13ATOM 84 CG GLN 11 20715 18524 85917 100 060 13ATOM 85 CD GLN 11 19399 18311 85186 100 060 13ATOM 86 OE1 GLN 11 19367 18222 83960 100 060 13ATOM 87 NE2 GLN 11 18283 18211 85958 100 060 13ATOM 88 N ILE 12 22892 20352 88918 100 039 13ATOM 89 CA ILE 12 23801 21423 88616 100 039 13ATOM 90 C ILE 12 25121 21270 89305 100 039 13ATOM 91 O ILE 12 25212 20866 90463 100 039 13ATOM 92 CB ILE 12 23265 22771 89003 100 039 13ATOM 93 CG1 ILE 12 24112 23891 88377 100 039 13ATOM 94 CG2 ILE 12 23199 22826 90538 100 039 13ATOM 95 CD1 ILE 12 23983 23973 86856 100 039 13ATOM 96 N THR 13 26194 21575 88543 100 014 13ATOM 97 CA THR 13 27539 21578 89035 100 014 13ATOM 98 C THR 13 28045 22979 88852 100 014 13ATOM 99 O THR 13 27949 23554 87771 100 014 13ATOM 100 CB THR 13 28436 20635 88281 100 014 13ATOM 101 OG1 THR 13 29742 20640 88838 100 014 13ATOM 102 CG2 THR 13 28473 21044 86798 100 014 13ATOM 103 N VAL 14 28606 23570 89920 100 8315 13ATOM 104 CA VAL 14 29091 24920 89861 100 8315 13ATOM 105 C VAL 14 30544 24787 89548 100 8315 13ATOM 106 O VAL 14 31091 23687 89579 100 8315 13ATOM 107 CB VAL 14 28921 25639 91175 100 8315 13ATOM 108 CG1 VAL 14 29451 27081 91071 100 8315 13ATOM 109 CG2 VAL 14 27436 25557 91569 100 8315 13ATOM 110 N GLY 15 31259 25889 89277 100 037 13ATOM 111 CA GLY 15 32612 25603 88917 100 037 13ATOM 112 C GLY 15 33377 26856 88628 100 037 13ATOM 113 O GLY 15 34581 26894 88874 100 037 13ATOM 114 N GLN 16 32741 27916 88107 100 000 13ATOM 115 CA GLN 16 33491 29131 87941 100 000 13ATOM 116 C GLN 16 32694 30214 88595 100 000 13ATOM 117 O GLN 16 31477 30257 88445 100 000 13ATOM 118 CB GLN 16 33672 29560 86474 100 000 13ATOM 119 CG GLN 16 34469 30858 86320 100 000 13ATOM 120 CD GLN 16 34516 31206 84841 100 000 13ATOM 121 OE1 GLN 16 34828 30364 84000 100 000 13ATOM 122 NE2 GLN 16 34177 32482 84512 100 000 13ATOM 123 N ARG 17 33352 31121 89345 100 9629 13ATOM 124 CA ARG 17 32609 32180 89974 100 9629 13ATOM 125 C ARG 17 32791 33417 89153 100 9629 13ATOM 126 O ARG 17 33899 33937 89031 100 9629 13ATOM 127 CB ARG 17 33093 32524 91392 100 9629 13ATOM 128 CG ARG 17 32317 33681 92023 100 9629 13ATOM 129 CD ARG 17 32912 34182 93340 100 9629 13ATOM 130 NE ARG 17 32683 33130 94368 100 9629 13ATOM 131 CZ ARG 17 32587 33489 95680 100 9629 13ATOM 132 NH1 ARG 17 32702 34802 96035 100 9629 13ATOM 133 NH2 ARG 17 32363 32541 96635 100 9629 13ATOM 134 N ILE 18 31697 33885 88519 100 8503 13ATOM 135 CA ILE 18 31720 35088 87734 100 8503 13ATOM 136 C ILE 18 31801 36293 88614 100 8503 13ATOM 137 O ILE 18 32602 37193 88367 100 8503 13ATOM 138 CB ILE 18 30494 35270 86888 100 8503 13ATOM 139 CG1 ILE 18 30375 34145 85845 100 8503 13ATOM 140 CG2 ILE 18 30564 36679 86276 100 8503 13ATOM 141 CD1 ILE 18 30103 32776 86459 100 8503 13ATOM 142 N GLY 19 30968 36346 89674 100 2472 13ATOM 143 CA GLY 19 30962 37535 90474 100 2472 13ATOM 144 C GLY 19 30405 37216 91823 100 2472 13ATOM 145 O GLY 19 29806 36162 92035 100 2472 13ATOM 146 N SER 20 30602 38152 92774 100 2432 13ATOM 147 CA SER 20 30131 37975 94114 100 2432 13ATOM 148 C SER 20 29593 39290 94575 100 2432 13ATOM 149 O SER 20 29965 40346 94060 100 2432 13ATOM 150 CB SER 20 31245 37578 95096 100 2432 13ATOM 151 OG SER 20 32200 38625 95189 100 2432 13ATOM 152 N GLY 21 28687 39245 95572 100 1208 13ATOM 153 CA GLY 21 28104 40448 96085 100 1208 13ATOM 154 C GLY 21 27410 40102 97365 100 1208 13ATOM 155 O GLY 21 27395 38947 97789 100 1208 13ATOM 156 N SER 22 26813 41120 98013 100 3507 13ATOM 157 CA SER 22 26142 40933 99266 100 3507 13ATOM 158 C SER 22 24950 40052 99051 100 3507 13ATOM 159 O SER 22 24651 39190 99874 100 3507 13ATOM 160 CB SER 22 25628 42256 99860 100 3507 13ATOM 161 OG SER 22 24984 42016 101102 100 3507 13ATOM 162 N PHE 23 24230 40275 97936 100 7279 13ATOM 163 CA PHE 23 23034 39564 97588 100 7279 13ATOM 164 C PHE 23 23348 38151 97220 100 7279 13ATOM 165 O PHE 23 22592 37233 97533 100 7279 13ATOM 166 CB PHE 23 22296 40244 96429 100 7279 13ATOM 167 CG PHE 23 21953 41591 96964 100 7279 13ATOM 168 CD1 PHE 23 20908 41737 97846 100 7279 13ATOM 169 CD2 PHE 23 22675 42704 96596 100 7279 13ATOM 170 CE1 PHE 23 20584 42973 98351 100 7279 13ATOM 171 CE2 PHE 23 22355 43944 97099 100 7279 13ATOM 172 CZ PHE 23 21307 44081 97978 100 7279 13ATOM 173 N GLY 24 24463 37917 96512 100 3326 13ATOM 174 CA GLY 24 24717 36551 96170 100 3326 13ATOM 175 C GLY 24 25847 36477 95200 100 3326 13ATOM 176 O GLY 24 26619 37420 95028 100 3326 13ATOM 177 N THR 25 25962 35310 94537 100 098 13ATOM 178 CA THR 25 27038 35067 93627 100 098 13ATOM 179 C THR 25 26486 34562 92334 100 098 13ATOM 180 O THR 25 25355 34083 92264 100 098 13ATOM 181 CB THR 25 27970 34020 94148 100 098 13ATOM 182 OG1 THR 25 29031 33785 93233 100 098 13ATOM 183 CG2 THR 25 27148 32744 94394 100 098 13ATOM 184 N VAL 26 27290 34702 91261 100 4196 13ATOM 185 CA VAL 26 26931 34198 89970 100 4196 13ATOM 186 C VAL 26 28015 33240 89597 100 4196 13ATOM 187 O VAL 26 29197 33556 89726 100 4196 13ATOM 188 CB VAL 26 26892 35252 88901 100 4196 13ATOM 189 CG1 VAL 26 26617 34570 87549 100 4196 13ATOM 190 CG2 VAL 26 25849 36313 89291 100 4196 13ATOM 191 N TYR 27 27642 32032 89130 100 048 13ATOM 192 CA TYR 27 28648 31073 88775 100 048 13ATOM 193 C TYR 27 28360 30542 87416 100 048 13ATOM 194 O TYR 27 27231 30579 86930 100 048 13ATOM 195 CB TYR 27 28683 29796 89637 100 048 13ATOM 196 CG TYR 27 29227 30069 90993 100 048 13ATOM 197 CD1 TYR 27 28400 30449 92022 100 048 13ATOM 198 CD2 TYR 27 30575 29929 91232 100 048 13ATOM 199 CE1 TYR 27 28921 30688 93270 100 048 13ATOM 200 CE2 TYR 27 31098 30170 92480 100 048 13ATOM 201 CZ TYR 27 30266 30551 93503 100 048 13ATOM 202 OH TYR 27 30788 30801 94790 100 048 13ATOM 203 N LYS 28 29424 30049 86763 100 091 13ATOM 204 CA LYS 28 29274 29355 85527 100 091 13ATOM 205 C LYS 28 29287 27920 85937 100 091 13ATOM 206 O LYS 28 30099 27509 86766 100 091 13ATOM 207 CB LYS 28 30414 29636 84529 100 091 13ATOM 208 CG LYS 28 30307 28889 83198 100 091 13ATOM 209 CD LYS 28 30663 27407 83303 100 091 13ATOM 210 CE LYS 28 32177 27184 83271 100 091 13ATOM 211 NZ LYS 28 32491 25750 83465 100 091 13ATOM 212 N GLY 29 28356 27115 85398 100 3269 13ATOM 213 CA GLY 29 28315 25754 85844 100 3269 13ATOM 214 C GLY 29 27702 24934 84762 100 3269 13ATOM 215 O GLY 29 27527 25396 83636 100 3269 13ATOM 216 N LYS 30 27375 23669 85086 100 027 13ATOM 217 CA LYS 30 26815 22793 84107 100 027 13ATOM 218 C LYS 30 25533 22250 84669 100 027 13ATOM 219 O LYS 30 25528 21706 85771 100 027 13ATOM 220 CB LYS 30 27706 21563 83850 100 027 13ATOM 221 CG LYS 30 29182 21877 83568 100 027 13ATOM 222 CD LYS 30 29439 22852 82416 100 027 13ATOM 223 CE LYS 30 30891 22856 81934 100 027 13ATOM 224 NZ LYS 30 31807 22989 83089 100 027 13ATOM 225 N TRP 31 24403 22403 83940 100 005 13ATOM 226 CA TRP 31 23182 21784 84382 100 005 13ATOM 227 C TRP 31 23047 20656 83440 100 005 13ATOM 228 O TRP 31 22206 20671 82543 100 005 13ATOM 229 CB TRP 31 21936 22681 84210 100 005 13ATOM 230 CG TRP 31 20642 22118 84763 100 005 13ATOM 231 CD1 TRP 31 20192 22136 86052 100 005 13ATOM 232 CD2 TRP 31 19615 21468 83989 100 005 13ATOM 233 NE1 TRP 31 18956 21538 86131 100 005 13ATOM 234 CE2 TRP 31 18589 21123 84869 100 005 13ATOM 235 CE3 TRP 31 19526 21183 82657 100 005 13ATOM 236 CZ2 TRP 31 17464 20486 84427 100 005 13ATOM 237 CZ3 TRP 31 18393 20538 82214 100 005 13ATOM 238 CH2 TRP 31 17380 20196 83083 100 005 13ATOM 239 N HIS 32 23907 19640 83633 100 9570 13ATOM 240 CA HIS 32 24003 18607 82654 100 9570 13ATOM 241 C HIS 32 24274 19341 81373 100 9570 13ATOM 242 O HIS 32 23838 18913 80307 100 9570 13ATOM 243 CB HIS 32 22714 17783 82478 100 9570 13ATOM 244 CG HIS 32 22358 16984 83696 100 9570 13ATOM 245 ND1 HIS 32 21239 16188 83798 100 9570 13ATOM 246 CD2 HIS 32 23000 16877 84891 100 9570 13ATOM 247 CE1 HIS 32 21259 15643 85041 100 9570 13ATOM 248 NE2 HIS 32 22309 16033 85741 100 9570 13ATOM 249 N GLY 33 25015 20474 81452 100 8194 13ATOM 250 CA GLY 33 25195 21289 80279 100 8194 13ATOM 251 C GLY 33 25964 22531 80636 100 8194 13ATOM 252 O GLY 33 26931 22464 81386 100 8194 13ATOM 253 N ASP 34 25608 23699 80046 100 021 13ATOM 254 CA ASP 34 26298 24920 80385 100 021 13ATOM 255 C ASP 34 25268 25929 80791 100 021 13ATOM 256 O ASP 34 24316 26187 80056 100 021 13ATOM 257 CB ASP 34 27100 25526 79221 100 021 13ATOM 258 CG ASP 34 28023 26606 79774 100 021 13ATOM 259 OD1 ASP 34 27716 27166 80860 100 021 13ATOM 260 OD2 ASP 34 29060 26880 79113 100 021 13ATOM 261 N VAL 35 25431 26537 81984 100 5910 13ATOM 262 CA VAL 35 24444 27477 82422 100 5910 13ATOM 263 C VAL 35 25089 28484 83317 100 5910 13ATOM 264 O VAL 35 26236 28333 83738 100 5910 13ATOM 265 CB VAL 35 23363 26839 83243 100 5910 13ATOM 266 CG1 VAL 35 22628 25799 82379 100 5910 13ATOM 267 CG2 VAL 35 24006 26248 84511 100 5910 13ATOM 268 N ALA 36 24343 29568 83605 100 4474 13ATOM 269 CA ALA 36 24791 30562 84533 100 4474 13ATOM 270 C ALA 36 23888 30410 85711 100 4474 13ATOM 271 O ALA 36 22677 30261 85551 100 4474 13ATOM 272 CB ALA 36 24630 32004 84022 100 4474 13ATOM 273 N VAL 37 24452 30419 86933 100 4284 13ATOM 274 CA VAL 37 23607 30241 88074 100 4284 13ATOM 275 C VAL 37 23792 31410 88980 100 4284 13ATOM 276 O VAL 37 24913 31842 89241 100 4284 13ATOM 277 CB VAL 37 23928 29009 88866 100 4284 13ATOM 278 CG1 VAL 37 23020 28970 90106 100 4284 13ATOM 279 CG2 VAL 37 23778 27787 87944 100 4284 13ATOM 280 N LYS 38 22669 31957 89478 100 054 13ATOM 281 CA LYS 38 22718 33062 90384 100 054 13ATOM 282 C LYS 38 22204 32526 91681 100 054 13ATOM 283 O LYS 38 21086 32017 91747 100 054 13ATOM 284 CB LYS 38 21816 34218 89910 100 054 13ATOM 285 CG LYS 38 21982 35557 90632 100 054 13ATOM 286 CD LYS 38 21463 35587 92066 100 054 13ATOM 287 CE LYS 38 21397 37005 92637 100 054 13ATOM 288 NZ LYS 38 20584 37020 93872 100 054 13ATOM 289 N MET 39 23015 32610 92752 100 5092 13ATOM 290 CA MET 39 22572 32058 93997 100 5092 13ATOM 291 C MET 39 22293 33183 94926 100 5092 13ATOM 292 O MET 39 22958 34217 94893 100 5092 13ATOM 293 CB MET 39 23595 31130 94682 100 5092 13ATOM 294 CG MET 39 23848 29828 93916 100 5092 13ATOM 295 SD MET 39 24927 28635 94768 100 5092 13ATOM 296 CE MET 39 26474 29545 94491 100 5092 13ATOM 297 N LEU 40 21266 32998 95774 100 8622 13ATOM 298 CA LEU 40 20900 34016 96702 100 8622 13ATOM 299 C LEU 40 21615 33712 97974 100 8622 13ATOM 300 O LEU 40 21737 32558 98382 100 8622 13ATOM 301 CB LEU 40 19380 34078 96951 100 8622 13ATOM 302 CG LEU 40 18942 35168 97945 100 8622 13ATOM 303 CD1 LEU 40 19478 36538 97512 100 8622 13ATOM 304 CD2 LEU 40 17411 35199 98105 100 8622 13ATOM 305 N ASN 41 22170 34755 98610 100 7628 13ATOM 306 CA ASN 41 22832 34534 99854 100 7628 13ATOM 307 C ASN 41 21730 34386 100838 100 7628 13ATOM 308 O ASN 41 20738 35109 100769 100 7628 13ATOM 309 CB ASN 41 23719 35709 100288 100 7628 13ATOM 310 CG ASN 41 24842 35823 99269 100 7628 13ATOM 311 OD1 ASN 41 25416 36895 99083 100 7628 13ATOM 312 ND2 ASN 41 25163 34692 98586 100 7628 13ATOM 313 N VAL 42 21871 33448 101791 100 025 13ATOM 314 CA VAL 42 20773 33231 102680 100 025 13ATOM 315 C VAL 42 20511 34477 103459 100 025 13ATOM 316 O VAL 42 21415 35072 104044 100 025 13ATOM 317 CB VAL 42 20986 32109 103657 100 025 13ATOM 318 CG1 VAL 42 22146 32483 104593 100 025 13ATOM 319 CG2 VAL 42 19657 31839 104384 100 025 13ATOM 320 N THR 43 19230 34891 103426 100 090 13ATOM 321 CA THR 43 18614 35986 104120 100 090 13ATOM 322 C THR 43 19210 37310 103760 100 090 13ATOM 323 O THR 43 18880 38310 104397 100 090 13ATOM 324 CB THR 43 18612 35845 105618 100 090 13ATOM 325 OG1 THR 43 17711 36785 106184 100 090 13ATOM 326 CG2 THR 43 20026 36095 106168 100 090 13ATOM 327 N ALA 44 20091 37398 102742 100 7946 13ATOM 328 CA ALA 44 20525 38735 102465 100 7946 13ATOM 329 C ALA 44 19326 39475 101931 100 7946 13ATOM 330 O ALA 44 19007 40549 102433 100 7946 13ATOM 331 CB ALA 44 21746 38820 101519 100 7946 13ATOM 332 N PRO 45 18650 38967 100922 100 038 13ATOM 333 CA PRO 45 17359 39519 100596 100 038 13ATOM 334 C PRO 45 16474 38490 101214 100 038 13ATOM 335 O PRO 45 17008 37478 101663 100 038 13ATOM 336 CB PRO 45 17229 39497 99072 100 038 13ATOM 337 CG PRO 45 18669 39376 98567 100 038 13ATOM 338 CD PRO 45 19372 38627 99703 100 038 13ATOM 339 N THR 46 15145 38670 101264 100 034 13ATOM 340 CA THR 46 14442 37522 101750 100 034 13ATOM 341 C THR 46 13783 36869 100580 100 034 13ATOM 342 O THR 46 12937 37437 99898 100 034 13ATOM 343 CB THR 46 13470 37802 102862 100 034 13ATOM 344 OG1 THR 46 12828 36599 103258 100 034 13ATOM 345 CG2 THR 46 12461 38871 102442 100 034 13ATOM 346 N PRO 47 14254 35686 100307 100 056 13ATOM 347 CA PRO 47 13807 34913 99178 100 056 13ATOM 348 C PRO 47 12327 34715 99140 100 056 13ATOM 349 O PRO 47 11708 35033 98124 100 056 13ATOM 350 CB PRO 47 14483 33557 99330 100 056 13ATOM 351 CG PRO 47 14556 33400 100861 100 056 13ATOM 352 CD PRO 47 14766 34837 101369 100 056 13ATOM 353 N GLN 48 11773 34093 100204 100 023 13ATOM 354 CA GLN 48 10366 33839 100300 100 023 13ATOM 355 C GLN 48 9621 35066 100709 100 023 13ATOM 356 O GLN 48 8620 35425 100093 100 023 13ATOM 357 CB GLN 48 10038 32712 101297 100 023 13ATOM 358 CG GLN 48 10363 33036 102756 100 023 13ATOM 359 CD GLN 48 9124 33663 103384 100 023 13ATOM 360 OE1 GLN 48 8120 33886 102708 100 023 13ATOM 361 NE2 GLN 48 9180 33939 104715 100 023 13ATOM 362 N GLN 49 10116 35766 101751 100 9406 13ATOM 363 CA GLN 49 9367 36884 102239 100 9406 13ATOM 364 C GLN 49 9337 37909 101170 100 9406 13ATOM 365 O GLN 49 8289 38475 100861 100 9406 13ATOM 366 CB GLN 49 9969 37547 103488 100 9406 13ATOM 367 CG GLN 49 9113 38716 103983 100 9406 13ATOM 368 CD GLN 49 9775 39314 105215 100 9406 13ATOM 369 OE1 GLN 49 9179 40121 105928 100 9406 13ATOM 370 NE2 GLN 49 11044 38906 105480 100 9406 13ATOM 371 N LEU 50 10502 38159 100555 100 073 13ATOM 372 CA LEU 50 10508 39127 99517 100 073 13ATOM 373 C LEU 50 10466 38365 98259 100 073 13ATOM 374 O LEU 50 11311 37524 97966 100 073 13ATOM 375 CB LEU 50 11739 40049 99461 100 073 13ATOM 376 CG LEU 50 11804 41071 100608 100 073 13ATOM 377 CD1 LEU 50 12927 42093 100382 100 073 13ATOM 378 CD2 LEU 50 10434 41717 100869 100 073 13ATOM 379 N GLN 51 9456 38671 97457 100 047 13ATOM 380 CA GLN 51 9306 37999 96220 100 047 13ATOM 381 C GLN 51 10514 38338 95426 100 047 13ATOM 382 O GLN 51 10953 37537 94613 100 047 13ATOM 383 CB GLN 51 8060 38449 95435 100 047 13ATOM 384 CG GLN 51 6733 38027 96075 100 047 13ATOM 385 CD GLN 51 6479 38895 97300 100 047 13ATOM 386 OE1 GLN 51 7143 39907 97516 100 047 13ATOM 387 NE2 GLN 51 5481 38489 98130 100 047 13ATOM 388 N ALA 52 11158 39479 95740 100 6822 13ATOM 389 CA ALA 52 12132 40098 94886 100 6822 13ATOM 390 C ALA 52 13169 39138 94385 100 6822 13ATOM 391 O ALA 52 13512 39205 93206 100 6822 13ATOM 392 CB ALA 52 12875 41253 95579 100 6822 13ATOM 393 N PHE 53 13713 38227 95210 100 6452 13ATOM 394 CA PHE 53 14685 37342 94627 100 6452 13ATOM 395 C PHE 53 14023 36536 93544 100 6452 13ATOM 396 O PHE 53 14493 36494 92408 100 6452 13ATOM 397 CB PHE 53 15284 36356 95645 100 6452 13ATOM 398 CG PHE 53 16169 35418 94897 100 6452 13ATOM 399 CD1 PHE 53 17445 35784 94535 100 6452 13ATOM 400 CD2 PHE 53 15720 34160 94564 100 6452 13ATOM 401 CE1 PHE 53 18255 34912 93845 100 6452 13ATOM 402 CE2 PHE 53 16527 33286 93875 100 6452 13ATOM 403 CZ PHE 53 17798 33660 93512 100 6452 13ATOM 404 N LYS 54 12878 35911 93875 100 6478 13ATOM 405 CA LYS 54 12128 35049 92999 100 6478 13ATOM 406 C LYS 54 11516 35818 91864 100 6478 13ATOM 407 O LYS 54 11341 35280 90775 100 6478 13ATOM 408 CB LYS 54 10961 34343 93716 100 6478 13ATOM 409 CG LYS 54 11392 33396 94838 100 6478 13ATOM 410 CD LYS 54 10231 32926 95716 100 6478 13ATOM 411 CE LYS 54 10622 31840 96715 100 6478 13ATOM 412 NZ LYS 54 9443 31456 97521 100 6478 13ATOM 413 N ASN 55 11177 37099 92080 100 6792 13ATOM 414 CA ASN 55 10412 37871 91144 100 6792 13ATOM 415 C ASN 55 11096 37885 89825 100 6792 13ATOM 416 O ASN 55 10450 37790 88783 100 6792 13ATOM 417 CB ASN 55 10233 39344 91559 100 6792 13ATOM 418 CG ASN 55 9192 39426 92662 100 6792 13ATOM 419 OD1 ASN 55 9367 40154 93639 100 6792 13ATOM 420 ND2 ASN 55 8068 38678 92492 100 6792 13ATOM 421 N GLU 56 12428 37994 89829 100 9432 13ATOM 422 CA GLU 56 13109 38076 88576 100 9432 13ATOM 423 C GLU 56 12825 36831 87786 100 9432 13ATOM 424 O GLU 56 12533 36907 86593 100 9432 13ATOM 425 CB GLU 56 14630 38237 88779 100 9432 13ATOM 426 CG GLU 56 15432 36970 88470 100 9432 13ATOM 427 CD GLU 56 16537 37323 87481 100 9432 13ATOM 428 OE1 GLU 56 16242 38025 86478 100 9432 13ATOM 429 OE2 GLU 56 17696 36894 87723 100 9432 13ATOM 430 N VAL 57 12875 35642 88419 100 022 13ATOM 431 CA VAL 57 12639 34452 87648 100 022 13ATOM 432 C VAL 57 11222 34429 87173 100 022 13ATOM 433 O VAL 57 10947 34065 86030 100 022 13ATOM 434 CB VAL 57 12878 33156 88380 100 022 13ATOM 435 CG1 VAL 57 14326 33152 88881 100 022 13ATOM 436 CG2 VAL 57 11809 32931 89460 100 022 13ATOM 437 N GLY 58 10281 34837 88042 100 2701 13ATOM 438 CA GLY 58 8891 34755 87705 100 2701 13ATOM 439 C GLY 58 8614 35594 86502 100 2701 13ATOM 440 O GLY 58 7861 35184 85619 100 2701 13ATOM 441 N VAL 59 9211 36797 86434 100 088 13ATOM 442 CA VAL 59 8930 37647 85317 100 088 13ATOM 443 C VAL 59 9426 36970 84084 100 088 13ATOM 444 O VAL 59 8765 36969 83049 100 088 13ATOM 445 CB VAL 59 9591 39002 85396 100 088 13ATOM 446 CG1 VAL 59 9108 39703 86676 100 088 13ATOM 447 CG2 VAL 59 11118 38867 85294 100 088 13ATOM 448 N LEU 60 10598 36327 84189 100 037 13ATOM 449 CA LEU 60 11257 35751 83061 100 037 13ATOM 450 C LEU 60 10412 34657 82486 100 037 13ATOM 451 O LEU 60 10441 34413 81280 100 037 13ATOM 452 CB LEU 60 12671 35257 83415 100 037 13ATOM 453 CG LEU 60 13542 34909 82196 100 037 13ATOM 454 CD1 LEU 60 13559 36070 81188 100 037 13ATOM 455 CD2 LEU 60 14975 34568 82637 100 037 13ATOM 456 N ARG 61 9631 33962 83333 100 6795 13ATOM 457 CA ARG 61 8799 32890 82863 100 6795 13ATOM 458 C ARG 61 7823 33443 81866 100 6795 13ATOM 459 O ARG 61 7558 32823 80837 100 6795 13ATOM 460 CB ARG 61 7981 32247 83997 100 6795 13ATOM 461 CG ARG 61 8845 31581 85072 100 6795 13ATOM 462 CD ARG 61 8038 30938 86203 100 6795 13ATOM 463 NE ARG 61 9007 30335 87161 100 6795 13ATOM 464 CZ ARG 61 8559 29811 88339 100 6795 13ATOM 465 NH1 ARG 61 7225 29834 88624 100 6795 13ATOM 466 NH2 ARG 61 9441 29275 89234 100 6795 13ATOM 467 N LYS 62 7254 34628 82155 100 9772 13ATOM 468 CA LYS 62 6265 35242 81319 100 9772 13ATOM 469 C LYS 62 6874 35663 80013 100 9772 13ATOM 470 O LYS 62 6231 35576 78968 100 9772 13ATOM 471 CB LYS 62 5639 36485 81973 100 9772 13ATOM 472 CG LYS 62 4985 36189 83327 100 9772 13ATOM 473 CD LYS 62 3852 35159 83272 100 9772 13ATOM 474 CE LYS 62 3267 34828 84649 100 9772 13ATOM 475 NZ LYS 62 2190 33821 84514 100 9772 13ATOM 476 N THR 63 8130 36150 80032 100 023 13ATOM 477 CA THR 63 8713 36636 78814 100 023 13ATOM 478 C THR 63 9703 35647 78273 100 023 13ATOM 479 O THR 63 10872 35642 78651 100 023 13ATOM 480 CB THR 63 9421 37949 79006 100 023 13ATOM 481 OG1 THR 63 10472 37823 79954 100 023 13ATOM 482 CG2 THR 63 8396 38982 79502 100 023 13ATOM 483 N ARG 64 9274 34806 77311 100 7846 13ATOM 484 CA ARG 64 10185 33819 76801 100 7846 13ATOM 485 C ARG 64 10528 34202 75402 100 7846 13ATOM 486 O ARG 64 9639 34373 74570 100 7846 13ATOM 487 CB ARG 64 9570 32412 76709 100 7846 13ATOM 488 CG ARG 64 9055 31852 78035 100 7846 13ATOM 489 CD ARG 64 8436 30459 77894 100 7846 13ATOM 490 NE ARG 64 7877 30067 79218 100 7846 13ATOM 491 CZ ARG 64 6994 29029 79294 100 7846 13ATOM 492 NH1 ARG 64 6624 28361 78163 100 7846 13ATOM 493 NH2 ARG 64 6469 28664 80499 100 7846 13ATOM 494 N HIS 65 11834 34348 75104 100 6844 13ATOM 495 CA HIS 65 12211 34702 73770 100 6844 13ATOM 496 C HIS 65 13668 34368 73632 100 6844 13ATOM 497 O HIS 65 14345 34084 74616 100 6844 13ATOM 498 CB HIS 65 12016 36201 73481 100 6844 13ATOM 499 CG HIS 65 11968 36539 72022 100 6844 13ATOM 500 ND1 HIS 65 13076 36703 71223 100 6844 13ATOM 501 CD2 HIS 65 10893 36751 71215 100 6844 13ATOM 502 CE1 HIS 65 12621 37003 69980 100 6844 13ATOM 503 NE2 HIS 65 11302 37044 69926 100 6844 13ATOM 504 N VAL 66 14187 34408 72393 100 085 13ATOM 505 CA VAL 66 15557 34096 72078 100 085 13ATOM 506 C VAL 66 16429 35126 72732 100 085 13ATOM 507 O VAL 66 17626 34928 72930 100 085 13ATOM 508 CB VAL 66 15825 34107 70599 100 085 13ATOM 509 CG1 VAL 66 15623 35535 70070 100 085 13ATOM 510 CG2 VAL 66 17230 33537 70344 100 085 13ATOM 511 N ASN 67 15807 36262 73078 100 081 13ATOM 512 CA ASN 67 16363 37472 73606 100 081 13ATOM 513 C ASN 67 17086 37308 74941 100 081 13ATOM 514 O ASN 67 17722 38305 75277 100 081 13ATOM 515 CB ASN 67 15281 38541 73816 100 081 13ATOM 516 CG ASN 67 14654 38825 72458 100 081 13ATOM 517 OD1 ASN 67 15297 38704 71417 100 081 13ATOM 518 ND2 ASN 67 13349 39209 72469 100 081 13ATOM 519 N ILE 68 16885 36192 75748 100 003 13ATOM 520 CA ILE 68 17446 35713 77030 100 003 13ATOM 521 C ILE 68 16505 34682 77612 100 003 13ATOM 522 O ILE 68 15291 34873 77615 100 003 13ATOM 523 CB ILE 68 17731 36761 78068 100 003 13ATOM 524 CG1 ILE 68 16568 37766 78242 100 003 13ATOM 525 CG2 ILE 68 19128 37308 77777 100 003 13ATOM 526 CD1 ILE 68 15325 37247 78966 100 003 13ATOM 527 N LEU 69 17030 33536 78116 100 088 13ATOM 528 CA LEU 69 16140 32498 78593 100 088 13ATOM 529 C LEU 69 16376 32151 80039 100 088 13ATOM 530 O LEU 69 17502 32211 80530 100 088 13ATOM 531 CB LEU 69 16278 31177 77810 100 088 13ATOM 532 CG LEU 69 15366 30047 78330 100 088 13ATOM 533 CD1 LEU 69 13881 30409 78172 100 088 13ATOM 534 CD2 LEU 69 15721 28690 77694 100 088 13ATOM 535 N LEU 70 15287 31767 80755 100 025 13ATOM 536 CA LEU 70 15362 31301 82117 100 025 13ATOM 537 C LEU 70 15328 29811 82025 100 025 13ATOM 538 O LEU 70 14344 29234 81563 100 025 13ATOM 539 CB LEU 70 14174 31764 82994 100 025 13ATOM 540 CG LEU 70 14191 31299 84468 100 025 13ATOM 541 CD1 LEU 70 13186 32094 85311 100 025 13ATOM 542 CD2 LEU 70 13884 29802 84588 100 025 13ATOM 543 N PHE 71 16452 29157 82390 100 8903 13ATOM 544 CA PHE 71 16511 27730 82289 100 8903 13ATOM 545 C PHE 71 15693 27058 83352 100 8903 13ATOM 546 O PHE 71 14782 26298 83032 100 8903 13ATOM 547 CB PHE 71 17946 27185 82377 100 8903 13ATOM 548 CG PHE 71 17917 25798 81828 100 8903 13ATOM 549 CD1 PHE 71 18064 25592 80473 100 8903 13ATOM 550 CD2 PHE 71 17730 24706 82645 100 8903 13ATOM 551 CE1 PHE 71 18036 24325 79938 100 8903 13ATOM 552 CE2 PHE 71 17702 23436 82118 100 8903 13ATOM 553 CZ PHE 71 17855 23243 80766 100 8903 13ATOM 554 N MET 72 15959 27361 84647 100 047 13ATOM 555 CA MET 72 15285 26657 85708 100 047 13ATOM 556 C MET 72 15539 27380 87006 100 047 13ATOM 557 O MET 72 16193 28422 87024 100 047 13ATOM 558 CB MET 72 15796 25221 85872 100 047 13ATOM 559 CG MET 72 15572 24281 84687 100 047 13ATOM 560 SD MET 72 13833 23887 84326 100 047 13ATOM 561 CE MET 72 13781 22411 85383 100 047 13ATOM 562 N GLY 73 15012 26844 88136 100 3639 13ATOM 563 CA GLY 73 15200 27485 89415 100 3639 13ATOM 564 C GLY 73 15287 26434 90487 100 3639 13ATOM 565 O GLY 73 14800 25317 90321 100 3639 13ATOM 566 N TYR 74 15931 26781 91624 100 029 13ATOM 567 CA TYR 74 16072 25887 92744 100 029 13ATOM 568 C TYR 74 15466 26600 93919 100 029 13ATOM 569 O TYR 74 15956 27642 94349 100 029 13ATOM 570 CB TYR 74 17553 25599 93053 100 029 13ATOM 571 CG TYR 74 17655 24541 94094 100 029 13ATOM 572 CD1 TYR 74 17339 23239 93786 100 029 13ATOM 573 CD2 TYR 74 18098 24844 95361 100 029 13ATOM 574 CE1 TYR 74 17438 22252 94734 100 029 13ATOM 575 CE2 TYR 74 18200 23861 96315 100 029 13ATOM 576 CZ TYR 74 17867 22565 96000 100 029 13ATOM 577 OH TYR 74 17972 21553 96977 100 029 13ATOM 578 N SER 75 14361 26049 94454 100 060 13ATOM 579 CA SER 75 13588 26638 95515 100 060 13ATOM 580 C SER 75 14261 26605 96859 100 060 13ATOM 581 O SER 75 14199 27582 97605 100 060 13ATOM 582 CB SER 75 12244 25914 95699 100 060 13ATOM 583 OG SER 75 11551 26444 96817 100 060 13ATOM 584 N THR 76 14918 25483 97217 100 006 13ATOM 585 CA THR 76 15404 25342 98564 100 006 13ATOM 586 C THR 76 16773 25921 98716 100 006 13ATOM 587 O THR 76 17404 26331 97744 100 006 13ATOM 588 CB THR 76 15437 23917 99042 100 006 13ATOM 589 OG1 THR 76 16337 23151 98259 100 006 13ATOM 590 CG2 THR 76 14022 23329 98922 100 006 13ATOM 591 N LYS 77 17249 25977 99983 100 044 13ATOM 592 CA LYS 77 18527 26537 100324 100 044 13ATOM 593 C LYS 77 19602 25602 99844 100 044 13ATOM 594 O LYS 77 19353 24399 99861 100 044 13ATOM 595 CB LYS 77 18721 26788 101829 100 044 13ATOM 596 CG LYS 77 19966 27617 102148 100 044 13ATOM 597 CD LYS 77 19941 28223 103554 100 044 13ATOM 598 CE LYS 77 21189 29032 103913 100 044 13ATOM 599 NZ LYS 77 22308 28127 104263 100 044 13ATOM 600 N PRO 78 20790 26010 99409 100 000 13ATOM 601 CA PRO 78 21279 27371 99398 100 000 13ATOM 602 C PRO 78 20260 28190 98727 100 000 13ATOM 603 O PRO 78 19580 27633 97863 100 000 13ATOM 604 CB PRO 78 22580 27343 98599 100 000 13ATOM 605 CG PRO 78 22365 26173 97626 100 000 13ATOM 606 CD PRO 78 21481 25199 98419 100 000 13ATOM 607 N GLN 79 20134 29461 99168 100 052 13ATOM 608 CA GLN 79 19059 30316 98783 100 052 13ATOM 609 C GLN 79 18904 30174 97322 100 052 13ATOM 610 O GLN 79 19875 29905 96617 100 052 13ATOM 611 CB GLN 79 19243 31789 99157 100 052 13ATOM 612 CG GLN 79 17918 32531 99227 100 052 13ATOM 613 CD GLN 79 17293 32157 100560 100 052 13ATOM 614 OE1 GLN 79 17546 32794 101582 100 052 13ATOM 615 NE2 GLN 79 16448 31092 100555 100 052 13ATOM 616 N LEU 80 17652 30330 96870 100 055 13ATOM 617 CA LEU 80 17205 29949 95570 100 055 13ATOM 618 C LEU 80 18188 30317 94522 100 055 13ATOM 619 O LEU 80 18846 31355 94574 100 055 13ATOM 620 CB LEU 80 15850 30572 95197 100 055 13ATOM 621 CG LEU 80 14691 30093 96089 100 055 13ATOM 622 CD1 LEU 80 14918 30484 97558 100 055 13ATOM 623 CD2 LEU 80 13338 30582 95546 100 055 13ATOM 624 N ALA 81 18345 29385 93566 100 4364 13ATOM 625 CA ALA 81 19247 29586 92483 100 4364 13ATOM 626 C ALA 81 18414 29843 91282 100 4364 13ATOM 627 O ALA 81 17385 29203 91071 100 4364 13ATOM 628 CB ALA 81 20129 28366 92168 100 4364 13ATOM 629 N ILE 82 18835 30830 90479 100 5618 13ATOM 630 CA ILE 82 18145 31130 89269 100 5618 13ATOM 631 C ILE 82 19117 30773 88198 100 5618 13ATOM 632 O ILE 82 20223 31309 88155 100 5618 13ATOM 633 CB ILE 82 17814 32591 89143 100 5618 13ATOM 634 CG1 ILE 82 16899 33018 90304 100 5618 13ATOM 635 CG2 ILE 82 17204 32829 87752 100 5618 13ATOM 636 CD1 ILE 82 16745 34531 90459 100 5618 13ATOM 637 N VAL 83 18745 29823 87319 100 058 13ATOM 638 CA VAL 83 19674 29434 86302 100 058 13ATOM 639 C VAL 83 19148 29915 84990 100 058 13ATOM 640 O VAL 83 17952 29824 84719 100 058 13ATOM 641 CB VAL 83 19877 27950 86206 100 058 13ATOM 642 CG1 VAL 83 18543 27295 85816 100 058 13ATOM 643 CG2 VAL 83 21026 27676 85220 100 058 13ATOM 644 N THR 84 20039 30467 84141 100 5108 13ATOM 645 CA THR 84 19583 30941 82867 100 5108 13ATOM 646 C THR 84 20603 30598 81836 100 5108 13ATOM 647 O THR 84 21655 30043 82147 100 5108 13ATOM 648 CB THR 84 19383 32426 82793 100 5108 13ATOM 649 OG1 THR 84 20613 33099 83005 100 5108 13ATOM 650 CG2 THR 84 18359 32838 83861 100 5108 13ATOM 651 N GLN 85 20301 30928 80563 100 5516 13ATOM 652 CA GLN 85 21210 30627 79496 100 5516 13ATOM 653 C GLN 85 22464 31395 79751 100 5516 13ATOM 654 O GLN 85 22428 32556 80154 100 5516 13ATOM 655 CB GLN 85 20707 31031 78098 100 5516 13ATOM 656 CG GLN 85 21677 30635 76982 100 5516 13ATOM 657 CD GLN 85 21099 31080 75647 100 5516 13ATOM 658 OE1 GLN 85 20247 31965 75597 100 5516 13ATOM 659 NE2 GLN 85 21574 30453 74537 100 5516 13ATOM 660 N TRP 86 23624 30752 79532 100 053 13ATOM 661 CA TRP 86 24845 31432 79836 100 053 13ATOM 662 C TRP 86 25349 32123 78611 100 053 13ATOM 663 O TRP 86 25211 31625 77494 100 053 13ATOM 664 CB TRP 86 25923 30494 80405 100 053 13ATOM 665 CG TRP 86 27186 31180 80860 100 053 13ATOM 666 CD1 TRP 86 27373 32129 81824 100 053 13ATOM 667 CD2 TRP 86 28484 30857 80351 100 053 13ATOM 668 NE1 TRP 86 28711 32428 81933 100 053 13ATOM 669 CE2 TRP 86 29408 31646 81036 100 053 13ATOM 670 CE3 TRP 86 28873 29967 79397 100 053 13ATOM 671 CZ2 TRP 86 30746 31558 80771 100 053 13ATOM 672 CZ3 TRP 86 30218 29883 79132 100 053 13ATOM 673 CH2 TRP 86 31139 30662 79803 100 053 13ATOM 674 N CYS 87 25930 33325 78810 100 6054 13ATOM 675 CA CYS 87 26452 34108 77725 100 6054 13ATOM 676 C CYS 87 27927 34235 77955 100 6054 13ATOM 677 O CYS 87 28366 34912 78883 100 6054 13ATOM 678 CB CYS 87 25874 35530 77693 100 6054 13ATOM 679 SG CYS 87 24074 35526 77448 100 6054 13ATOM 680 N GLU 88 28732 33620 77069 100 9833 13ATOM 681 CA GLU 88 30157 33543 77243 100 9833 13ATOM 682 C GLU 88 30790 34897 77243 100 9833 13ATOM 683 O GLU 88 31703 35162 78025 100 9833 13ATOM 684 CB GLU 88 30840 32716 76142 100 9833 13ATOM 685 CG GLU 88 30723 31207 76354 100 9833 13ATOM 686 CD GLU 88 32029 30741 76982 100 9833 13ATOM 687 OE1 GLU 88 32866 31616 77331 100 9833 13ATOM 688 OE2 GLU 88 32207 29503 77130 100 9833 13ATOM 689 N GLY 89 30310 35779 76355 100 7793 13ATOM 690 CA GLY 89 30860 37088 76133 100 7793 13ATOM 691 C GLY 89 30742 37976 77334 100 7793 13ATOM 692 O GLY 89 31558 38877 77495 100 7793 13ATOM 693 N SER 90 29678 37833 78146 100 079 13ATOM 694 CA SER 90 29496 38644 79325 100 079 13ATOM 695 C SER 90 28783 39896 78914 100 079 13ATOM 696 O SER 90 28123 39931 77877 100 079 13ATOM 697 CB SER 90 30770 39008 80144 100 079 13ATOM 698 OG SER 90 31422 40185 79685 100 079 13ATOM 699 N SER 91 28885 40963 79731 100 009 13ATOM 700 CA SER 91 28192 42185 79439 100 009 13ATOM 701 C SER 91 28802 42835 78236 100 009 13ATOM 702 O SER 91 30012 42783 78020 100 009 13ATOM 703 CB SER 91 28236 43199 80592 100 009 13ATOM 704 OG SER 91 27532 44376 80227 100 009 13ATOM 705 N LEU 92 27939 43494 77436 100 6451 13ATOM 706 CA LEU 92 28293 44144 76205 100 6451 13ATOM 707 C LEU 92 29226 45279 76490 100 6451 13ATOM 708 O LEU 92 30155 45538 75728 100 6451 13ATOM 709 CB LEU 92 27065 44714 75474 100 6451 13ATOM 710 CG LEU 92 27387 45384 74126 100 6451 13ATOM 711 CD1 LEU 92 27950 44370 73113 100 6451 13ATOM 712 CD2 LEU 92 26164 46148 73595 100 6451 13ATOM 713 N TYR 93 29011 45989 77608 100 018 13ATOM 714 CA TYR 93 29819 47136 77898 100 018 13ATOM 715 C TYR 93 31248 46696 77963 100 018 13ATOM 716 O TYR 93 32122 47330 77372 100 018 13ATOM 717 CB TYR 93 29453 47749 79261 100 018 13ATOM 718 CG TYR 93 30331 48927 79491 100 018 13ATOM 719 CD1 TYR 93 29994 50164 78999 100 018 13ATOM 720 CD2 TYR 93 31496 48785 80209 100 018 13ATOM 721 CE1 TYR 93 30816 51242 79219 100 018 13ATOM 722 CE2 TYR 93 32325 49860 80432 100 018 13ATOM 723 CZ TYR 93 31981 51093 79933 100 018 13ATOM 724 OH TYR 93 32820 52207 80152 100 018 13ATOM 725 N HIS 94 31519 45596 78693 100 7099 13ATOM 726 CA HIS 94 32852 45087 78847 100 7099 13ATOM 727 C HIS 94 33357 44482 77571 100 7099 13ATOM 728 O HIS 94 34521 44650 77213 100 7099 13ATOM 729 CB HIS 94 32958 44042 79968 100 7099 13ATOM 730 CG HIS 94 32737 44659 81319 100 7099 13ATOM 731 ND1 HIS 94 32422 43955 82460 100 7099 13ATOM 732 CD2 HIS 94 32792 45966 81695 100 7099 13ATOM 733 CE1 HIS 94 32300 44865 83461 100 7099 13ATOM 734 NE2 HIS 94 32515 46099 83044 100 7099 13ATOM 735 N HIS 95 32479 43780 76835 100 6832 13ATOM 736 CA HIS 95 32855 43035 75667 100 6832 13ATOM 737 C HIS 95 33400 43947 74607 100 6832 13ATOM 738 O HIS 95 34362 43602 73925 100 6832 13ATOM 739 CB HIS 95 31662 42236 75109 100 6832 13ATOM 740 CG HIS 95 32046 41127 74177 100 6832 13ATOM 741 ND1 HIS 95 32995 40173 74473 100 6832 13ATOM 742 CD2 HIS 95 31561 40799 72949 100 6832 13ATOM 743 CE1 HIS 95 33038 39324 73417 100 6832 13ATOM 744 NE2 HIS 95 32185 39663 72467 100 6832 13ATOM 745 N LEU 96 32839 45157 74450 100 091 13ATOM 746 CA LEU 96 33293 46006 73382 100 091 13ATOM 747 C LEU 96 34758 46302 73502 100 091 13ATOM 748 O LEU 96 35445 46373 72485 100 091 13ATOM 749 CB LEU 96 32551 47357 73269 100 091 13ATOM 750 CG LEU 96 31206 47323 72505 100 091 13ATOM 751 CD1 LEU 96 31424 47069 71005 100 091 13ATOM 752 CD2 LEU 96 30202 46338 73110 100 091 13ATOM 753 N HIS 97 35306 46482 74718 100 090 13ATOM 754 CA HIS 97 36686 46885 74741 100 090 13ATOM 755 C HIS 97 37602 45694 74754 100 090 13ATOM 756 O HIS 97 38309 45475 75735 100 090 13ATOM 757 CB HIS 97 37041 47731 75975 100 090 13ATOM 758 CG HIS 97 36250 49001 76068 100 090 13ATOM 759 ND1 HIS 97 35106 49147 76822 100 090 13ATOM 760 CD2 HIS 97 36460 50209 75476 100 090 13ATOM 761 CE1 HIS 97 34685 50426 76651 100 090 13ATOM 762 NE2 HIS 97 35476 51108 75842 100 090 13ATOM 763 N ILE 98 37635 44917 73650 100 075 13ATOM 764 CA ILE 98 38488 43765 73492 100 075 13ATOM 765 C ILE 98 38576 43478 72023 100 075 13ATOM 766 O ILE 98 38880 44357 71220 100 075 13ATOM 767 CB ILE 98 38039 42518 74233 100 075 13ATOM 768 CG1 ILE 98 36620 42064 73847 100 075 13ATOM 769 CG2 ILE 98 38224 42742 75741 100 075 13ATOM 770 CD1 ILE 98 36281 40659 74341 100 075 13ATOM 771 N ILE 99 38360 42200 71657 100 032 13ATOM 772 CA ILE 99 38283 41742 70305 100 032 13ATOM 773 C ILE 99 37158 42524 69717 100 032 13ATOM 774 O ILE 99 37137 42827 68524 100 032 13ATOM 775 CB ILE 99 37961 40278 70210 100 032 13ATOM 776 CG1 ILE 99 38213 39756 68786 100 032 13ATOM 777 CG2 ILE 99 36522 40067 70713 100 032 13ATOM 778 CD1 ILE 99 38220 38230 68697 100 032 13ATOM 779 N GLU 100 36180 42873 70571 100 031 13ATOM 780 CA GLU 100 35071 43663 70132 100 031 13ATOM 781 C GLU 100 35614 44926 69565 100 031 13ATOM 782 O GLU 100 35013 45522 68676 100 031 13ATOM 783 CB GLU 100 34088 44060 71240 100 031 13ATOM 784 CG GLU 100 33081 42966 71575 100 031 13ATOM 785 CD GLU 100 32019 42977 70483 100 031 13ATOM 786 OE1 GLU 100 32135 43828 69562 100 031 13ATOM 787 OE2 GLU 100 31080 42139 70554 100 031 13ATOM 788 N THR 101 36771 45380 70069 100 017 13ATOM 789 CA THR 101 37326 46574 69519 100 017 13ATOM 790 C THR 101 37584 46276 68082 100 017 13ATOM 791 O THR 101 37784 45130 67692 100 017 13ATOM 792 CB THR 101 38618 46990 70163 100 017 13ATOM 793 OG1 THR 101 38413 47218 71549 100 017 13ATOM 794 CG2 THR 101 39129 48274 69487 100 017 13ATOM 795 N LYS 102 37498 47318 67243 100 061 13ATOM 796 CA LYS 102 37678 47200 65830 100 061 13ATOM 797 C LYS 102 36724 46185 65277 100 061 13ATOM 798 O LYS 102 37036 45476 64321 100 061 13ATOM 799 CB LYS 102 39127 46911 65379 100 061 13ATOM 800 CG LYS 102 39719 45568 65807 100 061 13ATOM 801 CD LYS 102 41061 45281 65133 100 061 13ATOM 802 CE LYS 102 41758 44020 65645 100 061 13ATOM 803 NZ LYS 102 43048 43834 64943 100 061 13ATOM 804 N PHE 103 35512 46096 65861 100 078 13ATOM 805 CA PHE 103 34532 45220 65290 100 078 13ATOM 806 C PHE 103 33984 46063 64189 100 078 13ATOM 807 O PHE 103 34106 47286 64245 100 078 13ATOM 808 CB PHE 103 33360 44850 66219 100 078 13ATOM 809 CG PHE 103 32989 43457 65828 100 078 13ATOM 810 CD1 PHE 103 32354 43168 64642 100 078 13ATOM 811 CD2 PHE 103 33297 42419 66679 100 078 13ATOM 812 CE1 PHE 103 32040 41865 64322 100 078 13ATOM 813 CE2 PHE 103 32983 41119 66364 100 078 13ATOM 814 CZ PHE 103 32351 40837 65179 100 078 13ATOM 815 N GLU 104 33401 45448 63145 100 3518 13ATOM 816 CA GLU 104 32916 46224 62038 100 3518 13ATOM 817 C GLU 104 31713 46999 62475 100 3518 13ATOM 818 O GLU 104 31001 46604 63396 100 3518 13ATOM 819 CB GLU 104 32513 45369 60821 100 3518 13ATOM 820 CG GLU 104 33699 44648 60178 100 3518 13ATOM 821 CD GLU 104 33194 43840 58994 100 3518 13ATOM 822 OE1 GLU 104 31996 43999 58638 100 3518 13ATOM 823 OE2 GLU 104 34001 43055 58429 100 3518 13ATOM 824 N MET 105 31474 48153 61819 100 036 13ATOM 825 CA MET 105 30343 48980 62129 100 036 13ATOM 826 C MET 105 29128 48183 61800 100 036 13ATOM 827 O MET 105 28102 48280 62471 100 036 13ATOM 828 CB MET 105 30283 50279 61308 100 036 13ATOM 829 CG MET 105 29072 51152 61653 100 036 13ATOM 830 SD MET 105 29135 51909 63307 100 036 13ATOM 831 CE MET 105 27461 52612 63241 100 036 13ATOM 832 N ILE 106 29221 47364 60738 100 8112 13ATOM 833 CA ILE 106 28097 46583 60324 100 8112 13ATOM 834 C ILE 106 27725 45638 61423 100 8112 13ATOM 835 O ILE 106 26543 45455 61710 100 8112 13ATOM 836 CB ILE 106 28353 45783 59078 100 8112 13ATOM 837 CG1 ILE 106 27034 45173 58576 100 8112 13ATOM 838 CG2 ILE 106 29459 44752 59360 100 8112 13ATOM 839 CD1 ILE 106 27127 44608 57161 100 8112 13ATOM 840 N LYS 107 28714 44995 62072 100 065 13ATOM 841 CA LYS 107 28365 44062 63104 100 065 13ATOM 842 C LYS 107 27759 44800 64249 100 065 13ATOM 843 O LYS 107 26844 44313 64910 100 065 13ATOM 844 CB LYS 107 29540 43253 63649 100 065 13ATOM 845 CG LYS 107 29070 42141 64589 100 065 13ATOM 846 CD LYS 107 28231 41070 63885 100 065 13ATOM 847 CE LYS 107 27646 40014 64826 100 065 13ATOM 848 NZ LYS 107 26339 40467 65354 100 065 13ATOM 849 N LEU 108 28281 46004 64513 100 025 13ATOM 850 CA LEU 108 27831 46822 65596 100 025 13ATOM 851 C LEU 108 26392 47159 65336 100 025 13ATOM 852 O LEU 108 25569 47177 66250 100 025 13ATOM 853 CB LEU 108 28709 48090 65669 100 025 13ATOM 854 CG LEU 108 28476 49043 66851 100 025 13ATOM 855 CD1 LEU 108 29503 50186 66839 100 025 13ATOM 856 CD2 LEU 108 27049 49593 66863 100 025 13ATOM 857 N ILE 109 26025 47440 64073 100 021 13ATOM 858 CA ILE 109 24644 47737 63867 100 021 13ATOM 859 C ILE 109 23841 46510 64122 100 021 13ATOM 860 O ILE 109 22814 46579 64789 100 021 13ATOM 861 CB ILE 109 24290 48282 62525 100 021 13ATOM 862 CG1 ILE 109 22879 48865 62650 100 021 13ATOM 863 CG2 ILE 109 24421 47177 61465 100 021 13ATOM 864 CD1 ILE 109 22529 49870 61571 100 021 13ATOM 865 N ASP 110 24320 45343 63651 100 5010 13ATOM 866 CA ASP 110 23588 44113 63765 100 5010 13ATOM 867 C ASP 110 23273 43864 65210 100 5010 13ATOM 868 O ASP 110 22145 43506 65545 100 5010 13ATOM 869 CB ASP 110 24397 42910 63250 100 5010 13ATOM 870 CG ASP 110 23540 41656 63345 100 5010 13ATOM 871 OD1 ASP 110 22337 41771 63702 100 5010 13ATOM 872 OD2 ASP 110 24087 40557 63064 100 5010 13ATOM 873 N ILE 111 24251 44079 66109 100 016 13ATOM 874 CA ILE 111 24040 43845 67511 100 016 13ATOM 875 C ILE 111 22951 44772 67971 100 016 13ATOM 876 O ILE 111 22097 44391 68772 100 016 13ATOM 877 CB ILE 111 25288 44066 68336 100 016 13ATOM 878 CG1 ILE 111 25167 43408 69713 100 016 13ATOM 879 CG2 ILE 111 25558 45567 68453 100 016 13ATOM 880 CD1 ILE 111 24097 44026 70612 100 016 13ATOM 881 N ALA 112 22949 46020 67463 100 3981 13ATOM 882 CA ALA 112 21966 46996 67844 100 3981 13ATOM 883 C ALA 112 20600 46498 67462 100 3981 13ATOM 884 O ALA 112 19649 46659 68223 100 3981 13ATOM 885 CB ALA 112 22181 48356 67155 100 3981 13ATOM 886 N ARG 113 20468 45879 66271 100 8964 13ATOM 887 CA ARG 113 19214 45341 65810 100 8964 13ATOM 888 C ARG 113 18776 44286 66771 100 8964 13ATOM 889 O ARG 113 17605 44206 67130 100 8964 13ATOM 890 CB ARG 113 19348 44600 64468 100 8964 13ATOM 891 CG ARG 113 18138 43733 64116 100 8964 13ATOM 892 CD ARG 113 18406 42715 63003 100 8964 13ATOM 893 NE ARG 113 17185 41868 62863 100 8964 13ATOM 894 CZ ARG 113 17078 40713 63583 100 8964 13ATOM 895 NH1 ARG 113 18100 40329 64401 100 8964 13ATOM 896 NH2 ARG 113 15953 39945 63488 100 8964 13ATOM 897 N GLN 114 19727 43453 67226 100 9919 13ATOM 898 CA GLN 114 19415 42331 68063 100 9919 13ATOM 899 C GLN 114 18766 42825 69315 100 9919 13ATOM 900 O GLN 114 17770 42270 69772 100 9919 13ATOM 901 CB GLN 114 20683 41601 68541 100 9919 13ATOM 902 CG GLN 114 21601 41103 67423 100 9919 13ATOM 903 CD GLN 114 21044 39798 66886 100 9919 13ATOM 904 OE1 GLN 114 20326 39080 67581 100 9919 13ATOM 905 NE2 GLN 114 21387 39480 65610 100 9919 13ATOM 906 N THR 115 19324 43887 69914 100 018 13ATOM 907 CA THR 115 18784 44380 71144 100 018 13ATOM 908 C THR 115 17427 44947 70898 100 018 13ATOM 909 O THR 115 16531 44799 71725 100 018 13ATOM 910 CB THR 115 19620 45441 71798 100 018 13ATOM 911 OG1 THR 115 19144 45692 73112 100 018 13ATOM 912 CG2 THR 115 19550 46721 70958 100 018 13ATOM 913 N ALA 116 17244 45621 69748 100 4008 13ATOM 914 CA ALA 116 15986 46221 69418 100 4008 13ATOM 915 C ALA 116 14971 45127 69315 100 4008 13ATOM 916 O ALA 116 13833 45294 69751 100 4008 13ATOM 917 CB ALA 116 16013 46962 68070 100 4008 13ATOM 918 N GLN 117 15355 43971 68731 100 9047 13ATOM 919 CA GLN 117 14390 42923 68565 100 9047 13ATOM 920 C GLN 117 13960 42418 69911 100 9047 13ATOM 921 O GLN 117 12778 42147 70115 100 9047 13ATOM 922 CB GLN 117 14858 41726 67709 100 9047 13ATOM 923 CG GLN 117 15951 40841 68305 100 9047 13ATOM 924 CD GLN 117 16214 39725 67303 100 9047 13ATOM 925 OE1 GLN 117 15666 39727 66202 100 9047 13ATOM 926 NE2 GLN 117 17074 38744 67686 100 9047 13ATOM 927 N GLY 118 14900 42278 70871 100 3542 13ATOM 928 CA GLY 118 14531 41809 72174 100 3542 13ATOM 929 C GLY 118 13593 42791 72803 100 3542 13ATOM 930 O GLY 118 12607 42420 73437 100 3542 13ATOM 931 N MET 119 13880 44089 72640 100 6343 13ATOM 932 CA MET 119 13067 45120 73225 100 6343 13ATOM 933 C MET 119 11696 45087 72626 100 6343 13ATOM 934 O MET 119 10715 45382 73308 100 6343 13ATOM 935 CB MET 119 13629 46535 73007 100 6343 13ATOM 936 CG MET 119 14936 46785 73759 100 6343 13ATOM 937 SD MET 119 14797 46622 75565 100 6343 13ATOM 938 CE MET 119 13557 47934 75763 100 6343 13ATOM 939 N ASP 120 11594 44749 71326 100 7711 13ATOM 940 CA ASP 120 10324 44740 70647 100 7711 13ATOM 941 C ASP 120 9437 43734 71313 100 7711 13ATOM 942 O ASP 120 8240 43964 71487 100 7711 13ATOM 943 CB ASP 120 10460 44335 69167 100 7711 13ATOM 944 CG ASP 120 9135 44571 68459 100 7711 13ATOM 945 OD1 ASP 120 8149 44948 69147 100 7711 13ATOM 946 OD2 ASP 120 9097 44383 67214 100 7711 13ATOM 947 N TYR 121 10009 42581 71704 100 6225 13ATOM 948 CA TYR 121 9245 41544 72340 100 6225 13ATOM 949 C TYR 121 8714 42063 73647 100 6225 13ATOM 950 O TYR 121 7544 41872 73978 100 6225 13ATOM 951 CB TYR 121 10121 40308 72625 100 6225 13ATOM 952 CG TYR 121 9349 39297 73400 100 6225 13ATOM 953 CD1 TYR 121 8461 38453 72776 100 6225 13ATOM 954 CD2 TYR 121 9535 39181 74759 100 6225 13ATOM 955 CE1 TYR 121 7759 37518 73500 100 6225 13ATOM 956 CE2 TYR 121 8837 38248 75488 100 6225 13ATOM 957 CZ TYR 121 7945 37415 74858 100 6225 13ATOM 958 OH TYR 121 7223 36456 75600 100 6225 13ATOM 959 N LEU 122 9571 42771 74406 100 086 13ATOM 960 CA LEU 122 9233 43310 75694 100 086 13ATOM 961 C LEU 122 8065 44226 75487 100 086 13ATOM 962 O LEU 122 7078 44196 76224 100 086 13ATOM 963 CB LEU 122 10406 44176 76205 100 086 13ATOM 964 CG LEU 122 10374 44608 77680 100 086 13ATOM 965 CD1 LEU 122 10809 43457 78602 100 086 13ATOM 966 CD2 LEU 122 11190 45893 77898 100 086 13ATOM 967 N HIS 123 8155 45059 74438 100 8254 13ATOM 968 CA HIS 123 7136 46020 74123 100 8254 13ATOM 969 C HIS 123 5855 45313 73788 100 8254 13ATOM 970 O HIS 123 4796 45652 74318 100 8254 13ATOM 971 CB HIS 123 7527 46865 72898 100 8254 13ATOM 972 CG HIS 123 6511 47897 72511 100 8254 13ATOM 973 ND1 HIS 123 6577 49222 72878 100 8254 13ATOM 974 CD2 HIS 123 5385 47775 71757 100 8254 13ATOM 975 CE1 HIS 123 5496 49833 72331 100 8254 13ATOM 976 NE2 HIS 123 4743 48995 71642 100 8254 13ATOM 977 N ALA 124 5941 44263 72944 100 5497 13ATOM 978 CA ALA 124 4788 43577 72426 100 5497 13ATOM 979 C ALA 124 4000 43037 73560 100 5497 13ATOM 980 O ALA 124 2771 43093 73558 100 5497 13ATOM 981 CB ALA 124 5155 42391 71515 100 5497 13ATOM 982 N LYS 125 4686 42500 74578 100 061 13ATOM 983 CA LYS 125 3969 42050 75726 100 061 13ATOM 984 C LYS 125 3544 43306 76414 100 061 13ATOM 985 O LYS 125 3029 44233 75804 100 061 13ATOM 986 CB LYS 125 4828 41193 76671 100 061 13ATOM 987 CG LYS 125 5058 39771 76145 100 061 13ATOM 988 CD LYS 125 5815 39704 74817 100 061 13ATOM 989 CE LYS 125 4903 39706 73588 100 061 13ATOM 990 NZ LYS 125 5711 39580 72354 100 061 13ATOM 991 N SER 126 3642 43389 77733 100 082 13ATOM 992 CA SER 126 3315 44682 78247 100 082 13ATOM 993 C SER 126 4292 44892 79328 100 082 13ATOM 994 O SER 126 3939 44988 80503 100 082 13ATOM 995 CB SER 126 1918 44749 78883 100 082 13ATOM 996 OG SER 126 1668 46060 79370 100 082 13ATOM 997 N ILE 127 5567 45003 78944 100 091 13ATOM 998 CA ILE 127 6543 45100 79971 100 091 13ATOM 999 C ILE 127 7462 46223 79660 100 091 13ATOM 1000 O ILE 127 7570 46666 78517 100 091 13ATOM 1001 CB ILE 127 7339 43838 80124 100 091 13ATOM 1002 CG1 ILE 127 8390 43974 81240 100 091 13ATOM 1003 CG2 ILE 127 7875 43452 78740 100 091 13ATOM 1004 CD1 ILE 127 9050 42651 81630 100 091 13ATOM 1005 N ILE 128 8087 46752 80724 100 001 13ATOM 1006 CA ILE 128 9045 47814 80631 100 001 13ATOM 1007 C ILE 128 10269 47308 81331 100 001 13ATOM 1008 O ILE 128 10160 46594 82327 100 001 13ATOM 1009 CB ILE 128 8658 49044 81397 100 001 13ATOM 1010 CG1 ILE 128 7355 49653 80880 100 001 13ATOM 1011 CG2 ILE 128 9830 50022 81274 100 001 13ATOM 1012 CD1 ILE 128 7505 50203 79470 100 001 13ATOM 1013 N HIS 129 11459 47565 80755 100 082 13ATOM 1014 CA HIS 129 12713 47173 81339 100 082 13ATOM 1015 C HIS 129 13160 48101 82445 100 082 13ATOM 1016 O HIS 129 13472 47671 83555 100 082 13ATOM 1017 CB HIS 129 13818 47161 80277 100 082 13ATOM 1018 CG HIS 129 15029 46395 80702 100 082 13ATOM 1019 ND1 HIS 129 15920 46810 81666 100 082 13ATOM 1020 CD2 HIS 129 15494 45197 80253 100 082 13ATOM 1021 CE1 HIS 129 16873 45849 81753 100 082 13ATOM 1022 NE2 HIS 129 16657 44851 80914 100 082 13ATOM 1023 N ARG 130 13188 49419 82145 100 038 13ATOM 1024 CA ARG 130 13597 50498 83010 100 038 13ATOM 1025 C ARG 130 15073 50507 83303 100 038 13ATOM 1026 O ARG 130 15542 51413 83992 100 038 13ATOM 1027 CB ARG 130 12882 50526 84375 100 038 13ATOM 1028 CG ARG 130 11377 50813 84322 100 038 13ATOM 1029 CD ARG 130 10508 49578 84108 100 038 13ATOM 1030 NE ARG 130 9080 50003 84116 100 038 13ATOM 1031 CZ ARG 130 8436 50220 85301 100 038 13ATOM 1032 NH1 ARG 130 9127 50164 86477 100 038 13ATOM 1033 NH2 ARG 130 7100 50498 85309 100 038 13ATOM 1034 N ASP 131 15870 49550 82787 100 022 13ATOM 1035 CA ASP 131 17276 49631 83080 100 022 13ATOM 1036 C ASP 131 18013 49218 81848 100 022 13ATOM 1037 O ASP 131 18897 48364 81894 100 022 13ATOM 1038 CB ASP 131 17713 48688 84213 100 022 13ATOM 1039 CG ASP 131 19127 49072 84617 100 022 13ATOM 1040 OD1 ASP 131 19766 49865 83875 100 022 13ATOM 1041 OD2 ASP 131 19585 48584 85683 100 022 13ATOM 1042 N LEU 132 17699 49853 80706 100 7840 13ATOM 1043 CA LEU 132 18312 49433 79480 100 7840 13ATOM 1044 C LEU 132 19578 50210 79305 100 7840 13ATOM 1045 O LEU 132 19557 51433 79174 100 7840 13ATOM 1046 CB LEU 132 17408 49683 78256 100 7840 13ATOM 1047 CG LEU 132 17989 49206 76912 100 7840 13ATOM 1048 CD1 LEU 132 18172 47678 76885 100 7840 13ATOM 1049 CD2 LEU 132 17148 49719 75733 100 7840 13ATOM 1050 N LYS 133 20720 49487 79316 100 091 13ATOM 1051 CA LYS 133 22037 50046 79173 100 091 13ATOM 1052 C LYS 133 22891 48948 78608 100 091 13ATOM 1053 O LYS 133 22489 47786 78576 100 091 13ATOM 1054 CB LYS 133 22732 50346 80511 100 091 13ATOM 1055 CG LYS 133 21981 51281 81448 100 091 13ATOM 1056 CD LYS 133 22524 51269 82880 100 091 13ATOM 1057 CE LYS 133 23970 51761 82996 100 091 13ATOM 1058 NZ LYS 133 24396 51755 84414 100 091 13ATOM 1059 N SER 134 24120 49288 78175 100 8586 13ATOM 1060 CA SER 134 25019 48316 77618 100 8586 13ATOM 1061 C SER 134 25420 47344 78678 100 8586 13ATOM 1062 O SER 134 25754 46199 78378 100 8586 13ATOM 1063 CB SER 134 26316 48940 77077 100 8586 13ATOM 1064 OG SER 134 27154 47925 76544 100 8586 13ATOM 1065 N ASN 135 25412 47775 79950 100 083 13ATOM 1066 CA ASN 135 25829 46906 81011 100 083 13ATOM 1067 C ASN 135 24858 45771 81120 100 083 13ATOM 1068 O ASN 135 25234 44672 81526 100 083 13ATOM 1069 CB ASN 135 25913 47589 82393 100 083 13ATOM 1070 CG ASN 135 24518 47904 82910 100 083 13ATOM 1071 OD1 ASN 135 23604 48254 82166 100 083 13ATOM 1072 ND2 ASN 135 24351 47763 84253 100 083 13ATOM 1073 N ASN 136 23572 46004 80783 100 075 13ATOM 1074 CA ASN 136 22592 44969 80990 100 075 13ATOM 1075 C ASN 136 22271 44265 79706 100 075 13ATOM 1076 O ASN 136 21148 43813 79480 100 075 13ATOM 1077 CB ASN 136 21285 45548 81547 100 075 13ATOM 1078 CG ASN 136 21627 46176 82889 100 075 13ATOM 1079 OD1 ASN 136 22477 45671 83621 100 075 13ATOM 1080 ND2 ASN 136 20965 47320 83211 100 075 13ATOM 1081 N ILE 137 23279 44111 78840 100 9342 13ATOM 1082 CA ILE 137 23100 43383 77625 100 9342 13ATOM 1083 C ILE 137 24153 42322 77654 100 9342 13ATOM 1084 O ILE 137 25300 42600 77994 100 9342 13ATOM 1085 CB ILE 137 23272 44275 76441 100 9342 13ATOM 1086 CG1 ILE 137 22043 45195 76348 100 9342 13ATOM 1087 CG2 ILE 137 23570 43432 75204 100 9342 13ATOM 1088 CD1 ILE 137 22158 46293 75301 100 9342 13ATOM 1089 N PHE 138 23792 41068 77319 100 061 13ATOM 1090 CA PHE 138 24732 39987 77431 100 061 13ATOM 1091 C PHE 138 25126 39580 76040 100 061 13ATOM 1092 O PHE 138 24327 39675 75110 100 061 13ATOM 1093 CB PHE 138 24117 38750 78107 100 061 13ATOM 1094 CG PHE 138 23530 39207 79398 100 061 13ATOM 1095 CD1 PHE 138 24310 39631 80448 100 061 13ATOM 1096 CD2 PHE 138 22166 39191 79558 100 061 13ATOM 1097 CE1 PHE 138 23730 40042 81623 100 061 13ATOM 1098 CE2 PHE 138 21584 39603 80735 100 061 13ATOM 1099 CZ PHE 138 22366 40034 81777 100 061 13ATOM 1100 N LEU 139 26387 39123 75868 100 088 13ATOM 1101 CA LEU 139 26909 38751 74580 100 088 13ATOM 1102 C LEU 139 27274 37293 74600 100 088 13ATOM 1103 O LEU 139 27868 36802 75556 100 088 13ATOM 1104 CB LEU 139 28145 39610 74237 100 088 13ATOM 1105 CG LEU 139 28984 39163 73029 100 088 13ATOM 1106 CD1 LEU 139 29906 37997 73413 100 088 13ATOM 1107 CD2 LEU 139 28097 38867 71809 100 088 13ATOM 1108 N HIS 140 26917 36557 73525 100 9061 13ATOM 1109 CA HIS 140 27149 35139 73445 100 9061 13ATOM 1110 C HIS 140 28407 34901 72664 100 9061 13ATOM 1111 O HIS 140 28962 35816 72060 100 9061 13ATOM 1112 CB HIS 140 26005 34394 72734 100 9061 13ATOM 1113 CG HIS 140 26006 32910 72952 100 9061 13ATOM 1114 ND1 HIS 140 25286 32284 73943 100 9061 13ATOM 1115 CD2 HIS 140 26647 31917 72279 100 9061 13ATOM 1116 CE1 HIS 140 25521 30954 73821 100 9061 13ATOM 1117 NE2 HIS 140 26342 30682 72824 100 9061 13ATOM 1118 N GLU 141 28899 33646 72665 100 4621 13ATOM 1119 CA GLU 141 30082 33292 71929 100 4621 13ATOM 1120 C GLU 141 29777 33538 70489 100 4621 13ATOM 1121 O GLU 141 30647 33906 69700 100 4621 13ATOM 1122 CB GLU 141 30451 31804 72032 100 4621 13ATOM 1123 CG GLU 141 30898 31380 73424 100 4621 13ATOM 1124 CD GLU 141 31238 29902 73390 100 4621 13ATOM 1125 OE1 GLU 141 31449 29375 72267 100 4621 13ATOM 1126 OE2 GLU 141 31303 29281 74483 100 4621 13ATOM 1127 N ASP 142 28498 33331 70139 100 034 13ATOM 1128 CA ASP 142 27937 33457 68827 100 034 13ATOM 1129 C ASP 142 28021 34890 68403 100 034 13ATOM 1130 O ASP 142 27880 35191 67218 100 034 13ATOM 1131 CB ASP 142 26458 33039 68775 100 034 13ATOM 1132 CG ASP 142 26058 32855 67319 100 034 13ATOM 1133 OD1 ASP 142 26963 32913 66444 100 034 13ATOM 1134 OD2 ASP 142 24843 32642 67066 100 034 13ATOM 1135 N LEU 143 28259 35810 69361 100 045 13ATOM 1136 CA LEU 143 28282 37223 69093 100 045 13ATOM 1137 C LEU 143 26876 37697 68953 100 045 13ATOM 1138 O LEU 143 26627 38754 68373 100 045 13ATOM 1139 CB LEU 143 28998 37624 67788 100 045 13ATOM 1140 CG LEU 143 30493 37274 67757 100 045 13ATOM 1141 CD1 LEU 143 31138 37715 66432 100 045 13ATOM 1142 CD2 LEU 143 31219 37825 68992 100 045 13ATOM 1143 N THR 144 25911 36913 69466 100 026 13ATOM 1144 CA THR 144 24565 37395 69472 100 026 13ATOM 1145 C THR 144 24385 38109 70777 100 026 13ATOM 1146 O THR 144 25183 37951 71700 100 026 13ATOM 1147 CB THR 144 23526 36317 69364 100 026 13ATOM 1148 OG1 THR 144 22232 36889 69256 100 026 13ATOM 1149 CG2 THR 144 23608 35419 70608 100 026 13ATOM 1150 N VAL 145 23330 38932 70878 100 6536 13ATOM 1151 CA VAL 145 23106 39694 72065 100 6536 13ATOM 1152 C VAL 145 21746 39417 72596 100 6536 13ATOM 1153 O VAL 145 20803 39140 71855 100 6536 13ATOM 1154 CB VAL 145 23251 41154 71815 100 6536 13ATOM 1155 CG1 VAL 145 22524 41944 72909 100 6536 13ATOM 1156 CG2 VAL 145 24762 41423 71832 100 6536 13ATOM 1157 N LYS 146 21644 39474 73934 100 9801 13ATOM 1158 CA LYS 146 20419 39228 74617 100 9801 13ATOM 1159 C LYS 146 20316 40243 75717 100 9801 13ATOM 1160 O LYS 146 21307 40857 76110 100 9801 13ATOM 1161 CB LYS 146 20409 37816 75202 100 9801 13ATOM 1162 CG LYS 146 20259 36721 74146 100 9801 13ATOM 1163 CD LYS 146 20388 35304 74713 100 9801 13ATOM 1164 CE LYS 146 20218 34200 73669 100 9801 13ATOM 1165 NZ LYS 146 21418 34119 72809 100 9801 13ATOM 1166 N ILE 147 19100 40447 76259 100 068 13ATOM 1167 CA ILE 147 18933 41473 77261 100 068 13ATOM 1168 C ILE 147 18793 40858 78649 100 068 13ATOM 1169 O ILE 147 18414 39707 78821 100 068 13ATOM 1170 CB ILE 147 17746 42344 77011 100 068 13ATOM 1171 CG1 ILE 147 17782 42922 75589 100 068 13ATOM 1172 CG2 ILE 147 17748 43432 78096 100 068 13ATOM 1173 CD1 ILE 147 16431 43482 75172 100 068 13ATOM 1174 N GLY 148 19152 41599 79715 100 005 13ATOM 1175 CA GLY 148 18952 41116 81047 100 005 13ATOM 1176 C GLY 148 18915 42323 81886 100 005 13ATOM 1177 O GLY 148 18812 43437 81392 100 005 13ATOM 1178 N ASP 149 18953 42150 83198 100 055 13ATOM 1179 CA ASP 149 18962 43300 84015 100 055 13ATOM 1180 C ASP 149 19855 42865 85098 100 055 13ATOM 1181 O ASP 149 19409 42113 85961 100 055 13ATOM 1182 CB ASP 149 17540 43567 84559 100 055 13ATOM 1183 CG ASP 149 17393 44993 85041 100 055 13ATOM 1184 OD1 ASP 149 18408 45554 85525 100 055 13ATOM 1185 OD2 ASP 149 16266 45543 84918 100 055 13ATOM 1186 N PHE 150 21146 43266 85021 100 098 13ATOM 1187 CA PHE 150 22080 42900 86048 100 098 13ATOM 1188 C PHE 150 21594 43461 87337 100 098 13ATOM 1189 O PHE 150 21514 42750 88337 100 098 13ATOM 1190 CB PHE 150 23536 43404 85841 100 098 13ATOM 1191 CG PHE 150 24433 42280 85424 100 098 13ATOM 1192 CD1 PHE 150 24613 41867 84126 100 098 13ATOM 1193 CD2 PHE 150 25118 41623 86419 100 098 13ATOM 1194 CE1 PHE 150 25462 40815 83868 100 098 13ATOM 1195 CE2 PHE 150 25962 40575 86159 100 098 13ATOM 1196 CZ PHE 150 26136 40164 84870 100 098 13ATOM 1197 N GLY 151 21240 44756 87364 100 2429 13ATOM 1198 CA GLY 151 20699 45274 88586 100 2429 13ATOM 1199 C GLY 151 21796 45666 89528 100 2429 13ATOM 1200 O GLY 151 21547 45907 90709 100 2429 13ATOM 1201 N LEU 152 23052 45744 89052 100 5581 13ATOM 1202 CA LEU 152 24064 46161 89978 100 5581 13ATOM 1203 C LEU 152 24186 47644 89879 100 5581 13ATOM 1204 O LEU 152 25017 48160 89134 100 5581 13ATOM 1205 CB LEU 152 25462 45580 89699 100 5581 13ATOM 1206 CG LEU 152 25592 44077 90007 100 5581 13ATOM 1207 CD1 LEU 152 27040 43596 89820 100 5581 13ATOM 1208 CD2 LEU 152 25041 43745 91401 100 5581 13ATOM 1209 N ALA 153 23350 48373 90641 100 2709 13ATOM 1210 CA ALA 153 23445 49797 90632 100 2709 13ATOM 1211 C ALA 153 24775 50124 91213 100 2709 13ATOM 1212 O ALA 153 25509 50959 90688 100 2709 13ATOM 1213 CB ALA 153 22376 50475 91499 100 2709 13ATOM 1214 N THR 154 25128 49433 92314 100 078 13ATOM 1215 CA THR 154 26395 49673 92927 100 078 13ATOM 1216 C THR 154 27385 48829 92197 100 078 13ATOM 1217 O THR 154 27249 47611 92100 100 078 13ATOM 1218 CB THR 154 26423 49339 94395 100 078 13ATOM 1219 OG1 THR 154 27680 49692 94951 100 078 13ATOM 1220 CG2 THR 154 26142 47840 94597 100 078 13ATOM 1221 N VAL 155 28401 49491 91623 100 9865 13ATOM 1222 CA VAL 155 29390 48809 90850 100 9865 13ATOM 1223 C VAL 155 30330 49884 90404 100 9865 13ATOM 1224 O VAL 155 30460 50914 91063 100 9865 13ATOM 1225 CB VAL 155 28807 48125 89643 100 9865 13ATOM 1226 CG1 VAL 155 28328 49195 88646 100 9865 13ATOM 1227 CG2 VAL 155 29832 47123 89082 100 9865 13ATOM 1228 N LYS 156 31040 49663 89286 100 006 13ATOM 1229 CA LYS 156 31919 50674 88784 100 006 13ATOM 1230 C LYS 156 31056 51851 88449 100 006 13ATOM 1231 O LYS 156 31460 52998 88632 100 006 13ATOM 1232 CB LYS 156 32645 50241 87492 100 006 13ATOM 1233 CG LYS 156 33678 51243 86952 100 006 13ATOM 1234 CD LYS 156 33098 52580 86471 100 006 13ATOM 1235 CE LYS 156 34138 53566 85939 100 006 13ATOM 1236 NZ LYS 156 34219 53457 84467 100 006 13ATOM 1237 N SER 157 29841 51567 87941 100 9685 13ATOM 1238 CA SER 157 28887 52524 87447 100 9685 13ATOM 1239 C SER 157 28172 53242 88559 100 9685 13ATOM 1240 O SER 157 27150 53881 88320 100 9685 13ATOM 1241 CB SER 157 27823 51884 86534 100 9685 13ATOM 1242 OG SER 157 26997 52881 85950 100 9685 13ATOM 1243 N ARG 158 28671 53177 89805 100 034 13ATOM 1244 CA ARG 158 27976 53794 90904 100 034 13ATOM 1245 C ARG 158 27829 55263 90636 100 034 13ATOM 1246 O ARG 158 26827 55867 91015 100 034 13ATOM 1247 CB ARG 158 28705 53650 92249 100 034 13ATOM 1248 CG ARG 158 27927 54257 93420 100 034 13ATOM 1249 CD ARG 158 26594 53553 93689 100 034 13ATOM 1250 NE ARG 158 26046 54079 94972 100 034 13ATOM 1251 CZ ARG 158 25225 55170 94990 100 034 13ATOM 1252 NH1 ARG 158 24883 55799 93828 100 034 13ATOM 1253 NH2 ARG 158 24740 55627 96181 100 034 13ATOM 1254 N TRP 159 28820 55873 89962 100 086 13ATOM 1255 CA TRP 159 28821 57284 89686 100 086 13ATOM 1256 C TRP 159 27602 57589 88888 100 086 13ATOM 1257 O TRP 159 27069 58696 88944 100 086 13ATOM 1258 CB TRP 159 30025 57740 88837 100 086 13ATOM 1259 CG TRP 159 30055 59224 88535 100 086 13ATOM 1260 CD1 TRP 159 29344 60240 89104 100 086 13ATOM 1261 CD2 TRP 159 30886 59827 87527 100 086 13ATOM 1262 NE1 TRP 159 29689 61439 88523 100 086 13ATOM 1263 CE2 TRP 159 30635 61199 87548 100 086 13ATOM 1264 CE3 TRP 159 31784 59282 86656 100 086 13ATOM 1265 CZ2 TRP 159 31282 62048 86694 100 086 13ATOM 1266 CZ3 TRP 159 32433 60141 85797 100 086 13ATOM 1267 CH2 TRP 159 32189 61498 85815 100 086 13ATOM 1268 N SER 160 27140 56603 88105 100 8287 13ATOM 1269 CA SER 160 26024 56780 87233 100 8287 13ATOM 1270 C SER 160 24815 57219 88000 100 8287 13ATOM 1271 O SER 160 24054 58045 87504 100 8287 13ATOM 1272 CB SER 160 25644 55481 86505 100 8287 13ATOM 1273 OG SER 160 24511 55700 85680 100 8287 13ATOM 1274 N GLY 161 24584 56690 89218 100 3462 13ATOM 1275 CA GLY 161 23385 57049 89926 100 3462 13ATOM 1276 C GLY 161 23612 58265 90765 100 3462 13ATOM 1277 O GLY 161 24742 58577 91139 100 3462 13ATOM 1278 N SER 162 22509 58973 91097 100 8164 13ATOM 1279 CA SER 162 22586 60142 91923 100 8164 13ATOM 1280 C SER 162 21330 60187 92735 100 8164 13ATOM 1281 O SER 162 20280 59719 92298 100 8164 13ATOM 1282 CB SER 162 22668 61447 91116 100 8164 13ATOM 1283 OG SER 162 23868 61469 90358 100 8164 13ATOM 1284 N HIS 163 21405 60762 93951 100 7417 13ATOM 1285 CA HIS 163 20245 60814 94787 100 7417 13ATOM 1286 C HIS 163 19575 62118 94528 100 7417 13ATOM 1287 O HIS 163 20013 63162 95009 100 7417 13ATOM 1288 CB HIS 163 20583 60780 96284 100 7417 13ATOM 1289 CG HIS 163 21306 59534 96694 100 7417 13ATOM 1290 ND1 HIS 163 22640 59300 96444 100 7417 13ATOM 1291 CD2 HIS 163 20856 58437 97363 100 7417 13ATOM 1292 CE1 HIS 163 22930 58084 96969 100 7417 13ATOM 1293 NE2 HIS 163 21879 57521 97538 100 7417 13ATOM 1294 N GLN 164 18479 62091 93750 100 030 13ATOM 1295 CA GLN 164 17807 63324 93487 100 030 13ATOM 1296 C GLN 164 17066 63675 94736 100 030 13ATOM 1297 O GLN 164 16313 62865 95274 100 030 13ATOM 1298 CB GLN 164 16813 63242 92314 100 030 13ATOM 1299 CG GLN 164 16085 64556 92019 100 030 13ATOM 1300 CD GLN 164 14916 64678 92985 100 030 13ATOM 1301 OE1 GLN 164 14489 65779 93331 100 030 13ATOM 1302 NE2 GLN 164 14370 63513 93424 100 030 13ATOM 1303 N PHE 165 17281 64908 95236 100 092 13ATOM 1304 CA PHE 165 16653 65356 96444 100 092 13ATOM 1305 C PHE 165 17166 64481 97547 100 092 13ATOM 1306 O PHE 165 16600 64423 98637 100 092 13ATOM 1307 CB PHE 165 15115 65257 96372 100 092 13ATOM 1308 CG PHE 165 14520 65969 97540 100 092 13ATOM 1309 CD1 PHE 165 14465 67343 97558 100 092 13ATOM 1310 CD2 PHE 165 13994 65273 98605 100 092 13ATOM 1311 CE1 PHE 165 13915 68016 98623 100 092 13ATOM 1312 CE2 PHE 165 13442 65939 99674 100 092 13ATOM 1313 CZ PHE 165 13403 67312 99685 100 092 13ATOM 1314 N GLU 166 18296 63801 97276 100 062 13ATOM 1315 CA GLU 166 18958 62920 98193 100 062 13ATOM 1316 C GLU 166 18008 61814 98531 100 062 13ATOM 1317 O GLU 166 18206 61095 99510 100 062 13ATOM 1318 CB GLU 166 19418 63636 99481 100 062 13ATOM 1319 CG GLU 166 20473 62876 100295 100 062 13ATOM 1320 CD GLU 166 19805 62162 101462 100 062 13ATOM 1321 OE1 GLU 166 18582 62378 101672 100 062 13ATOM 1322 OE2 GLU 166 20514 61392 102163 100 062 13ATOM 1323 N GLN 167 16935 61651 97728 100 074 13ATOM 1324 CA GLN 167 15993 60617 98048 100 074 13ATOM 1325 C GLN 167 16491 59237 97744 100 074 13ATOM 1326 O GLN 167 16640 58410 98637 100 074 13ATOM 1327 CB GLN 167 14655 60760 97306 100 074 13ATOM 1328 CG GLN 167 13848 61991 97715 100 074 13ATOM 1329 CD GLN 167 12521 61939 96970 100 074 13ATOM 1330 OE1 GLN 167 13448 62742 97061 100 074 13ATOM 1331 NE2 GLN 167 12196 60715 97467 100 074 13ATOM 1332 N LEU 168 16835 58944 96479 100 073 13ATOM 1333 CA LEU 168 17153 57572 96203 100 073 13ATOM 1334 C LEU 168 18046 57588 95011 100 073 13ATOM 1335 O LEU 168 17925 58462 94154 100 073 13ATOM 1336 CB LEU 168 15869 56755 95915 100 073 13ATOM 1337 CG LEU 168 15988 55215 95843 100 073 13ATOM 1338 CD1 LEU 168 14599 54577 95684 100 073 13ATOM 1339 CD2 LEU 168 16928 54741 94729 100 073 13ATOM 1340 N SER 169 18994 56637 94932 100 9525 13ATOM 1341 CA SER 169 19887 56653 93813 100 9525 13ATOM 1342 C SER 169 19103 56391 92571 100 9525 13ATOM 1343 O SER 169 18350 55422 92482 100 9525 13ATOM 1344 CB SER 169 20997 55591 93890 100 9525 13ATOM 1345 OG SER 169 21844 55843 95002 100 9525 13ATOM 1346 N GLY 170 19280 57257 91557 100 2902 13ATOM 1347 CA GLY 170 18585 57066 90321 100 2902 13ATOM 1348 C GLY 170 19559 57370 89233 100 2902 13ATOM 1349 O GLY 170 20435 58219 89391 100 2902 13ATOM 1350 N SER 171 19431 56679 88085 100 9539 13ATOM 1351 CA SER 171 20339 56965 87017 100 9539 13ATOM 1352 C SER 171 19735 58105 86277 100 9539 13ATOM 1353 O SER 171 19006 57919 85305 100 9539 13ATOM 1354 CB SER 171 20498 55798 86027 100 9539 13ATOM 1355 OG SER 171 21408 56156 84999 100 9539 13ATOM 1356 N ILE 172 20057 59330 86722 100 084 13ATOM 1357 CA ILE 172 19464 60491 86137 100 084 13ATOM 1358 C ILE 172 19884 60645 84709 100 084 13ATOM 1359 O ILE 172 19054 60922 83847 100 084 13ATOM 1360 CB ILE 172 19793 61765 86865 100 084 13ATOM 1361 CG1 ILE 172 21306 62040 86866 100 084 13ATOM 1362 CG2 ILE 172 19183 61668 88272 100 084 13ATOM 1363 CD1 ILE 172 22121 61013 87652 100 084 13ATOM 1364 N LEU 173 21179 60450 84408 100 5163 13ATOM 1365 CA LEU 173 21644 60716 83075 100 5163 13ATOM 1366 C LEU 173 20977 59808 82085 100 5163 13ATOM 1367 O LEU 173 20529 60251 81030 100 5163 13ATOM 1368 CB LEU 173 23162 60508 82927 100 5163 13ATOM 1369 CG LEU 173 23997 61434 83829 100 5163 13ATOM 1370 CD1 LEU 173 25504 61213 83620 100 5163 13ATOM 1371 CD2 LEU 173 23576 62903 83665 100 5163 13ATOM 1372 N TRP 174 20908 58504 82397 100 8085 13ATOM 1373 CA TRP 174 20385 57523 81489 100 8085 13ATOM 1374 C TRP 174 18904 57626 81278 100 8085 13ATOM 1375 O TRP 174 18436 57497 80148 100 8085 13ATOM 1376 CB TRP 174 20750 56097 81924 100 8085 13ATOM 1377 CG TRP 174 22206 55787 81667 100 8085 13ATOM 1378 CD1 TRP 174 22761 54959 80735 100 8085 13ATOM 1379 CD2 TRP 174 23299 56412 82357 100 8085 13ATOM 1380 NE1 TRP 174 24131 55014 80815 100 8085 13ATOM 1381 CE2 TRP 174 24477 55909 81805 100 8085 13ATOM 1382 CE3 TRP 174 23321 57339 83359 100 8085 13ATOM 1383 CZ2 TRP 174 25698 56326 82251 100 8085 13ATOM 1384 CZ3 TRP 174 24554 57750 83815 100 8085 13ATOM 1385 CH2 TRP 174 25719 57250 83272 100 8085 13ATOM 1386 N MET 175 18123 57876 82344 100 7651 13ATOM 1387 CA MET 175 16690 57841 82231 100 7651 13ATOM 1388 C MET 175 16205 58919 81316 100 7651 13ATOM 1389 O MET 175 16849 59951 81141 100 7651 13ATOM 1390 CB MET 175 15971 58046 83570 100 7651 13ATOM 1391 CG MET 175 16261 56956 84599 100 7651 13ATOM 1392 SD MET 175 15458 57247 86201 100 7651 13ATOM 1393 CE MET 175 16395 58764 86542 100 7651 13ATOM 1394 N ALA 176 15042 58672 80675 100 5898 13ATOM 1395 CA ALA 176 14460 59661 79816 100 5898 13ATOM 1396 C ALA 176 13946 60748 80700 100 5898 13ATOM 1397 O ALA 176 13383 60472 81759 100 5898 13ATOM 1398 CB ALA 176 13278 59127 78991 100 5898 13ATOM 1399 N PRO 177 14157 61983 80331 100 041 13ATOM 1400 CA PRO 177 13643 63006 81192 100 041 13ATOM 1401 C PRO 177 12162 63082 81142 100 041 13ATOM 1402 O PRO 177 11621 63904 80414 100 041 13ATOM 1403 CB PRO 177 14388 64286 80821 100 041 13ATOM 1404 CG PRO 177 15743 63762 80307 100 041 13ATOM 1405 CD PRO 177 15423 62371 79731 100 041 13ATOM 1406 N GLU 178 11502 62282 81978 100 084 13ATOM 1407 CA GLU 178 10086 62191 82099 100 084 13ATOM 1408 C GLU 178 9974 61353 83315 100 084 13ATOM 1409 O GLU 178 9286 61693 84274 100 084 13ATOM 1410 CB GLU 178 9429 61499 80887 100 084 13ATOM 1411 CG GLU 178 9976 60110 80544 100 084 13ATOM 1412 CD GLU 178 9500 59758 79146 100 084 13ATOM 1413 OE1 GLU 178 9585 60649 78258 100 084 13ATOM 1414 OE2 GLU 178 9048 58601 78940 100 084 13ATOM 1415 N VAL 179 10672 60205 83268 100 001 13ATOM 1416 CA VAL 179 10819 59344 84393 100 001 13ATOM 1417 C VAL 179 11707 60040 85382 100 001 13ATOM 1418 O VAL 179 11475 59973 86587 100 001 13ATOM 1419 CB VAL 179 11443 58028 84044 100 001 13ATOM 1420 CG1 VAL 179 12873 58271 83534 100 001 13ATOM 1421 CG2 VAL 179 11372 57132 85287 100 001 13ATOM 1422 N ILE 180 12746 60751 84886 100 5984 13ATOM 1423 CA ILE 180 13685 61396 85763 100 5984 13ATOM 1424 C ILE 180 12926 62374 86579 100 5984 13ATOM 1425 O ILE 180 13103 62450 87794 100 5984 13ATOM 1426 CB ILE 180 14754 62162 85040 100 5984 13ATOM 1427 CG1 ILE 180 15618 61222 84186 100 5984 13ATOM 1428 CG2 ILE 180 15553 62949 86092 100 5984 13ATOM 1429 CD1 ILE 180 16539 61958 83213 100 5984 13ATOM 1430 N ARG 181 12039 63150 85936 100 055 13ATOM 1431 CA ARG 181 11256 64023 86747 100 055 13ATOM 1432 C ARG 181 10256 63112 87367 100 055 13ATOM 1433 O ARG 181 9754 62210 86703 100 055 13ATOM 1434 CB ARG 181 10472 65097 85967 100 055 13ATOM 1435 CG ARG 181 11350 66120 85243 100 055 13ATOM 1436 CD ARG 181 10554 67251 84582 100 055 13ATOM 1437 NE ARG 181 10589 68433 85493 100 055 13ATOM 1438 CZ ARG 181 9634 68602 86454 100 055 13ATOM 1439 NH1 ARG 181 8635 67683 86598 100 055 13ATOM 1440 NH2 ARG 181 9679 69694 87271 100 055 13ATOM 1441 N MET 182 9973 63296 88666 100 009 13ATOM 1442 CA MET 182 9034 62455 89349 100 009 13ATOM 1443 C MET 182 9359 61012 89118 100 009 13ATOM 1444 O MET 182 8513 60232 88688 100 009 13ATOM 1445 CB MET 182 7556 62727 88996 100 009 13ATOM 1446 CG MET 182 7147 62463 87543 100 009 13ATOM 1447 SD MET 182 7431 63840 86392 100 009 13ATOM 1448 CE MET 182 6052 64828 87032 100 009 13ATOM 1449 N GLN 183 10611 60615 89400 100 8335 13ATOM 1450 CA GLN 183 11028 59258 89197 100 8335 13ATOM 1451 C GLN 183 10172 58407 90078 100 8335 13ATOM 1452 O GLN 183 9756 57317 89685 100 8335 13ATOM 1453 CB GLN 183 12500 59056 89597 100 8335 13ATOM 1454 CG GLN 183 13455 59902 88750 100 8335 13ATOM 1455 CD GLN 183 14884 59660 89215 100 8335 13ATOM 1456 OE1 GLN 183 15788 60416 88864 100 8335 13ATOM 1457 NE2 GLN 183 15096 58585 90022 100 8335 13ATOM 1458 N ASP 184 9869 58907 91293 100 2079 13ATOM 1459 CA ASP 184 9050 58191 92229 100 2079 13ATOM 1460 C ASP 184 7712 58022 91590 100 2079 13ATOM 1461 O ASP 184 7043 57006 91767 100 2079 13ATOM 1462 CB ASP 184 8828 58949 93551 100 2079 13ATOM 1463 CG ASP 184 10148 58985 94311 100 2079 13ATOM 1464 OD1 ASP 184 11172 58522 93740 100 2079 13ATOM 1465 OD2 ASP 184 10149 59469 95475 100 2079 13ATOM 1466 N LYS 185 7307 59036 90809 100 063 13ATOM 1467 CA LYS 185 6053 59054 90118 100 063 13ATOM 1468 C LYS 185 6064 57916 89152 100 063 13ATOM 1469 O LYS 185 5018 57375 88800 100 063 13ATOM 1470 CB LYS 185 5851 60351 89325 100 063 13ATOM 1471 CG LYS 185 4495 60509 88642 100 063 13ATOM 1472 CD LYS 185 4290 61931 88112 100 063 13ATOM 1473 CE LYS 185 2961 62133 87382 100 063 13ATOM 1474 NZ LYS 185 2880 63514 86861 100 063 13ATOM 1475 N ASN 186 7272 57510 88719 100 082 13ATOM 1476 CA ASN 186 7419 56466 87742 100 082 13ATOM 1477 C ASN 186 6663 56783 86480 100 082 13ATOM 1478 O ASN 186 5702 56087 86155 100 082 13ATOM 1479 CB ASN 186 6906 55101 88235 100 082 13ATOM 1480 CG ASN 186 7771 54654 89404 100 082 13ATOM 1481 OD1 ASN 186 8989 54531 89287 100 082 13ATOM 1482 ND2 ASN 186 7122 54414 90574 100 082 13ATOM 1483 N PRO 187 7037 57806 85744 100 054 13ATOM 1484 CA PRO 187 6383 58065 84495 100 054 13ATOM 1485 C PRO 187 6799 57038 83491 100 054 13ATOM 1486 O PRO 187 6404 57168 82335 100 054 13ATOM 1487 CB PRO 187 6772 59483 84080 100 054 13ATOM 1488 CG PRO 187 7078 60179 85412 100 054 13ATOM 1489 CD PRO 187 7575 59037 86309 100 054 13ATOM 1490 N TYR 188 7608 56036 83896 100 092 13ATOM 1491 CA TYR 188 8147 55050 82992 100 092 13ATOM 1492 C TYR 188 7113 54544 82041 100 092 13ATOM 1493 O TYR 188 5986 54225 82416 100 092 13ATOM 1494 CB TYR 188 8678 53755 83637 100 092 13ATOM 1495 CG TYR 188 9855 53995 84517 100 092 13ATOM 1496 CD1 TYR 188 11137 53933 84019 100 092 13ATOM 1497 CD2 TYR 188 9674 54267 85852 100 092 13ATOM 1498 CE1 TYR 188 12222 54139 84840 100 092 13ATOM 1499 CE2 TYR 188 10753 54472 86677 100 092 13ATOM 1500 CZ TYR 188 12030 54407 86174 100 092 13ATOM 1501 OH TYR 188 13136 54619 87026 100 092 13ATOM 1502 N SER 189 7516 54460 80758 100 051 13ATOM 1503 CA SER 189 6720 53930 79693 100 051 13ATOM 1504 C SER 189 7682 53165 78850 100 051 13ATOM 1505 O SER 189 8887 53181 79093 100 051 13ATOM 1506 CB SER 189 6151 54992 78739 100 051 13ATOM 1507 OG SER 189 5325 55904 79440 100 051 13ATOM 1508 N PHE 190 7167 52470 77825 100 095 13ATOM 1509 CA PHE 190 8025 51725 76960 100 095 13ATOM 1510 C PHE 190 8828 52765 76233 100 095 13ATOM 1511 O PHE 190 9955 52532 75801 100 095 13ATOM 1512 CB PHE 190 7265 50858 75946 100 095 13ATOM 1513 CG PHE 190 8155 49695 75664 100 095 13ATOM 1514 CD1 PHE 190 8195 48652 76562 100 095 13ATOM 1515 CD2 PHE 190 8938 49626 74537 100 095 13ATOM 1516 CE1 PHE 190 9000 47557 76354 100 095 13ATOM 1517 CE2 PHE 190 9742 48528 74331 100 095 13ATOM 1518 CZ PHE 190 9782 47492 75230 100 095 13ATOM 1519 N GLN 191 8227 53959 76069 100 033 13ATOM 1520 CA GLN 191 8823 55086 75407 100 033 13ATOM 1521 C GLN 191 10062 55494 76152 100 033 13ATOM 1522 O GLN 191 11014 55982 75544 100 033 13ATOM 1523 CB GLN 191 7886 56306 75375 100 033 13ATOM 1524 CG GLN 191 6581 56056 74615 100 033 13ATOM 1525 CD GLN 191 6896 55964 73128 100 033 13ATOM 1526 OE1 GLN 191 6014 55704 72311 100 033 13ATOM 1527 NE2 GLN 191 8187 56185 72763 100 033 13ATOM 1528 N SER 192 10064 55353 77495 100 4338 13ATOM 1529 CA SER 192 11212 55709 78288 100 4338 13ATOM 1530 C SER 192 12339 54786 77923 100 4338 13ATOM 1531 O SER 192 13502 55189 77891 100 4338 13ATOM 1532 CB SER 192 10966 55552 79799 100 4338 13ATOM 1533 OG SER 192 9946 56444 80222 100 4338 13ATOM 1534 N ASP 193 12016 53509 77642 100 4390 13ATOM 1535 CA ASP 193 13005 52533 77274 100 4390 13ATOM 1536 C ASP 193 13611 52932 75966 100 4390 13ATOM 1537 O ASP 193 14795 52702 75728 100 4390 13ATOM 1538 CB ASP 193 12426 51121 77064 100 4390 13ATOM 1539 CG ASP 193 12098 50504 78416 100 4390 13ATOM 1540 OD1 ASP 193 12522 51077 79454 100 4390 13ATOM 1541 OD2 ASP 193 11426 49438 78424 100 4390 13ATOM 1542 N VAL 194 12795 53522 75073 100 4184 13ATOM 1543 CA VAL 194 13242 53936 73772 100 4184 13ATOM 1544 C VAL 194 14320 54961 73942 100 4184 13ATOM 1545 O VAL 194 15323 54938 73231 100 4184 13ATOM 1546 CB VAL 194 12151 54574 72962 100 4184 13ATOM 1547 CG1 VAL 194 12753 55084 71642 100 4184 13ATOM 1548 CG2 VAL 194 11017 53553 72773 100 4184 13ATOM 1549 N TYR 195 14139 55892 74897 100 9390 13ATOM 1550 CA TYR 195 15105 56931 75125 100 9390 13ATOM 1551 C TYR 195 16395 56285 75546 100 9390 13ATOM 1552 O TYR 195 17466 56631 75048 100 9390 13ATOM 1553 CB TYR 195 14655 57892 76242 100 9390 13ATOM 1554 CG TYR 195 15693 58944 76435 100 9390 13ATOM 1555 CD1 TYR 195 16729 58758 77320 100 9390 13ATOM 1556 CD2 TYR 195 15624 60124 75729 100 9390 13ATOM 1557 CE1 TYR 195 17683 59733 77498 100 9390 13ATOM 1558 CE2 TYR 195 16576 61102 75902 100 9390 13ATOM 1559 CZ TYR 195 17608 60907 76788 100 9390 13ATOM 1560 OH TYR 195 18587 61908 76969 100 9390 13ATOM 1561 N ALA 196 16316 55296 76457 100 3404 13ATOM 1562 CA ALA 196 17477 54625 76976 100 3404 13ATOM 1563 C ALA 196 18194 53931 75858 100 3404 13ATOM 1564 O ALA 196 19423 53892 75824 100 3404 13ATOM 1565 CB ALA 196 17129 53565 78035 100 3404 13ATOM 1566 N PHE 197 17432 53366 74906 100 7602 13ATOM 1567 CA PHE 197 17990 52652 73793 100 7602 13ATOM 1568 C PHE 197 18866 53586 73018 100 7602 13ATOM 1569 O PHE 197 19943 53197 72570 100 7602 13ATOM 1570 CB PHE 197 16893 52117 72851 100 7602 13ATOM 1571 CG PHE 197 17532 51412 71704 100 7602 13ATOM 1572 CD1 PHE 197 18118 50180 71875 100 7602 13ATOM 1573 CD2 PHE 197 17522 51975 70448 100 7602 13ATOM 1574 CE1 PHE 197 18702 49537 70810 100 7602 13ATOM 1575 CE2 PHE 197 18104 51333 69380 100 7602 13ATOM 1576 CZ PHE 197 18700 50109 69561 100 7602 13ATOM 1577 N GLY 198 18433 54849 72840 100 2982 13ATOM 1578 CA GLY 198 19220 55785 72086 100 2982 13ATOM 1579 C GLY 198 20548 55972 72760 100 2982 13ATOM 1580 O GLY 198 21576 56075 72091 100 2982 13ATOM 1581 N ILE 199 20559 56052 74106 100 8739 13ATOM 1582 CA ILE 199 21787 56197 74840 100 8739 13ATOM 1583 C ILE 199 22612 54967 74622 100 8739 13ATOM 1584 O ILE 199 23830 55044 74470 100 8739 13ATOM 1585 CB ILE 199 21595 56364 76323 100 8739 13ATOM 1586 CG1 ILE 199 21010 57749 76646 100 8739 13ATOM 1587 CG2 ILE 199 22948 56110 77010 100 8739 13ATOM 1588 CD1 ILE 199 19604 57981 76107 100 8739 13ATOM 1589 N VAL 200 21961 53790 74603 100 4917 13ATOM 1590 CA VAL 200 22661 52548 74429 100 4917 13ATOM 1591 C VAL 200 23361 52559 73097 100 4917 13ATOM 1592 O VAL 200 24465 52032 72973 100 4917 13ATOM 1593 CB VAL 200 21752 51353 74445 100 4917 13ATOM 1594 CG1 VAL 200 22581 50104 74100 100 4917 13ATOM 1595 CG2 VAL 200 21061 51284 75819 100 4917 13ATOM 1596 N LEU 201 22757 53156 72049 100 080 13ATOM 1597 CA LEU 201 23431 53125 70777 100 080 13ATOM 1598 C LEU 201 24742 53846 70903 100 080 13ATOM 1599 O LEU 201 25729 53468 70277 100 080 13ATOM 1600 CB LEU 201 22723 53805 69584 100 080 13ATOM 1601 CG LEU 201 21381 53199 69127 100 080 13ATOM 1602 CD1 LEU 201 20196 53779 69908 100 080 13ATOM 1603 CD2 LEU 201 21207 53301 67607 100 080 13ATOM 1604 N TYR 202 24784 54924 71706 100 054 13ATOM 1605 CA TYR 202 25983 55701 71872 100 054 13ATOM 1606 C TYR 202 27055 54815 72424 100 054 13ATOM 1607 O TYR 202 28188 54821 71945 100 054 13ATOM 1608 CB TYR 202 25757 56851 72875 100 054 13ATOM 1609 CG TYR 202 27051 57508 73222 100 054 13ATOM 1610 CD1 TYR 202 27593 58479 72411 100 054 13ATOM 1611 CD2 TYR 202 27713 57158 74377 100 054 13ATOM 1612 CE1 TYR 202 28782 59081 72748 100 054 13ATOM 1613 CE2 TYR 202 28903 57758 74716 100 054 13ATOM 1614 CZ TYR 202 29440 58723 73899 100 054 13ATOM 1615 OH TYR 202 30661 59343 74239 100 054 13ATOM 1616 N GLU 203 26712 54019 73450 100 064 13ATOM 1617 CA GLU 203 27660 53146 74076 100 064 13ATOM 1618 C GLU 203 28051 52062 73121 100 064 13ATOM 1619 O GLU 203 29149 51522 73183 100 064 13ATOM 1620 CB GLU 203 27159 52570 75417 100 064 13ATOM 1621 CG GLU 203 25803 51877 75352 100 064 13ATOM 1622 CD GLU 203 25273 51747 76770 100 064 13ATOM 1623 OE1 GLU 203 26087 51831 77729 100 064 13ATOM 1624 OE2 GLU 203 24033 51573 76907 100 064 13ATOM 1625 N LEU 204 27160 51666 72210 100 084 13ATOM 1626 CA LEU 204 27565 50620 71327 100 084 13ATOM 1627 C LEU 204 28691 51129 70484 100 084 13ATOM 1628 O LEU 204 29710 50458 70328 100 084 13ATOM 1629 CB LEU 204 26376 50151 70467 100 084 13ATOM 1630 CG LEU 204 26593 48841 69695 100 084 13ATOM 1631 CD1 LEU 204 27029 47709 70638 100 084 13ATOM 1632 CD2 LEU 204 25309 48462 68938 100 084 13ATOM 1633 N MET 205 28560 52337 69906 100 8884 13ATOM 1634 CA MET 205 29673 52748 69109 100 8884 13ATOM 1635 C MET 205 30890 53012 69957 100 8884 13ATOM 1636 O MET 205 31964 52490 69670 100 8884 13ATOM 1637 CB MET 205 29424 54002 68264 100 8884 13ATOM 1638 CG MET 205 30574 54238 67285 100 8884 13ATOM 1639 SD MET 205 30217 55445 65984 100 8884 13ATOM 1640 CE MET 205 30262 56877 67101 100 8884 13ATOM 1641 N THR 206 30769 53854 71009 100 7132 13ATOM 1642 CA THR 206 31913 54215 71815 100 7132 13ATOM 1643 C THR 206 32344 53173 72821 100 7132 13ATOM 1644 O THR 206 33507 52777 72856 100 7132 13ATOM 1645 CB THR 206 31703 55498 72565 100 7132 13ATOM 1646 OG1 THR 206 30613 55375 73467 100 7132 13ATOM 1647 CG2 THR 206 31431 56617 71548 100 7132 13ATOM 1648 N GLY 207 31395 52694 73655 100 6259 13ATOM 1649 CA GLY 207 31616 51792 74754 100 6259 13ATOM 1650 C GLY 207 31963 52673 75912 100 6259 13ATOM 1651 O GLY 207 32159 52207 77032 100 6259 13ATOM 1652 N GLN 208 32040 53992 75645 100 8360 13ATOM 1653 CA GLN 208 32380 54966 76640 100 8360 13ATOM 1654 C GLN 208 31125 55455 77289 100 8360 13ATOM 1655 O GLN 208 30019 55136 76854 100 8360 13ATOM 1656 CB GLN 208 33128 56187 76077 100 8360 13ATOM 1657 CG GLN 208 32302 57005 75084 100 8360 13ATOM 1658 CD GLN 208 33163 58168 74615 100 8360 13ATOM 1659 OE1 GLN 208 33677 58935 75429 100 8360 13ATOM 1660 NE2 GLN 208 33330 58305 73272 100 8360 13ATOM 1661 N LEU 209 31282 56230 78382 100 8434 13ATOM 1662 CA LEU 209 30168 56752 79122 100 8434 13ATOM 1663 C LEU 209 29557 57817 78256 100 8434 13ATOM 1664 O LEU 209 30262 58547 77560 100 8434 13ATOM 1665 CB LEU 209 30615 57369 80466 100 8434 13ATOM 1666 CG LEU 209 29517 57607 81529 100 8434 13ATOM 1667 CD1 LEU 209 30119 58247 82791 100 8434 13ATOM 1668 CD2 LEU 209 28307 58384 80996 100 8434 13ATOM 1669 N PRO 210 28256 57901 78258 100 088 13ATOM 1670 CA PRO 210 27566 58868 77442 100 088 13ATOM 1671 C PRO 210 27659 60252 77997 100 088 13ATOM 1672 O PRO 210 27939 60404 79185 100 088 13ATOM 1673 CB PRO 210 26130 58352 77293 100 088 13ATOM 1674 CG PRO 210 26002 57229 78339 100 088 13ATOM 1675 CD PRO 210 27441 56726 78510 100 088 13ATOM 1676 N TYR 211 27419 61273 77147 100 061 13ATOM 1677 CA TYR 211 27495 62637 77578 100 061 13ATOM 1678 C TYR 211 28833 62797 78210 100 061 13ATOM 1679 O TYR 211 28961 63299 79323 100 061 13ATOM 1680 CB TYR 211 26417 63005 78617 100 061 13ATOM 1681 CG TYR 211 25070 62747 78023 100 061 13ATOM 1682 CD1 TYR 211 24497 63627 77132 100 061 13ATOM 1683 CD2 TYR 211 24370 61615 78375 100 061 13ATOM 1684 CE1 TYR 211 23254 63375 76598 100 061 13ATOM 1685 CE2 TYR 211 23127 61357 77846 100 061 13ATOM 1686 CZ TYR 211 22566 62238 76954 100 061 13ATOM 1687 OH TYR 211 21291 61975 76409 100 061 13ATOM 1688 N SER 212 29881 62344 77505 100 080 13ATOM 1689 CA SER 212 31186 62425 78075 100 080 13ATOM 1690 C SER 212 31638 63843 78041 100 080 13ATOM 1691 O SER 212 31065 64692 77359 100 080 13ATOM 1692 CB SER 212 32237 61579 77338 100 080 13ATOM 1693 OG SER 212 33502 61723 77967 100 080 13ATOM 1694 N ASN 213 32689 64119 78830 100 9066 13ATOM 1695 CA ASN 213 33286 65413 78908 100 9066 13ATOM 1696 C ASN 213 32290 66361 79488 100 9066 13ATOM 1697 O ASN 213 32457 67576 79408 100 9066 13ATOM 1698 CB ASN 213 33743 65929 77530 100 9066 13ATOM 1699 CG ASN 213 34737 67067 77722 100 9066 13ATOM 1700 OD1 ASN 213 34372 68229 77885 100 9066 13ATOM 1701 ND2 ASN 213 36052 66720 77697 100 9066 13ATOM 1702 N ILE 214 31216 65845 80113 100 099 13ATOM 1703 CA ILE 214 30364 66799 80749 100 099 13ATOM 1704 C ILE 214 30335 66453 82201 100 099 13ATOM 1705 O ILE 214 29910 65371 82604 100 099 13ATOM 1706 CB ILE 214 28961 66894 80192 100 099 13ATOM 1707 CG1 ILE 214 28292 68179 80702 100 099 13ATOM 1708 CG2 ILE 214 28164 65624 80521 100 099 13ATOM 1709 CD1 ILE 214 28950 69452 80170 100 099 13ATOM 1710 N ASN 215 30846 67373 83033 100 000 13ATOM 1711 CA ASN 215 30803 67163 84444 100 000 13ATOM 1712 C ASN 215 29420 67587 84782 100 000 13ATOM 1713 O ASN 215 28699 68012 83884 100 000 13ATOM 1714 CB ASN 215 31797 68048 85219 100 000 13ATOM 1715 CG ASN 215 32054 67441 86593 100 000 13ATOM 1716 OD1 ASN 215 31258 66669 87126 100 000 13ATOM 1717 ND2 ASN 215 33220 67806 87191 100 000 13ATOM 1718 N ASN 216 29019 67519 86065 100 006 13ATOM 1719 CA ASN 216 27678 67884 86420 100 006 13ATOM 1720 C ASN 216 26677 67145 85587 100 006 13ATOM 1721 O ASN 216 26306 67549 84487 100 006 13ATOM 1722 CB ASN 216 27389 69392 86292 100 006 13ATOM 1723 CG ASN 216 28124 70117 87411 100 006 13ATOM 1724 OD1 ASN 216 28543 69506 88393 100 006 13ATOM 1725 ND2 ASN 216 28267 71462 87269 100 006 13ATOM 1726 N ARG 217 26226 65989 86101 100 043 13ATOM 1727 CA ARG 217 25233 65231 85403 100 043 13ATOM 1728 C ARG 217 24056 66140 85258 100 043 13ATOM 1729 O ARG 217 23356 66114 84248 100 043 13ATOM 1730 CB ARG 217 24764 63981 86171 100 043 13ATOM 1731 CG ARG 217 24082 64278 87510 100 043 13ATOM 1732 CD ARG 217 24980 64102 88738 100 043 13ATOM 1733 NE ARG 217 25609 65419 89040 100 043 13ATOM 1734 CZ ARG 217 26007 65703 90315 100 043 13ATOM 1735 NH1 ARG 217 25837 64776 91303 100 043 13ATOM 1736 NH2 ARG 217 26575 66911 90605 100 043 13ATOM 1737 N ASP 218 23822 66994 86268 100 3429 13ATOM 1738 CA ASP 218 22718 67906 86215 100 3429 13ATOM 1739 C ASP 218 22905 68755 85002 100 3429 13ATOM 1740 O ASP 218 21938 69181 84373 100 3429 13ATOM 1741 CB ASP 218 22648 68849 87426 100 3429 13ATOM 1742 CG ASP 218 22145 68048 88614 100 3429 13ATOM 1743 OD1 ASP 218 21696 66891 88397 100 3429 13ATOM 1744 OD2 ASP 218 22201 68582 89753 100 3429 13ATOM 1745 N GLN 219 24170 69021 84644 100 8165 13ATOM 1746 CA GLN 219 24495 69830 83509 100 8165 13ATOM 1747 C GLN 219 23940 69175 82281 100 8165 13ATOM 1748 O GLN 219 23454 69859 81383 100 8165 13ATOM 1749 CB GLN 219 26013 70023 83339 100 8165 13ATOM 1750 CG GLN 219 26396 71046 82268 100 8165 13ATOM 1751 CD GLN 219 27911 71202 82305 100 8165 13ATOM 1752 OE1 GLN 219 28616 70409 82928 100 8165 13ATOM 1753 NE2 GLN 219 28429 72257 81622 100 8165 13ATOM 1754 N ILE 220 23985 67831 82194 100 084 13ATOM 1755 CA ILE 220 23426 67219 81022 100 084 13ATOM 1756 C ILE 220 21968 67564 81034 100 084 13ATOM 1757 O ILE 220 21375 67863 80000 100 084 13ATOM 1758 CB ILE 220 23592 65719 80950 100 084 13ATOM 1759 CG1 ILE 220 23487 65245 79490 100 084 13ATOM 1760 CG2 ILE 220 22558 65035 81859 100 084 13ATOM 1761 CD1 ILE 220 22131 65499 78834 100 084 13ATOM 1762 N ILE 221 21360 67557 82236 100 3703 13ATOM 1763 CA ILE 221 19967 67861 82397 100 3703 13ATOM 1764 C ILE 221 19738 69266 81931 100 3703 13ATOM 1765 O ILE 221 18729 69565 81295 100 3703 13ATOM 1766 CB ILE 221 19510 67769 83823 100 3703 13ATOM 1767 CG1 ILE 221 19685 66334 84349 100 3703 13ATOM 1768 CG2 ILE 221 18060 68276 83890 100 3703 13ATOM 1769 CD1 ILE 221 19533 66211 85864 100 3703 13ATOM 1770 N PHE 222 20673 70178 82242 100 3119 13ATOM 1771 CA PHE 222 20505 71544 81841 100 3119 13ATOM 1772 C PHE 222 20450 71602 80343 100 3119 13ATOM 1773 O PHE 222 19598 72279 79769 100 3119 13ATOM 1774 CB PHE 222 21677 72434 82288 100 3119 13ATOM 1775 CG PHE 222 21399 73821 81822 100 3119 13ATOM 1776 CD1 PHE 222 20681 74691 82608 100 3119 13ATOM 1777 CD2 PHE 222 21853 74250 80595 100 3119 13ATOM 1778 CE1 PHE 222 20422 75972 82182 100 3119 13ATOM 1779 CE2 PHE 222 21596 75530 80165 100 3119 13ATOM 1780 CZ PHE 222 20880 76394 80958 100 3119 13ATOM 1781 N MET 223 21363 70877 79671 100 9538 13ATOM 1782 CA MET 223 21459 70899 78238 100 9538 13ATOM 1783 C MET 223 20204 70362 77622 100 9538 13ATOM 1784 O MET 223 19710 70906 76635 100 9538 13ATOM 1785 CB MET 223 22608 70029 77708 100 9538 13ATOM 1786 CG MET 223 22724 70042 76183 100 9538 13ATOM 1787 SD MET 223 24072 69020 75521 100 9538 13ATOM 1788 CE MET 223 23309 67442 75996 100 9538 13ATOM 1789 N VAL 224 19647 69277 78190 100 8339 13ATOM 1790 CA VAL 224 18489 68665 77600 100 8339 13ATOM 1791 C VAL 224 17358 69642 77629 100 8339 13ATOM 1792 O VAL 224 16553 69703 76700 100 8339 13ATOM 1793 CB VAL 224 18037 67411 78295 100 8339 13ATOM 1794 CG1 VAL 224 17449 67771 79668 100 8339 13ATOM 1795 CG2 VAL 224 17044 66682 77372 100 8339 13ATOM 1796 N GLY 225 17271 70440 78708 100 2030 13ATOM 1797 CA GLY 225 16208 71393 78856 100 2030 13ATOM 1798 C GLY 225 16285 72371 77727 100 2030 13ATOM 1799 O GLY 225 15261 72834 77227 100 2030 13ATOM 1800 N ARG 226 17516 72722 77314 100 091 13ATOM 1801 CA ARG 226 17730 73669 76257 100 091 13ATOM 1802 C ARG 226 17185 73092 74991 100 091 13ATOM 1803 O ARG 226 16737 73827 74112 100 091 13ATOM 1804 CB ARG 226 19209 74029 76030 100 091 13ATOM 1805 CG ARG 226 19848 74736 77228 100 091 13ATOM 1806 CD ARG 226 21116 75528 76890 100 091 13ATOM 1807 NE ARG 226 22103 74591 76283 100 091 13ATOM 1808 CZ ARG 226 23189 75093 75625 100 091 13ATOM 1809 NH1 ARG 226 23379 76443 75560 100 091 13ATOM 1810 NH2 ARG 226 24078 74250 75023 100 091 13ATOM 1811 N GLY 227 17192 71752 74865 100 2783 13ATOM 1812 CA GLY 227 16710 71150 73656 100 2783 13ATOM 1813 C GLY 227 17877 70558 72938 100 2783 13ATOM 1814 O GLY 227 17738 70019 71841 100 2783 13ATOM 1815 N TYR 228 19069 70649 73551 100 7496 13ATOM 1816 CA TYR 228 20254 70104 72956 100 7496 13ATOM 1817 C TYR 228 20330 68654 73316 100 7496 13ATOM 1818 O TYR 228 19870 68236 74377 100 7496 13ATOM 1819 CB TYR 228 21548 70794 73417 100 7496 13ATOM 1820 CG TYR 228 21431 72218 72994 100 7496 13ATOM 1821 CD1 TYR 228 21659 72597 71691 100 7496 13ATOM 1822 CD2 TYR 228 21083 73179 73913 100 7496 13ATOM 1823 CE1 TYR 228 21545 73915 71314 100 7496 13ATOM 1824 CE2 TYR 228 20967 74497 73542 100 7496 13ATOM 1825 CZ TYR 228 21198 74868 72241 100 7496 13ATOM 1826 OH TYR 228 21079 76222 71862 100 7496 13ATOM 1827 N LEU 229 20862 67835 72390 100 5125 13ATOM 1828 CA LEU 229 20997 66425 72611 100 5125 13ATOM 1829 C LEU 229 22459 66120 72706 100 5125 13ATOM 1830 O LEU 229 23300 67008 72570 100 5125 13ATOM 1831 CB LEU 229 20407 65580 71469 100 5125 13ATOM 1832 CG LEU 229 18894 65806 71289 100 5125 13ATOM 1833 CD1 LEU 229 18322 64942 70154 100 5125 13ATOM 1834 CD2 LEU 229 18141 65630 72617 100 5125 13ATOM 1835 N SER 230 22803 64841 72959 100 4041 13ATOM 1836 CA SER 230 24185 64484 73101 100 4041 13ATOM 1837 C SER 230 24874 64846 71827 100 4041 13ATOM 1838 O SER 230 24304 64778 70740 100 4041 13ATOM 1839 CB SER 230 24411 62984 73359 100 4041 13ATOM 1840 OG SER 230 25798 62711 73491 100 4041 13ATOM 1841 N PRO 231 26108 65239 71953 100 8936 13ATOM 1842 CA PRO 231 26867 65644 70807 100 8936 13ATOM 1843 C PRO 231 27138 64454 69959 100 8936 13ATOM 1844 O PRO 231 27259 63355 70498 100 8936 13ATOM 1845 CB PRO 231 28118 66345 71348 100 8936 13ATOM 1846 CG PRO 231 28097 66073 72866 100 8936 13ATOM 1847 CD PRO 231 26611 65835 73176 100 8936 13ATOM 1848 N ASP 232 27235 64643 68632 100 3399 13ATOM 1849 CA ASP 232 27468 63503 67808 100 3399 13ATOM 1850 C ASP 232 28766 62933 68239 100 3399 13ATOM 1851 O ASP 232 29726 63658 68494 100 3399 13ATOM 1852 CB ASP 232 27566 63816 66305 100 3399 13ATOM 1853 CG ASP 232 26171 64151 65800 100 3399 13ATOM 1854 OD1 ASP 232 25210 64043 66607 100 3399 13ATOM 1855 OD2 ASP 232 26047 64512 64598 100 3399 13ATOM 1856 N LEU 233 28811 61596 68358 100 005 13ATOM 1857 CA LEU 233 30044 60998 68746 100 005 13ATOM 1858 C LEU 233 30984 61374 67651 100 005 13ATOM 1859 O LEU 233 30837 60919 66518 100 005 13ATOM 1860 CB LEU 233 29944 59464 68794 100 005 13ATOM 1861 CG LEU 233 28925 58922 69812 100 005 13ATOM 1862 CD1 LEU 233 27491 59352 69461 100 005 13ATOM 1863 CD2 LEU 233 29064 57401 69982 100 005 13ATOM 1864 N SER 234 31979 62233 67951 100 078 13ATOM 1865 CA SER 234 32854 62641 66893 100 078 13ATOM 1866 C SER 234 34266 62451 67331 100 078 13ATOM 1867 O SER 234 34781 63195 68163 100 078 13ATOM 1868 CB SER 234 32702 64124 66520 100 078 13ATOM 1869 OG SER 234 33595 64465 65469 100 078 13ATOM 1870 N LYS 235 34927 61436 66749 100 020 13ATOM 1871 CA LYS 235 36306 61159 67003 100 020 13ATOM 1872 C LYS 235 36761 60394 65810 100 020 13ATOM 1873 O LYS 235 35940 59940 65015 100 020 13ATOM 1874 CB LYS 235 36572 60256 68223 100 020 13ATOM 1875 CG LYS 235 36279 60908 69575 100 020 13ATOM 1876 CD LYS 235 34790 61064 69888 100 020 13ATOM 1877 CE LYS 235 34198 59874 70645 100 020 13ATOM 1878 NZ LYS 235 32773 60130 70949 100 020 13ATOM 1879 N VAL 236 38086 60247 65630 100 094 13ATOM 1880 CA VAL 236 38516 59474 64506 100 094 13ATOM 1881 C VAL 236 37965 58105 64726 100 094 13ATOM 1882 O VAL 236 37333 57525 63845 100 094 13ATOM 1883 CB VAL 236 40010 59346 64415 100 094 13ATOM 1884 CG1 VAL 236 40350 58407 63245 100 094 13ATOM 1885 CG2 VAL 236 40626 60750 64283 100 094 13ATOM 1886 N ARG 237 38181 57568 65941 100 8275 13ATOM 1887 CA ARG 237 37690 56271 66297 100 8275 13ATOM 1888 C ARG 237 36200 56321 66366 100 8275 13ATOM 1889 O ARG 237 35522 55383 65946 100 8275 13ATOM 1890 CB ARG 237 38207 55790 67661 100 8275 13ATOM 1891 CG ARG 237 39692 55428 67632 100 8275 13ATOM 1892 CD ARG 237 40268 55072 69004 100 8275 13ATOM 1893 NE ARG 237 41701 54714 68810 100 8275 13ATOM 1894 CZ ARG 237 42643 55696 68699 100 8275 13ATOM 1895 NH1 ARG 237 42267 57008 68718 100 8275 13ATOM 1896 NH2 ARG 237 43960 55367 68556 100 8275 13ATOM 1897 N SER 238 35653 57433 66894 100 045 13ATOM 1898 CA SER 238 34239 57543 67076 100 045 13ATOM 1899 C SER 238 33551 57281 65777 100 045 13ATOM 1900 O SER 238 32948 56224 65599 100 045 13ATOM 1901 CB SER 238 33829 58944 67536 100 045 13ATOM 1902 OG SER 238 32419 58991 67648 100 045 13ATOM 1903 N ASN 239 33661 58231 64828 100 6225 13ATOM 1904 CA ASN 239 33061 58099 63530 100 6225 13ATOM 1905 C ASN 239 31643 57630 63651 100 6225 13ATOM 1906 O ASN 239 31295 56582 63111 100 6225 13ATOM 1907 CB ASN 239 33795 57120 62595 100 6225 13ATOM 1908 CG ASN 239 35131 57735 62202 100 6225 13ATOM 1909 OD1 ASN 239 35411 58896 62496 100 6225 13ATOM 1910 ND2 ASN 239 35980 56930 61507 100 6225 13ATOM 1911 N CYS 240 30788 58377 64377 100 4390 13ATOM 1912 CA CYS 240 29412 57979 64474 100 4390 13ATOM 1913 C CYS 240 28786 58236 63143 100 4390 13ATOM 1914 O CYS 240 28891 59333 62599 100 4390 13ATOM 1915 CB CYS 240 28626 58786 65521 100 4390 13ATOM 1916 SG CYS 240 26887 58280 65649 100 4390 13ATOM 1917 N PRO 241 28130 57254 62593 100 6317 13ATOM 1918 CA PRO 241 27517 57491 61320 100 6317 13ATOM 1919 C PRO 241 26366 58423 61498 100 6317 13ATOM 1920 O PRO 241 25708 58369 62536 100 6317 13ATOM 1921 CB PRO 241 27164 56115 60765 100 6317 13ATOM 1922 CG PRO 241 28229 55198 61394 100 6317 13ATOM 1923 CD PRO 241 28582 55879 62729 100 6317 13ATOM 1924 N LYS 242 26108 59289 60502 100 046 13ATOM 1925 CA LYS 242 25056 60252 60617 100 046 13ATOM 1926 C LYS 242 23752 59534 60696 100 046 13ATOM 1927 O LYS 242 22878 59907 61475 100 046 13ATOM 1928 CB LYS 242 24981 61208 59415 100 046 13ATOM 1929 CG LYS 242 23986 62354 59604 100 046 13ATOM 1930 CD LYS 242 24411 63372 60664 100 046 13ATOM 1931 CE LYS 242 25660 64164 60278 100 046 13ATOM 1932 NZ LYS 242 26841 63272 60272 100 046 13ATOM 1933 N ALA 243 23592 58461 59902 100 2583 13ATOM 1934 CA ALA 243 22328 57793 59885 100 2583 13ATOM 1935 C ALA 243 22035 57295 61261 100 2583 13ATOM 1936 O ALA 243 20923 57466 61757 100 2583 13ATOM 1937 CB ALA 243 22304 56581 58938 100 2583 13ATOM 1938 N MET 244 23032 56692 61934 100 9951 13ATOM 1939 CA MET 244 22776 56153 63239 100 9951 13ATOM 1940 C MET 244 22464 57269 64185 100 9951 13ATOM 1941 O MET 244 21608 57131 65058 100 9951 13ATOM 1942 CB MET 244 23938 55329 63824 100 9951 13ATOM 1943 CG MET 244 25134 56154 64293 100 9951 13ATOM 1944 SD MET 244 26454 55166 65060 100 9951 13ATOM 1945 CE MET 244 25449 54632 66476 100 9951 13ATOM 1946 N LYS 245 23153 58414 64031 100 8439 13ATOM 1947 CA LYS 245 22939 59524 64913 100 8439 13ATOM 1948 C LYS 245 21510 59967 64789 100 8439 13ATOM 1949 O LYS 245 20875 60308 65786 100 8439 13ATOM 1950 CB LYS 245 23845 60726 64584 100 8439 13ATOM 1951 CG LYS 245 23843 61835 65643 100 8439 13ATOM 1952 CD LYS 245 22495 62541 65822 100 8439 13ATOM 1953 CE LYS 245 22507 63642 66885 100 8439 13ATOM 1954 NZ LYS 245 21207 64348 66898 100 8439 13ATOM 1955 N ARG 246 20963 59977 63557 100 076 13ATOM 1956 CA ARG 246 19603 60405 63372 100 076 13ATOM 1957 C ARG 246 18689 59465 64093 100 076 13ATOM 1958 O ARG 246 17674 59891 64641 100 076 13ATOM 1959 CB ARG 246 19138 60473 61905 100 076 13ATOM 1960 CG ARG 246 19556 61753 61176 100 076 13ATOM 1961 CD ARG 246 21016 61776 60735 100 076 13ATOM 1962 NE ARG 246 21086 61050 59436 100 076 13ATOM 1963 CZ ARG 246 20858 61719 58268 100 076 13ATOM 1964 NH1 ARG 246 20621 63063 58285 100 076 13ATOM 1965 NH2 ARG 246 20871 61043 57083 100 076 13ATOM 1966 N LEU 247 19010 58156 64107 100 028 13ATOM 1967 CA LEU 247 18163 57216 64792 100 028 13ATOM 1968 C LEU 247 18123 57575 66239 100 028 13ATOM 1969 O LEU 247 17064 57550 66864 100 028 13ATOM 1970 CB LEU 247 18668 55756 64800 100 028 13ATOM 1971 CG LEU 247 18402 54925 63535 100 028 13ATOM 1972 CD1 LEU 247 16898 54676 63345 100 028 13ATOM 1973 CD2 LEU 247 19069 55528 62298 100 028 13ATOM 1974 N MET 248 19289 57926 66807 100 4298 13ATOM 1975 CA MET 248 19370 58225 68204 100 4298 13ATOM 1976 C MET 248 18470 59393 68463 100 4298 13ATOM 1977 O MET 248 17739 59414 69448 100 4298 13ATOM 1978 CB MET 248 20785 58651 68636 100 4298 13ATOM 1979 CG MET 248 21849 57562 68478 100 4298 13ATOM 1980 SD MET 248 23550 58119 68817 100 4298 13ATOM 1981 CE MET 248 24334 56520 68455 100 4298 13ATOM 1982 N ALA 249 18466 60379 67549 100 3229 13ATOM 1983 CA ALA 249 17713 61588 67730 100 3229 13ATOM 1984 C ALA 249 16258 61264 67869 100 3229 13ATOM 1985 O ALA 249 15555 61888 68661 100 3229 13ATOM 1986 CB ALA 249 17861 62564 66550 100 3229 13ATOM 1987 N GLU 250 15763 60279 67099 100 3629 13ATOM 1988 CA GLU 250 14370 59928 67134 100 3629 13ATOM 1989 C GLU 250 14027 59467 68521 100 3629 13ATOM 1990 O GLU 250 12961 59781 69047 100 3629 13ATOM 1991 CB GLU 250 14038 58760 66190 100 3629 13ATOM 1992 CG GLU 250 14267 59067 64710 100 3629 13ATOM 1993 CD GLU 250 13920 57819 63910 100 3629 13ATOM 1994 OE1 GLU 250 13281 56901 64490 100 3629 13ATOM 1995 OE2 GLU 250 14288 57766 62706 100 3629 13ATOM 1996 N CYS 251 14936 58701 69150 100 5674 13ATOM 1997 CA CYS 251 14724 58138 70457 100 5674 13ATOM 1998 C CYS 251 14654 59219 71502 100 5674 13ATOM 1999 O CYS 251 14048 59035 72557 100 5674 13ATOM 2000 CB CYS 251 15841 57163 70860 100 5674 13ATOM 2001 SG CYS 251 15883 55699 69783 100 5674 13ATOM 2002 N LEU 252 15369 60329 71251 100 056 13ATOM 2003 CA LEU 252 15522 61515 72055 100 056 13ATOM 2004 C LEU 252 14354 62451 72047 100 056 13ATOM 2005 O LEU 252 14363 63412 72817 100 056 13ATOM 2006 CB LEU 252 16770 62346 71698 100 056 13ATOM 2007 CG LEU 252 18082 61826 72321 100 056 13ATOM 2008 CD1 LEU 252 18377 60372 71931 100 056 13ATOM 2009 CD2 LEU 252 19251 62773 72008 100 056 13ATOM 2010 N LYS 253 13394 62277 71118 100 076 13ATOM 2011 CA LYS 253 12299 63196 70944 100 076 13ATOM 2012 C LYS 253 11695 63565 72268 100 076 13ATOM 2013 O LYS 253 11639 62768 73204 100 076 13ATOM 2014 CB LYS 253 11194 62637 70037 100 076 13ATOM 2015 CG LYS 253 10273 63714 69474 100 076 13ATOM 2016 CD LYS 253 10943 64609 68429 100 076 13ATOM 2017 CE LYS 253 12082 65462 68989 100 076 13ATOM 2018 NZ LYS 253 12627 66340 67929 100 076 13ATOM 2019 N LYS 254 11256 64838 72365 100 7349 13ATOM 2020 CA LYS 254 10758 65415 73583 100 7349 13ATOM 2021 C LYS 254 9541 64688 74067 100 7349 13ATOM 2022 O LYS 254 9492 64271 75225 100 7349 13ATOM 2023 CB LYS 254 10372 66890 73379 100 7349 13ATOM 2024 CG LYS 254 9830 67595 74623 100 7349 13ATOM 2025 CD LYS 254 9746 69113 74450 100 7349 13ATOM 2026 CE LYS 254 9177 69848 75665 100 7349 13ATOM 2027 NZ LYS 254 10180 69879 76752 100 7349 13ATOM 2028 N LYS 255 8528 64490 73196 100 009 13ATOM 2029 CA LYS 255 7350 63829 73679 100 009 13ATOM 2030 C LYS 255 7566 62362 73529 100 009 13ATOM 2031 O LYS 255 8172 61910 72561 100 009 13ATOM 2032 CB LYS 255 6060 64188 72922 100 009 13ATOM 2033 CG LYS 255 5628 65644 73100 100 009 13ATOM 2034 CD LYS 255 4476 66048 72179 100 009 13ATOM 2035 CE LYS 255 4742 65756 70702 100 009 13ATOM 2036 NZ LYS 255 5932 66508 70248 100 009 13ATOM 2037 N ARG 256 7056 61578 74497 100 083 13ATOM 2038 CA ARG 256 7252 60158 74513 100 083 13ATOM 2039 C ARG 256 6602 59593 73288 100 083 13ATOM 2040 O ARG 256 7146 58705 72636 100 083 13ATOM 2041 CB ARG 256 6560 59446 75692 100 083 13ATOM 2042 CG ARG 256 7034 59806 77105 100 083 13ATOM 2043 CD ARG 256 6427 61086 77690 100 083 13ATOM 2044 NE ARG 256 7177 62253 77150 100 083 13ATOM 2045 CZ ARG 256 7059 63471 77756 100 083 13ATOM 2046 NH1 ARG 256 6247 63616 78844 100 083 13ATOM 2047 NH2 ARG 256 7754 64542 77276 100 083 13ATOM 2048 N ASP 257 5422 60135 72934 100 5658 13ATOM 2049 CA ASP 257 4597 59647 71865 100 5658 13ATOM 2050 C ASP 257 5350 59719 70576 100 5658 13ATOM 2051 O ASP 257 5183 58871 69702 100 5658 13ATOM 2052 CB ASP 257 3354 60524 71642 100 5658 13ATOM 2053 CG ASP 257 2498 60487 72893 100 5658 13ATOM 2054 OD1 ASP 257 2764 59622 73770 100 5658 13ATOM 2055 OD2 ASP 257 1566 61329 72992 100 5658 13ATOM 2056 N GLU 258 6180 60762 70423 100 8580 13ATOM 2057 CA GLU 258 6906 61009 69210 100 8580 13ATOM 2058 C GLU 258 7917 59928 68961 100 8580 13ATOM 2059 O GLU 258 8230 59628 67811 100 8580 13ATOM 2060 CB GLU 258 7656 62350 69230 100 8580 13ATOM 2061 CG GLU 258 6724 63563 69259 100 8580 13ATOM 2062 CD GLU 258 6011 63656 67916 100 8580 13ATOM 2063 OE1 GLU 258 6713 63656 66869 100 8580 13ATOM 2064 OE2 GLU 258 4753 63726 67922 100 8580 13ATOM 2065 N ARG 259 8471 59324 70028 100 8909 13ATOM 2066 CA ARG 259 9523 58356 69871 100 8909 13ATOM 2067 C ARG 259 9007 57155 69137 100 8909 13ATOM 2068 O ARG 259 7831 56805 69211 100 8909 13ATOM 2069 CB ARG 259 10100 57871 71209 100 8909 13ATOM 2070 CG ARG 259 10686 59005 72049 100 8909 13ATOM 2071 CD ARG 259 11260 58531 73383 100 8909 13ATOM 2072 NE ARG 259 11634 59741 74164 100 8909 13ATOM 2073 CZ ARG 259 11384 59775 75505 100 8909 13ATOM 2074 NH1 ARG 259 10801 58701 76113 100 8909 13ATOM 2075 NH2 ARG 259 11713 60879 76237 100 8909 13ATOM 2076 N PRO 260 9899 56533 68407 100 053 13ATOM 2077 CA PRO 260 9548 55378 67621 100 053 13ATOM 2078 C PRO 260 9373 54152 68453 100 053 13ATOM 2079 O PRO 260 9823 54121 69597 100 053 13ATOM 2080 CB PRO 260 10662 55214 66590 100 053 13ATOM 2081 CG PRO 260 11231 56632 66433 100 053 13ATOM 2082 CD PRO 260 10982 57290 67798 100 053 13ATOM 2083 N LEU 261 8684 53138 67901 100 035 13ATOM 2084 CA LEU 261 8533 51895 68589 100 035 13ATOM 2085 C LEU 261 9742 51061 68312 100 035 13ATOM 2086 O LEU 261 10492 51319 67372 100 035 13ATOM 2087 CB LEU 261 7234 51145 68229 100 035 13ATOM 2088 CG LEU 261 6722 51405 66800 100 035 13ATOM 2089 CD1 LEU 261 7682 50857 65743 100 035 13ATOM 2090 CD2 LEU 261 5285 50892 66617 100 035 13ATOM 2091 N PHE 262 9979 50056 69175 100 077 13ATOM 2092 CA PHE 262 11101 49174 69048 100 077 13ATOM 2093 C PHE 262 10987 48374 67791 100 077 13ATOM 2094 O PHE 262 12009 48052 67189 100 077 13ATOM 2095 CB PHE 262 11306 48258 70262 100 077 13ATOM 2096 CG PHE 262 11938 49130 71295 100 077 13ATOM 2097 CD1 PHE 262 11168 49879 72150 100 077 13ATOM 2098 CD2 PHE 262 13307 49226 71390 100 077 13ATOM 2099 CE1 PHE 262 11747 50686 73101 100 077 13ATOM 2100 CE2 PHE 262 13895 50032 72337 100 077 13ATOM 2101 CZ PHE 262 13115 50764 73199 100 077 13ATOM 2102 N PRO 263 9821 47993 67355 100 7214 13ATOM 2103 CA PRO 263 9768 47289 66113 100 7214 13ATOM 2104 C PRO 263 10278 48145 64997 100 7214 13ATOM 2105 O PRO 263 10893 47610 64077 100 7214 13ATOM 2106 CB PRO 263 8322 46799 65965 100 7214 13ATOM 2107 CG PRO 263 7585 47365 67201 100 7214 13ATOM 2108 CD PRO 263 8710 47633 68214 100 7214 13ATOM 2109 N GLN 264 10033 49468 65047 100 4468 13ATOM 2110 CA GLN 264 10495 50335 64003 100 4468 13ATOM 2111 C GLN 264 11987 50409 64062 100 4468 13ATOM 2112 O GLN 264 12657 50349 63033 100 4468 13ATOM 2113 CB GLN 264 9958 51772 64107 100 4468 13ATOM 2114 CG GLN 264 10430 52675 62964 100 4468 13ATOM 2115 CD GLN 264 9839 54060 63178 100 4468 13ATOM 2116 OE1 GLN 264 9219 54332 64204 100 4468 13ATOM 2117 NE2 GLN 264 10035 54963 62180 100 4468 13ATOM 2118 N ILE 265 12552 50519 65282 100 9129 13ATOM 2119 CA ILE 265 13975 50657 65408 100 9129 13ATOM 2120 C ILE 265 14614 49416 64870 100 9129 13ATOM 2121 O ILE 265 15631 49480 64183 100 9129 13ATOM 2122 CB ILE 265 14466 50855 66816 100 9129 13ATOM 2123 CG1 ILE 265 14210 49608 67671 100 9129 13ATOM 2124 CG2 ILE 265 13809 52130 67370 100 9129 13ATOM 2125 CD1 ILE 265 14980 49614 68989 100 9129 13ATOM 2126 N LEU 266 14027 48245 65171 100 093 13ATOM 2127 CA LEU 266 14559 46988 64726 100 093 13ATOM 2128 C LEU 266 14558 46966 63228 100 093 13ATOM 2129 O LEU 266 15515 46523 62594 100 093 13ATOM 2130 CB LEU 266 13676 45815 65190 100 093 13ATOM 2131 CG LEU 266 14154 44424 64744 100 093 13ATOM 2132 CD1 LEU 266 15498 44056 65385 100 093 13ATOM 2133 CD2 LEU 266 13069 43366 64996 100 093 13ATOM 2134 N ALA 267 13469 47442 62608 100 3027 13ATOM 2135 CA ALA 267 13402 47384 61180 100 3027 13ATOM 2136 C ALA 267 14473 48239 60575 100 3027 13ATOM 2137 O ALA 267 15176 47797 59670 100 3027 13ATOM 2138 CB ALA 267 12055 47877 60627 100 3027 13ATOM 2139 N SER 268 14649 49476 61080 100 3187 13ATOM 2140 CA SER 268 15582 50404 60496 100 3187 13ATOM 2141 C SER 268 16979 49899 60645 100 3187 13ATOM 2142 O SER 268 17775 49965 59710 100 3187 13ATOM 2143 CB SER 268 15523 51798 61146 100 3187 13ATOM 2144 OG SER 268 15907 51722 62511 100 3187 13ATOM 2145 N ILE 269 17309 49360 61832 100 007 13ATOM 2146 CA ILE 269 18641 48899 62074 100 007 13ATOM 2147 C ILE 269 18925 47805 61103 100 007 13ATOM 2148 O ILE 269 20040 47705 60593 100 007 13ATOM 2149 CB ILE 269 18841 48412 63495 100 007 13ATOM 2150 CG1 ILE 269 20298 48012 63778 100 007 13ATOM 2151 CG2 ILE 269 17859 47271 63774 100 007 13ATOM 2152 CD1 ILE 269 20766 46745 63055 100 007 13ATOM 2153 N GLU 270 17947 46923 60855 100 3532 13ATOM 2154 CA GLU 270 18203 45821 59978 100 3532 13ATOM 2155 C GLU 270 18548 46358 58625 100 3532 13ATOM 2156 O GLU 270 19453 45847 57967 100 3532 13ATOM 2157 CB GLU 270 16994 44879 59836 100 3532 13ATOM 2158 CG GLU 270 16640 44180 61150 100 3532 13ATOM 2159 CD GLU 270 15530 43170 60889 100 3532 13ATOM 2160 OE1 GLU 270 15380 42739 59713 100 3532 13ATOM 2161 OE2 GLU 270 14823 42807 61867 100 3532 13ATOM 2162 N LEU 271 17845 47414 58169 100 8439 13ATOM 2163 CA LEU 271 18141 47946 56867 100 8439 13ATOM 2164 C LEU 271 19546 48445 56841 100 8439 13ATOM 2165 O LEU 271 20282 48185 55890 100 8439 13ATOM 2166 CB LEU 271 17269 49150 56448 100 8439 13ATOM 2167 CG LEU 271 15854 48830 55924 100 8439 13ATOM 2168 CD1 LEU 271 14988 48107 56960 100 8439 13ATOM 2169 CD2 LEU 271 15174 50103 55390 100 8439 13ATOM 2170 N LEU 272 19967 49172 57889 100 031 13ATOM 2171 CA LEU 272 21281 49740 57847 100 031 13ATOM 2172 C LEU 272 22310 48653 57855 100 031 13ATOM 2173 O LEU 272 23336 48762 57188 100 031 13ATOM 2174 CB LEU 272 21552 50761 58961 100 031 13ATOM 2175 CG LEU 272 22971 51361 58895 100 031 13ATOM 2176 CD1 LEU 272 23211 52050 57544 100 031 13ATOM 2177 CD2 LEU 272 23253 52305 60076 100 031 13ATOM 2178 N ALA 273 22075 47570 58617 100 2823 13ATOM 2179 CA ALA 273 23038 46505 58649 100 2823 13ATOM 2180 C ALA 273 23147 45905 57281 100 2823 13ATOM 2181 O ALA 273 24248 45657 56792 100 2823 13ATOM 2182 CB ALA 273 22647 45376 59618 100 2823 13ATOM 2183 N ARG 274 22003 45682 56607 100 052 13ATOM 2184 CA ARG 274 22064 45056 55318 100 052 13ATOM 2185 C ARG 274 22877 45925 54426 100 052 13ATOM 2186 O ARG 274 23746 45440 53703 100 052 13ATOM 2187 CB ARG 274 20689 44874 54654 100 052 13ATOM 2188 CG ARG 274 20776 44347 53219 100 052 13ATOM 2189 CD ARG 274 21389 42949 53102 100 052 13ATOM 2190 NE ARG 274 21382 42581 51657 100 052 13ATOM 2191 CZ ARG 274 20282 41975 51122 100 052 13ATOM 2192 NH1 ARG 274 19205 41700 51914 100 052 13ATOM 2193 NH2 ARG 274 20254 41652 49796 100 052 13ATOM 2194 N SER 275 22626 47246 54452 100 8357 13ATOM 2195 CA SER 275 23422 48080 53608 100 8357 13ATOM 2196 C SER 275 24367 48833 54483 100 8357 13ATOM 2197 O SER 275 24037 49887 55023 100 8357 13ATOM 2198 CB SER 275 22600 49098 52790 100 8357 13ATOM 2199 OG SER 275 21861 49957 53647 100 8357 13ATOM 2200 N LEU 276 25598 48303 54615 100 019 13ATOM 2201 CA LEU 276 26600 48931 55421 100 019 13ATOM 2202 C LEU 276 26737 50368 54940 100 019 13ATOM 2203 O LEU 276 27030 50564 53731 100 019 13ATOM 2204 CB LEU 276 27980 48265 55296 100 019 13ATOM 2205 CG LEU 276 29086 48972 56104 100 019 13ATOM 2206 CD1 LEU 276 28821 48913 57617 100 019 13ATOM 2207 CD2 LEU 276 30475 48443 55714 100 019 13ATOM 2208 OXT LEU 276 26549 51288 55779 100 019 13TER13ATOM 2209 C1 EPO 1 18691 38893 85113 100 2078 13ATOM 2210 N9 EPO 1 17400 39045 85460 100 3522 13ATOM 2211 H39 EPO 1 17061 38592 86284 100 000 13ATOM 2212 N2 EPO 1 19463 38104 85882 100 1871 13ATOM 2213 H38 EPO 1 19077 37692 86708 100 000 13ATOM 2214 C3 EPO 1 20745 37860 85558 100 2320 13ATOM 2215 C8 EPO 1 21589 38848 85287 100 2366 13ATOM 2216 H8 EPO 1 21288 39801 85327 100 000 13ATOM 2217 C7 EPO 1 22909 38530 84944 100 1990 13ATOM 2218 H7 EPO 1 23567 39257 84747 100 000 13ATOM 2219 C6 EPO 1 23296 37203 84880 100 2170 13ATOM 2220 C5 EPO 1 22407 36217 85168 100 2200 13ATOM 2221 C4 EPO 1 21123 36555 85498 100 2118 13ATOM 2222 O1 EPO 1 19191 39492 84150 100 1573 13ATOM 2223 C29 EPO 1 24693 36790 84526 100 000 13ATOM 2224 H31 EPO 1 22687 35257 85139 100 000 13ATOM 2225 H33 EPO 1 20457 35836 85697 100 000 13ATOM 2226 H35 EPO 1 25272 36838 85339 100 000 13ATOM 2227 H37 EPO 1 24686 35854 84176 100 000 13ATOM 2228 C39 EPO 1 25205 37728 83461 100 000 13ATOM 2229 N41 EPO 1 25233 36954 82226 100 000 13ATOM 2230 H43 EPO 1 24581 38501 83348 100 000 13ATOM 2231 H45 EPO 1 26115 38066 83704 100 000 13ATOM 2232 H47 EPO 1 26113 36571 81876 100 000 13ATOM 2233 C49 EPO 1 24076 36535 81613 100 000 13ATOM 2234 C51 EPO 1 24207 35628 80445 100 000 13ATOM 2235 O55 EPO 1 22971 36867 82061 100 000 13ATOM 2236 O55 EPO 1 25375 34870 80911 100 000 13ATOM 2237 C56 EPO 1 25491 35439 79648 100 000 13ATOM 2238 H60 EPO 1 26107 36213 79504 100 000 13ATOM 2239 H62 EPO 1 25492 34855 78837 100 000 13ATOM 2240 H64 EPO 1 23408 34897 80297 100 000 13ATOM 2241 C EPO 1 16509 39830 84688 100 000 13ATOM 2242 C EPO 1 16542 39757 83297 100 000 13ATOM 2243 H11 EPO 1 17195 39150 82844 100 000 13ATOM 2244 C EPO 1 15665 40530 82537 100 000 13ATOM 2245 C2 EPO 1 15701 40449 81015 100 000 13ATOM 2246 F3 EPO 1 14490 40034 80554 100 000 13ATOM 2247 F2 EPO 1 15983 41678 80502 100 000 13ATOM 2248 F1 EPO 1 16660 39565 80629 100 000 13ATOM 2249 C EPO 1 14756 41376 83170 100 000 13ATOM 2250 CL1 EPO 1 13654 42346 82216 100 000 13ATOM 2251 C EPO 1 14723 41449 84561 100 000 13ATOM 2252 H10 EPO 1 14070 42056 85015 100 000 13ATOM 2253 C EPO 1 15600 40676 85320 100 000 13ATOM 2254 H9 EPO 1 15577 40729 86318 100 000 13TER13

scriptstxt

script

__MACOSX_scriptstxt

CLUSTAL W (183) multiple sequence alignmentNLK -EEIYV--VTE--LMp38beta -SEVYL--VTT--LMp38alpha -NDVYL--VTH--LMMOK -GSLAL--ICE--LMSpeg -RYLVL--IAE-SCGYes -EPIYI--VTE-FMSSrc -EPIYI--VTE-YMSFgr -EPIYI--VTE-FMCFyn -EPIYI--VTE-YMNHCK -EPIYI--ITE-FMABLK -EPIYI--VTE-YMALyn -EPIYI--ITE-YMABRK -DPVYI--ITE-LMASrm -EPVYI--VTE-LMRPDGFRb -GPIYI--ITE-YCRPDGFRa -GPIYI--ITE-YCFFRK -DPIYI--ITE-LMRTEC -KPIYI--VTE-FMETXK -KPLYI--VTE-FMEBTK -RPIFI--ITE-YMAEtkBMK -YPIYI--VTE-YISAbl -PPFYI--ITE-FMTArg -PPFYI--VTE-YMPKit -GPTLV--ITE-YCCFmsCSFR -GPVLV--ITE-YCCEphA1 -KPIMI--ITE-FMEEphB2 -TPVMI--ITE-FMEEphB3 -RPVMI--LTE-FMEEphB4 -MPVMI--LTE-FMEEphB1 -RPVMI--ITE-FMEEphA3 -KPVMI--VTE-YMEEphA5 -KPVMI--VTE-YMEEphA4 -KPVMI--ITE-YMEEphA2 -KPMMI--ITE-YMEEphA8 -RLAMI--VTE-YMEEphB6 -RPLMV--LTE-FMETESK1 -GQLHA--LTE-YMNTESK2 -GQLHA--LTE-YINDDR1 -DPLCM--ITD-YMEDDR2 -DPLCM--ITE-YMELIMK1 -KRLNF--ITE-YIKLIMK2 -KKLNL--LTE-YIETGFbetaR1 -TQLWL--VSD-YHEALK4 -TQLWL--VSD-YHEALK7 -TQLWL--VSE-YHEBMPR1A -TQLYL--ITD-YHEBMPR1B -TQLYL--ITD-YHEALK1 -TQLWL--ITH-YHEALK2 -TQLWL--ITH-YHEActR2 -VDLWL--ITA-FHEActR2B -VELWL--ITA-FHDTGFbetaR2 -KQYWL--ITA-FHASTLK6 -SWLWV--ISP-FMASTLK5 -NELWV--VTS-FMARIPK2 -EFLGI--VTE-YMPCSK -GGLYI--VTE-YMAHER2 --TVQL--VTQ-LMPEGFR --TVQL--ITQ-LMPHER4 --TIQL--VTQ-LMPHER3 --SLQL--VTQ-YLPILK -PHPTL--ITH-WMPAck -PPMKM--VTE-LAPBRAF --QLAI--VTQ-WCERAF1 --NLAI--VTQ-WCEARAF --GFAI--ITQ-WCEHH498 --QFAI--VTQ-YISKSR --HLAI--ITS-FCKANPa PPNICI--LTE-YCPANPb PPNICI--VTE-YCPCYGF SGMFAI--VTE-FCSCYGD EGNLAV--VSE-HCTSIK KDMLYI--VTE-FAKPKN3 SSHARF--VTE-FVPNek11 QDNFCI--ITE-YCEMLKL PPNYGI--VTE-YASZAK PPNYGI--VTE-YASMAP2K5 ENRISI--CTE-FMDRIPK3 -PKPAL--VTK-FMEMYT1 -GILYL--QTE--LC

94 Appendix A Supplementary material

010

2030

0

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= 8

Activation X(t)

Tim

e t (

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1020

30020406080

α =

80

β =

8

Activation X(t)

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e t (

min

utes

)0

1020

30050100

150

200

α =

1000

β

= 8

Activation X(t)

Tim

e t (

min

utes

)0

1020

30050100

150

200α

= 50

000

β =

8

Activation X(t)

Tim

e t (

min

utes

)

010

2030

0

051

α =

1 β

= 7

0

Activation X(t)

Tim

e t (

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utes

)0

1020

30051015

α =

80

β =

70

Activation X(t)

Tim

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min

utes

)0

1020

30050100α

= 10

00

β =

70

Activation X(t)

Tim

e t (

min

utes

)0

1020

30050100

150

200α

= 50

000

β =

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Activation X(t)

Tim

e t (

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RA

SR

AF

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RK

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10

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Figure A1 Scanning different parameter values

Erklarung

Ich erklare dass ich die vorgelegte Dissertation selbstandig angefertigt undkeine anderen als die angegebenen Quellen und Hilfsmittel benutzt habe

Weiterhin erklare ich dass die vorgelegte Arbeit noch in keinem anderenPrufungsverfahren in gleicher oder ahnlicher Form vorgelegen hat

Auszliger den in dem Zulassungsgesuch urkundlich vorgelegten Graden habeich keine weiteren Grade erworben oder zu erwerben versucht

Wurzburg 12 Dezember 2007

(Armin Robubi)

Title page

Dedication

Quotation

Abstract

Zusammenfassung

Introduction

History and nomenclature of RAF kinases

RAF kinase signaling

Mouse knockout models

RAF kinases in cancer

Architecture of Raf kinases

Kinase domain

Development of a novel RAF kinase inhibitor

Dynamic pathway modeling

DiRas3


Compound characterization

Cell culture

Conditions for inhibitor studies

Conditions used for modeling studies

Immuno blot analysis

Kinase assay (immuno blot)

Kinase assay (ELISA)

Kinase assay (DiRas3)

Biosensor measurements

Mass spectrometry measurements

Gel filtration

Bioinformatics

Molecular modeling


Results


Homology modeling

Activity of compound 1 in vitro

Activation in cell culture

Other compounds

Dynamic modeling

DiRas3

DiRas3 interacts in vitro efficiently with active C-RAF and MEK

Inhibition of MEK activity by DiRas3 in vitro

Discussion

Developing a novel RAF kinase inhibitor

Dynamic modeling

DiRas3

Bibliography

Acknowledgments

Curriculum vitae


Poster Abstracts

Oral presentations


Erklaumlrung

Eingereicht am

Mitglieder der Promotionskommission

Vorsitzender Prof Dr Martin J MullerGutachter Prof Dr Thomas DandekarGutachter Prof Dr Ulf R Rapp

Tag des Promotionskolloquiums

Doktorurkunde ausgehandigt am




December 12 2007



Mark Twain

Abstract




VI Abstract



Zusammenfassung








Contents

Abstract V

Zusammenfassung VII

1 Introduction 19









18 DiRas3 30



22 Cell culture 33













X Contents







Bibliography 71

Acknowledgments 85

Curriculum vitae 87



Erklarung 95

List of Figures









XII List of Figures



List of Tables






XVI List of Tables


List of Tables XVII




Chapter 1

Introduction












1983 2007

Time



2006RAF germline






2000BAY 43-9006 [5]

2005BAY 43-9006







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151 Kinase domain




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18 DiRas3



Signal Response





18 DiRas3 31



Chapter 2




22 Cell culture


























29 Gel filtration


210 Bioinformatics







Chapter 3

Results


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314 Other compounds
















32 Dynamic modeling



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Conc 1 7 10 BAY ZM GW 1 7 10

3 nM 97 100 119 140 107 88 111 104 8610 nM 95 105 117 137 99 83 100 96 9333 nM 79 91 99 81 74 58 108 90 91

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100 microM 6 19 14 4 3 2 25 48 23


dXi

dt= αiXiminus1

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33 DiRas3



33 DiRas3 59







33 DiRas3 61



Chapter 4

Discussion










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42 Dynamic modeling












43 DiRas3


43 DiRas3 69



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Bibliography



101038ng1641 22




S0014-5793(00)02037-8 67



MCB23144778-47872003 23

72 Bibliography


org101038sjonc1202174 24






1471-4159200402530x 28 45



Bibliography 73



science1326789 22





1038383178a0 45





74 Bibliography



S1359-6349(05)81088-2 21





org101038sjonc1202603 28 45 64 65



S1097-2765(02)00528-2 29 49 52 55 66


Bibliography 75



200411105 24 64 65 66


1159000048259 30




101038372746a0 37 39




76 Bibliography









81636 29 30




1471-2407-4-24 66



Bibliography 77


1038358417a0 22




1381612023393125 20 21




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78 Bibliography


abstractuid=PII0092867495904018 29 30




org101093emboj2081952 23




28654431374 22


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Bibliography 79




cbic200400280 67




dxdoiorg101016S0960-9822(02)00548-1 23






M203010200 33

80 Bibliography






org1011581078-0432CCR-04-0698 30


org101128MCB2662262-22722006 45 46 64


Bibliography 81












82 Bibliography



S0092-8674(04)00215-6 25 27 39 40 45





11580008-5472CAN-04-1443 21 64 65



Bibliography 83


0014-5793(96)00363-8 29 30


dxdoiorg101038ng0797-293 23








S0076-6879(05)07037-0 30 69


0008-5472CAN-05-0115 22

84 Bibliography



onc1204614 33


S0969-2126(99)80086-0 37 39

Acknowledgments





Curriculum vitae


Personal data


88 Curriculum vitae

Education
















Poster Abstracts









Oral presentations




(Armin Robubi)

Appendix A






supplmodelpdb


scriptstxt

script

__MACOSX_scriptstxt



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utes

)

010

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α =

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= 7

0

Activation X(t)

Tim

e t (

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utes

)0

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30051015

α =

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β =

70

Activation X(t)

Tim

e t (

min

utes

)0

1020

30050100α

= 10

00

β =

70

Activation X(t)

Tim

e t (

min

utes

)0

1020

30050100

150

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= 50

000

β =

70

Activation X(t)

Tim

e t (

min

utes

)

RA

SR

AF

ME

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α =

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Tim

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Erklarung





(Armin Robubi)

Title page

Dedication

Quotation

Abstract

Zusammenfassung

Introduction






Kinase domain



DiRas3



Cell culture









Gel filtration

Bioinformatics

Molecular modeling


Results


Homology modeling



Other compounds

Dynamic modeling

DiRas3



Discussion


Dynamic modeling

DiRas3

Bibliography

Acknowledgments

Curriculum vitae


Poster Abstracts

Oral presentations


Erklaumlrung




December 12 2007



Mark Twain

Abstract




VI Abstract



Zusammenfassung








Contents

Abstract V

Zusammenfassung VII

1 Introduction 19









18 DiRas3 30



22 Cell culture 33













X Contents







Bibliography 71

Acknowledgments 85

Curriculum vitae 87



Erklarung 95

List of Figures









XII List of Figures



List of Tables






XVI List of Tables


List of Tables XVII




Chapter 1

Introduction












1983 2007

Time



2006RAF germline






2000BAY 43-9006 [5]

2005BAY 43-9006







braf


craf1






















Grb2

RTK

Sos

RasGDP

SHC

RasGTPGAPs

RAFRAF

Plasma membrane

Sig

na

l

MEK

ERK

MEK

ERK

PP

PP










151 Kinase domain




$

amp

(

)+-

0120134567863097+-

0+014+-lt13764=6713







hibitor



O

OH

NH

HN

N

CH3

H3C

CH3

O

S

F

O

NHN

N

H3C

I

Br

Br

O

HO

NH

H

ZM 336372 SB 203580 GW 5074









18 DiRas3



Signal Response





18 DiRas3 31



Chapter 2




22 Cell culture


























29 Gel filtration


210 Bioinformatics







Chapter 3

Results


hibitor





HN

HN

OCl

CF3

O

BAY 43-9006

N

HN

CH3

O

HN

HN

OCl

CF3

NH

O

O

1









COOHHO

NH2

HOCOOH

Br

COOK

O

COOH

O

NH2

NH

O

O

HN

HN

OCl

CF3

NH

O

O

1

2 3 4

5 6

a b

c d

e

4

6















($)

+++- ++ ++- + +- - + - +++

+

0+

+

1+

++

+230-45++24678

230-45++94678

9amplt=gt24678

9amplt=gt94678

$amp(amp)+

-$amp))amp0




amp()+

+ + + -+ + + ++

+

+

+

0+

++

+

+

+

++)12

-3)12

+)12

-3)12

)12

+3-)12

+3)12

+3+-)12

+3+)12

+3++-)12

$amp()$+-$

amp$$01












314 Other compounds
















32 Dynamic modeling



NH2

NH

O

HN

HN

OCl

CF3

NH

O

7

9 8

a

b8

NH2

H2N

NH2

HO

NHBoc

HO

NH2

O

O

HN

O

O

HN

OCl

CF3

NHBoc

O

O

11

10

12

13 14

13c d

e

f14



Conc 1 7 10 BAY ZM GW 1 7 10

3 nM 97 100 119 140 107 88 111 104 8610 nM 95 105 117 137 99 83 100 96 9333 nM 79 91 99 81 74 58 108 90 91

100 nM 81 96 104 4 51 44 87 91 95333 nM 63 86 117 4 17 33 87 93 97


100 microM 6 19 14 4 3 2 25 48 23


dXi

dt= αiXiminus1

(1minus Xi

Ci

)minus βiXi (31)




Grb2

RTK

Sos

RasGDP

SHC

RasGTPGAPs

RAFRAF

Plasma membrane

1

1

Sig

na

l

MEK

ERK

MEK

ERK

PP

2

PP3

2

3





0 10 20 30 40 50 60 70 80 90 100minus02

0

02

04

06

08

1


Inte

nsity

X(t)

Time t

RASRAFMEKERK




0 5 10 15 20 25 300

20

40

60

80

100

120

140

160BminusRAF

Activ

atio

n X(

t)

Time t (minutes)

RASRAFMEKERK

A

0 5 10 15 20 25 300

05

1

15CminusRAF

Activ

atio

n X(

t)Time t (minutes)

RASRAFMEKERK

B

0 05 1 15 20

50

100

150

BminusRAF

Activ

atio

n X(

t)

Time t (minutes)

RASRAFMEKERK

C

0 02 04 06 08 1 12 14 16 18 20

02

04

06

08

1

12

14

CminusRAF

Activ

atio

n X(

t)

Time t (minutes)

RASRAFMEKERK

D







$amp

$(

)

+

-+

(

0+1

2(

3$

45657$845-57$8



His

-B-R

afH

is-C

-Raf

-wt

GS

T-C

-Raf

-RL

GS

T-C

-Raf

-375

WG

ST-

C-R

af-3

403

41D

DG

ST-

C-R

af-w

t

P-ERK













33 DiRas3



33 DiRas3 59







33 DiRas3 61



Chapter 4

Discussion










Grb2

RTK

Sos

RasGDP

SHC

RasGTPGAPs

RAFRAF

Plasma membrane

Sig

na

l

MEK

ERK

MEK

ERK

PP

PP

Inhibitor

X

Grb2

RTK

Sos

RasGDP

SHC

RasGTPGAPs

RAFRAF

Plasma membrane

Sig

na

l

MEK

ERK

MEK

ERK

PP

PP

B

A





42 Dynamic modeling












43 DiRas3


43 DiRas3 69



Grb2

RTK

Sos

RasGDP

SHC

RasGTPGAPs

RAFRAF

Plasma membrane

Sig

na

l

MEK

ERK

MEK

ERK

PP

PP

DiRas3






Bibliography



101038ng1641 22




S0014-5793(00)02037-8 67



MCB23144778-47872003 23

72 Bibliography


org101038sjonc1202174 24






1471-4159200402530x 28 45



Bibliography 73



science1326789 22





1038383178a0 45





74 Bibliography



S1359-6349(05)81088-2 21





org101038sjonc1202603 28 45 64 65



S1097-2765(02)00528-2 29 49 52 55 66


Bibliography 75



200411105 24 64 65 66


1159000048259 30




101038372746a0 37 39




76 Bibliography









81636 29 30




1471-2407-4-24 66



Bibliography 77


1038358417a0 22




1381612023393125 20 21




dxdoiorg101038383181a0 45




78 Bibliography


abstractuid=PII0092867495904018 29 30




org101093emboj2081952 23




28654431374 22


1038nsb770 37 39 45


Bibliography 79




cbic200400280 67




dxdoiorg101016S0960-9822(02)00548-1 23






M203010200 33

80 Bibliography






org1011581078-0432CCR-04-0698 30


org101128MCB2662262-22722006 45 46 64


Bibliography 81












82 Bibliography



S0092-8674(04)00215-6 25 27 39 40 45





11580008-5472CAN-04-1443 21 64 65



Bibliography 83


0014-5793(96)00363-8 29 30


dxdoiorg101038ng0797-293 23








S0076-6879(05)07037-0 30 69


0008-5472CAN-05-0115 22

84 Bibliography



onc1204614 33


S0969-2126(99)80086-0 37 39

Acknowledgments





Curriculum vitae


Personal data


88 Curriculum vitae

Education
















Poster Abstracts









Oral presentations




(Armin Robubi)

Appendix A






supplmodelpdb


scriptstxt

script

__MACOSX_scriptstxt



010

2030

0

051

15

α =

1 β

= 8

Activation X(t)

Tim

e t (

min

utes

)0

1020

30020406080

α =

80

β =

8

Activation X(t)

Tim

e t (

min

utes

)0

1020

30050100

150

200

α =

1000

β

= 8

Activation X(t)

Tim

e t (

min

utes

)0

1020

30050100

150

200α

= 50

000

β =

8

Activation X(t)

Tim

e t (

min

utes

)

010

2030

0

051

α =

1 β

= 7

0

Activation X(t)

Tim

e t (

min

utes

)0

1020

30051015

α =

80

β =

70

Activation X(t)

Tim

e t (

min

utes

)0

1020

30050100α

= 10

00

β =

70

Activation X(t)

Tim

e t (

min

utes

)0

1020

30050100

150

200α

= 50

000

β =

70

Activation X(t)

Tim

e t (

min

utes

)

RA

SR

AF

ME

KE

RK

010

2030

0

051

152

α =

10

β =

80

Activation X(t)

Tim

e t (

min

utes

)0

1020

300

02

04

06

081

α =

10

β =

800

Activation X(t)

Tim

e t (

min

utes

)


Erklarung





(Armin Robubi)

Title page

Dedication

Quotation

Abstract

Zusammenfassung

Introduction






Kinase domain



DiRas3



Cell culture









Gel filtration

Bioinformatics

Molecular modeling


Results


Homology modeling



Other compounds

Dynamic modeling

DiRas3



Discussion


Dynamic modeling

DiRas3

Bibliography

Acknowledgments

Curriculum vitae


Poster Abstracts

Oral presentations


Erklaumlrung



Mark Twain

Abstract




VI Abstract



Zusammenfassung








Contents

Abstract V

Zusammenfassung VII

1 Introduction 19









18 DiRas3 30



22 Cell culture 33













X Contents







Bibliography 71

Acknowledgments 85

Curriculum vitae 87



Erklarung 95

List of Figures









XII List of Figures



List of Tables






XVI List of Tables


List of Tables XVII




Chapter 1

Introduction












1983 2007

Time



2006RAF germline






2000BAY 43-9006 [5]

2005BAY 43-9006







braf


craf1






















Grb2

RTK

Sos

RasGDP

SHC

RasGTPGAPs

RAFRAF

Plasma membrane

Sig

na

l

MEK

ERK

MEK

ERK

PP

PP










151 Kinase domain




$

amp

(

)+-

0120134567863097+-

0+014+-lt13764=6713







hibitor



O

OH

NH

HN

N

CH3

H3C

CH3

O

S

F

O

NHN

N

H3C

I

Br

Br

O

HO

NH

H

ZM 336372 SB 203580 GW 5074









18 DiRas3



Signal Response





18 DiRas3 31



Chapter 2




22 Cell culture


























29 Gel filtration


210 Bioinformatics







Chapter 3

Results


hibitor





HN

HN

OCl

CF3

O

BAY 43-9006

N

HN

CH3

O

HN

HN

OCl

CF3

NH

O

O

1









COOHHO

NH2

HOCOOH

Br

COOK

O

COOH

O

NH2

NH

O

O

HN

HN

OCl

CF3

NH

O

O

1

2 3 4

5 6

a b

c d

e

4

6















($)

+++- ++ ++- + +- - + - +++

+

0+

+

1+

++

+230-45++24678

230-45++94678

9amplt=gt24678

9amplt=gt94678

$amp(amp)+

-$amp))amp0




amp()+

+ + + -+ + + ++

+

+

+

0+

++

+

+

+

++)12

-3)12

+)12

-3)12

)12

+3-)12

+3)12

+3+-)12

+3+)12

+3++-)12

$amp()$+-$

amp$$01












314 Other compounds
















32 Dynamic modeling



NH2

NH

O

HN

HN

OCl

CF3

NH

O

7

9 8

a

b8

NH2

H2N

NH2

HO

NHBoc

HO

NH2

O

O

HN

O

O

HN

OCl

CF3

NHBoc

O

O

11

10

12

13 14

13c d

e

f14



Conc 1 7 10 BAY ZM GW 1 7 10

3 nM 97 100 119 140 107 88 111 104 8610 nM 95 105 117 137 99 83 100 96 9333 nM 79 91 99 81 74 58 108 90 91

100 nM 81 96 104 4 51 44 87 91 95333 nM 63 86 117 4 17 33 87 93 97


100 microM 6 19 14 4 3 2 25 48 23


dXi

dt= αiXiminus1

(1minus Xi

Ci

)minus βiXi (31)




Grb2

RTK

Sos

RasGDP

SHC

RasGTPGAPs

RAFRAF

Plasma membrane

1

1

Sig

na

l

MEK

ERK

MEK

ERK

PP

2

PP3

2

3





0 10 20 30 40 50 60 70 80 90 100minus02

0

02

04

06

08

1


Inte

nsity

X(t)

Time t

RASRAFMEKERK




0 5 10 15 20 25 300

20

40

60

80

100

120

140

160BminusRAF

Activ

atio

n X(

t)

Time t (minutes)

RASRAFMEKERK

A

0 5 10 15 20 25 300

05

1

15CminusRAF

Activ

atio

n X(

t)Time t (minutes)

RASRAFMEKERK

B

0 05 1 15 20

50

100

150

BminusRAF

Activ

atio

n X(

t)

Time t (minutes)

RASRAFMEKERK

C

0 02 04 06 08 1 12 14 16 18 20

02

04

06

08

1

12

14

CminusRAF

Activ

atio

n X(

t)

Time t (minutes)

RASRAFMEKERK

D







$amp

$(

)

+

-+

(

0+1

2(

3$

45657$845-57$8



His

-B-R

afH

is-C

-Raf

-wt

GS

T-C

-Raf

-RL

GS

T-C

-Raf

-375

WG

ST-

C-R

af-3

403

41D

DG

ST-

C-R

af-w

t

P-ERK













33 DiRas3



33 DiRas3 59







33 DiRas3 61



Chapter 4

Discussion










Grb2

RTK

Sos

RasGDP

SHC

RasGTPGAPs

RAFRAF

Plasma membrane

Sig

na

l

MEK

ERK

MEK

ERK

PP

PP

Inhibitor

X

Grb2

RTK

Sos

RasGDP

SHC

RasGTPGAPs

RAFRAF

Plasma membrane

Sig

na

l

MEK

ERK

MEK

ERK

PP

PP

B

A





42 Dynamic modeling












43 DiRas3


43 DiRas3 69



Grb2

RTK

Sos

RasGDP

SHC

RasGTPGAPs

RAFRAF

Plasma membrane

Sig

na

l

MEK

ERK

MEK

ERK

PP

PP

DiRas3






Bibliography



101038ng1641 22




S0014-5793(00)02037-8 67



MCB23144778-47872003 23

72 Bibliography


org101038sjonc1202174 24






1471-4159200402530x 28 45



Bibliography 73



science1326789 22





1038383178a0 45





74 Bibliography



S1359-6349(05)81088-2 21





org101038sjonc1202603 28 45 64 65



S1097-2765(02)00528-2 29 49 52 55 66


Bibliography 75



200411105 24 64 65 66


1159000048259 30




101038372746a0 37 39




76 Bibliography









81636 29 30




1471-2407-4-24 66



Bibliography 77


1038358417a0 22




1381612023393125 20 21




dxdoiorg101038383181a0 45




78 Bibliography


abstractuid=PII0092867495904018 29 30




org101093emboj2081952 23




28654431374 22


1038nsb770 37 39 45


Bibliography 79




cbic200400280 67




dxdoiorg101016S0960-9822(02)00548-1 23






M203010200 33

80 Bibliography






org1011581078-0432CCR-04-0698 30


org101128MCB2662262-22722006 45 46 64


Bibliography 81












82 Bibliography



S0092-8674(04)00215-6 25 27 39 40 45





11580008-5472CAN-04-1443 21 64 65



Bibliography 83


0014-5793(96)00363-8 29 30


dxdoiorg101038ng0797-293 23








S0076-6879(05)07037-0 30 69


0008-5472CAN-05-0115 22

84 Bibliography



onc1204614 33


S0969-2126(99)80086-0 37 39

Acknowledgments





Curriculum vitae


Personal data


88 Curriculum vitae

Education
















Poster Abstracts









Oral presentations




(Armin Robubi)

Appendix A






supplmodelpdb


scriptstxt

script

__MACOSX_scriptstxt



010

2030

0

051

15

α =

1 β

= 8

Activation X(t)

Tim

e t (

min

utes

)0

1020

30020406080

α =

80

β =

8

Activation X(t)

Tim

e t (

min

utes

)0

1020

30050100

150

200

α =

1000

β

= 8

Activation X(t)

Tim

e t (

min

utes

)0

1020

30050100

150

200α

= 50

000

β =

8

Activation X(t)

Tim

e t (

min

utes

)

010

2030

0

051

α =

1 β

= 7

0

Activation X(t)

Tim

e t (

min

utes

)0

1020

30051015

α =

80

β =

70

Activation X(t)

Tim

e t (

min

utes

)0

1020

30050100α

= 10

00

β =

70

Activation X(t)

Tim

e t (

min

utes

)0

1020

30050100

150

200α

= 50

000

β =

70

Activation X(t)

Tim

e t (

min

utes

)

RA

SR

AF

ME

KE

RK

010

2030

0

051

152

α =

10

β =

80

Activation X(t)

Tim

e t (

min

utes

)0

1020

300

02

04

06

081

α =

10

β =

800

Activation X(t)

Tim

e t (

min

utes

)


Erklarung





(Armin Robubi)

Title page

Dedication

Quotation

Abstract

Zusammenfassung

Introduction






Kinase domain



DiRas3



Cell culture









Gel filtration

Bioinformatics

Molecular modeling


Results


Homology modeling



Other compounds

Dynamic modeling

DiRas3



Discussion


Dynamic modeling

DiRas3

Bibliography

Acknowledgments

Curriculum vitae


Poster Abstracts

Oral presentations


Erklaumlrung

Abstract




VI Abstract



Zusammenfassung








Contents

Abstract V

Zusammenfassung VII

1 Introduction 19









18 DiRas3 30



22 Cell culture 33













X Contents







Bibliography 71

Acknowledgments 85

Curriculum vitae 87



Erklarung 95

List of Figures









XII List of Figures



List of Tables






XVI List of Tables


List of Tables XVII




Chapter 1

Introduction












1983 2007

Time



2006RAF germline






2000BAY 43-9006 [5]

2005BAY 43-9006







braf


craf1






















Grb2

RTK

Sos

RasGDP

SHC

RasGTPGAPs

RAFRAF

Plasma membrane

Sig

na

l

MEK

ERK

MEK

ERK

PP

PP










151 Kinase domain




$

amp

(

)+-

0120134567863097+-

0+014+-lt13764=6713







hibitor



O

OH

NH

HN

N

CH3

H3C

CH3

O

S

F

O

NHN

N

H3C

I

Br

Br

O

HO

NH

H

ZM 336372 SB 203580 GW 5074









18 DiRas3



Signal Response





18 DiRas3 31



Chapter 2




22 Cell culture


























29 Gel filtration


210 Bioinformatics







Chapter 3

Results


hibitor





HN

HN

OCl

CF3

O

BAY 43-9006

N

HN

CH3

O

HN

HN

OCl

CF3

NH

O

O

1









COOHHO

NH2

HOCOOH

Br

COOK

O

COOH

O

NH2

NH

O

O

HN

HN

OCl

CF3

NH

O

O

1

2 3 4

5 6

a b

c d

e

4

6















($)

+++- ++ ++- + +- - + - +++

+

0+

+

1+

++

+230-45++24678

230-45++94678

9amplt=gt24678

9amplt=gt94678

$amp(amp)+

-$amp))amp0




amp()+

+ + + -+ + + ++

+

+

+

0+

++

+

+

+

++)12

-3)12

+)12

-3)12

)12

+3-)12

+3)12

+3+-)12

+3+)12

+3++-)12

$amp()$+-$

amp$$01












314 Other compounds
















32 Dynamic modeling



NH2

NH

O

HN

HN

OCl

CF3

NH

O

7

9 8

a

b8

NH2

H2N

NH2

HO

NHBoc

HO

NH2

O

O

HN

O

O

HN

OCl

CF3

NHBoc

O

O

11

10

12

13 14

13c d

e

f14



Conc 1 7 10 BAY ZM GW 1 7 10

3 nM 97 100 119 140 107 88 111 104 8610 nM 95 105 117 137 99 83 100 96 9333 nM 79 91 99 81 74 58 108 90 91

100 nM 81 96 104 4 51 44 87 91 95333 nM 63 86 117 4 17 33 87 93 97


100 microM 6 19 14 4 3 2 25 48 23


dXi

dt= αiXiminus1

(1minus Xi

Ci

)minus βiXi (31)




Grb2

RTK

Sos

RasGDP

SHC

RasGTPGAPs

RAFRAF

Plasma membrane

1

1

Sig

na

l

MEK

ERK

MEK

ERK

PP

2

PP3

2

3





0 10 20 30 40 50 60 70 80 90 100minus02

0

02

04

06

08

1


Inte

nsity

X(t)

Time t

RASRAFMEKERK




0 5 10 15 20 25 300

20

40

60

80

100

120

140

160BminusRAF

Activ

atio

n X(

t)

Time t (minutes)

RASRAFMEKERK

A

0 5 10 15 20 25 300

05

1

15CminusRAF

Activ

atio

n X(

t)Time t (minutes)

RASRAFMEKERK

B

0 05 1 15 20

50

100

150

BminusRAF

Activ

atio

n X(

t)

Time t (minutes)

RASRAFMEKERK

C

0 02 04 06 08 1 12 14 16 18 20

02

04

06

08

1

12

14

CminusRAF

Activ

atio

n X(

t)

Time t (minutes)

RASRAFMEKERK

D







$amp

$(

)

+

-+

(

0+1

2(

3$

45657$845-57$8



His

-B-R

afH

is-C

-Raf

-wt

GS

T-C

-Raf

-RL

GS

T-C

-Raf

-375

WG

ST-

C-R

af-3

403

41D

DG

ST-

C-R

af-w

t

P-ERK













33 DiRas3



33 DiRas3 59







33 DiRas3 61



Chapter 4

Discussion










Grb2

RTK

Sos

RasGDP

SHC

RasGTPGAPs

RAFRAF

Plasma membrane

Sig

na

l

MEK

ERK

MEK

ERK

PP

PP

Inhibitor

X

Grb2

RTK

Sos

RasGDP

SHC

RasGTPGAPs

RAFRAF

Plasma membrane

Sig

na

l

MEK

ERK

MEK

ERK

PP

PP

B

A





42 Dynamic modeling












43 DiRas3


43 DiRas3 69



Grb2

RTK

Sos

RasGDP

SHC

RasGTPGAPs

RAFRAF

Plasma membrane

Sig

na

l

MEK

ERK

MEK

ERK

PP

PP

DiRas3






Bibliography



101038ng1641 22




S0014-5793(00)02037-8 67



MCB23144778-47872003 23

72 Bibliography


org101038sjonc1202174 24






1471-4159200402530x 28 45



Bibliography 73



science1326789 22





1038383178a0 45





74 Bibliography



S1359-6349(05)81088-2 21





org101038sjonc1202603 28 45 64 65



S1097-2765(02)00528-2 29 49 52 55 66


Bibliography 75



200411105 24 64 65 66


1159000048259 30




101038372746a0 37 39




76 Bibliography









81636 29 30




1471-2407-4-24 66



Bibliography 77


1038358417a0 22




1381612023393125 20 21




dxdoiorg101038383181a0 45




78 Bibliography


abstractuid=PII0092867495904018 29 30




org101093emboj2081952 23




28654431374 22


1038nsb770 37 39 45


Bibliography 79




cbic200400280 67




dxdoiorg101016S0960-9822(02)00548-1 23






M203010200 33

80 Bibliography






org1011581078-0432CCR-04-0698 30


org101128MCB2662262-22722006 45 46 64


Bibliography 81












82 Bibliography



S0092-8674(04)00215-6 25 27 39 40 45





11580008-5472CAN-04-1443 21 64 65



Bibliography 83


0014-5793(96)00363-8 29 30


dxdoiorg101038ng0797-293 23








S0076-6879(05)07037-0 30 69


0008-5472CAN-05-0115 22

84 Bibliography



onc1204614 33


S0969-2126(99)80086-0 37 39

Acknowledgments





Curriculum vitae


Personal data


88 Curriculum vitae

Education
















Poster Abstracts









Oral presentations




(Armin Robubi)

Appendix A






supplmodelpdb


scriptstxt

script

__MACOSX_scriptstxt



010

2030

0

051

15

α =

1 β

= 8

Activation X(t)

Tim

e t (

min

utes

)0

1020

30020406080

α =

80

β =

8

Activation X(t)

Tim

e t (

min

utes

)0

1020

30050100

150

200

α =

1000

β

= 8

Activation X(t)

Tim

e t (

min

utes

)0

1020

30050100

150

200α

= 50

000

β =

8

Activation X(t)

Tim

e t (

min

utes

)

010

2030

0

051

α =

1 β

= 7

0

Activation X(t)

Tim

e t (

min

utes

)0

1020

30051015

α =

80

β =

70

Activation X(t)

Tim

e t (

min

utes

)0

1020

30050100α

= 10

00

β =

70

Activation X(t)

Tim

e t (

min

utes

)0

1020

30050100

150

200α

= 50

000

β =

70

Activation X(t)

Tim

e t (

min

utes

)

RA

SR

AF

ME

KE

RK

010

2030

0

051

152

α =

10

β =

80

Activation X(t)

Tim

e t (

min

utes

)0

1020

300

02

04

06

081

α =

10

β =

800

Activation X(t)

Tim

e t (

min

utes

)


Erklarung





(Armin Robubi)

Title page

Dedication

Quotation

Abstract

Zusammenfassung

Introduction






Kinase domain



DiRas3



Cell culture









Gel filtration

Bioinformatics

Molecular modeling


Results


Homology modeling



Other compounds

Dynamic modeling

DiRas3



Discussion


Dynamic modeling

DiRas3

Bibliography

Acknowledgments

Curriculum vitae


Poster Abstracts

Oral presentations


Erklaumlrung

VI Abstract



Zusammenfassung








Contents

Abstract V

Zusammenfassung VII

1 Introduction 19









18 DiRas3 30



22 Cell culture 33













X Contents







Bibliography 71

Acknowledgments 85

Curriculum vitae 87



Erklarung 95

List of Figures









XII List of Figures



List of Tables






XVI List of Tables


List of Tables XVII




Chapter 1

Introduction












1983 2007

Time



2006RAF germline






2000BAY 43-9006 [5]

2005BAY 43-9006







braf


craf1






















Grb2

RTK

Sos

RasGDP

SHC

RasGTPGAPs

RAFRAF

Plasma membrane

Sig

na

l

MEK

ERK

MEK

ERK

PP

PP










151 Kinase domain




$

amp

(

)+-

0120134567863097+-

0+014+-lt13764=6713







hibitor



O

OH

NH

HN

N

CH3

H3C

CH3

O

S

F

O

NHN

N

H3C

I

Br

Br

O

HO

NH

H

ZM 336372 SB 203580 GW 5074









18 DiRas3



Signal Response





18 DiRas3 31



Chapter 2




22 Cell culture


























29 Gel filtration


210 Bioinformatics







Chapter 3

Results


hibitor





HN

HN

OCl

CF3

O

BAY 43-9006

N

HN

CH3

O

HN

HN

OCl

CF3

NH

O

O

1









COOHHO

NH2

HOCOOH

Br

COOK

O

COOH

O

NH2

NH

O

O

HN

HN

OCl

CF3

NH

O

O

1

2 3 4

5 6

a b

c d

e

4

6















($)

+++- ++ ++- + +- - + - +++

+

0+

+

1+

++

+230-45++24678

230-45++94678

9amplt=gt24678

9amplt=gt94678

$amp(amp)+

-$amp))amp0




amp()+

+ + + -+ + + ++

+

+

+

0+

++

+

+

+

++)12

-3)12

+)12

-3)12

)12

+3-)12

+3)12

+3+-)12

+3+)12

+3++-)12

$amp()$+-$

amp$$01












314 Other compounds
















32 Dynamic modeling



NH2

NH

O

HN

HN

OCl

CF3

NH

O

7

9 8

a

b8

NH2

H2N

NH2

HO

NHBoc

HO

NH2

O

O

HN

O

O

HN

OCl

CF3

NHBoc

O

O

11

10

12

13 14

13c d

e

f14



Conc 1 7 10 BAY ZM GW 1 7 10

3 nM 97 100 119 140 107 88 111 104 8610 nM 95 105 117 137 99 83 100 96 9333 nM 79 91 99 81 74 58 108 90 91

100 nM 81 96 104 4 51 44 87 91 95333 nM 63 86 117 4 17 33 87 93 97


100 microM 6 19 14 4 3 2 25 48 23


dXi

dt= αiXiminus1

(1minus Xi

Ci

)minus βiXi (31)




Grb2

RTK

Sos

RasGDP

SHC

RasGTPGAPs

RAFRAF

Plasma membrane

1

1

Sig

na

l

MEK

ERK

MEK

ERK

PP

2

PP3

2

3





0 10 20 30 40 50 60 70 80 90 100minus02

0

02

04

06

08

1


Inte

nsity

X(t)

Time t

RASRAFMEKERK




0 5 10 15 20 25 300

20

40

60

80

100

120

140

160BminusRAF

Activ

atio

n X(

t)

Time t (minutes)

RASRAFMEKERK

A

0 5 10 15 20 25 300

05

1

15CminusRAF

Activ

atio

n X(

t)Time t (minutes)

RASRAFMEKERK

B

0 05 1 15 20

50

100

150

BminusRAF

Activ

atio

n X(

t)

Time t (minutes)

RASRAFMEKERK

C

0 02 04 06 08 1 12 14 16 18 20

02

04

06

08

1

12

14

CminusRAF

Activ

atio

n X(

t)

Time t (minutes)

RASRAFMEKERK

D







$amp

$(

)

+

-+

(

0+1

2(

3$

45657$845-57$8



His

-B-R

afH

is-C

-Raf

-wt

GS

T-C

-Raf

-RL

GS

T-C

-Raf

-375

WG

ST-

C-R

af-3

403

41D

DG

ST-

C-R

af-w

t

P-ERK













33 DiRas3



33 DiRas3 59







33 DiRas3 61



Chapter 4

Discussion










Grb2

RTK

Sos

RasGDP

SHC

RasGTPGAPs

RAFRAF

Plasma membrane

Sig

na

l

MEK

ERK

MEK

ERK

PP

PP

Inhibitor

X

Grb2

RTK

Sos

RasGDP

SHC

RasGTPGAPs

RAFRAF

Plasma membrane

Sig

na

l

MEK

ERK

MEK

ERK

PP

PP

B

A





42 Dynamic modeling












43 DiRas3


43 DiRas3 69



Grb2

RTK

Sos

RasGDP

SHC

RasGTPGAPs

RAFRAF

Plasma membrane

Sig

na

l

MEK

ERK

MEK

ERK

PP

PP

DiRas3






Bibliography



101038ng1641 22




S0014-5793(00)02037-8 67



MCB23144778-47872003 23

72 Bibliography


org101038sjonc1202174 24






1471-4159200402530x 28 45



Bibliography 73



science1326789 22





1038383178a0 45





74 Bibliography



S1359-6349(05)81088-2 21





org101038sjonc1202603 28 45 64 65



S1097-2765(02)00528-2 29 49 52 55 66


Bibliography 75



200411105 24 64 65 66


1159000048259 30




101038372746a0 37 39




76 Bibliography









81636 29 30




1471-2407-4-24 66



Bibliography 77


1038358417a0 22




1381612023393125 20 21




dxdoiorg101038383181a0 45




78 Bibliography


abstractuid=PII0092867495904018 29 30




org101093emboj2081952 23




28654431374 22


1038nsb770 37 39 45


Bibliography 79




cbic200400280 67




dxdoiorg101016S0960-9822(02)00548-1 23






M203010200 33

80 Bibliography






org1011581078-0432CCR-04-0698 30


org101128MCB2662262-22722006 45 46 64


Bibliography 81












82 Bibliography



S0092-8674(04)00215-6 25 27 39 40 45





11580008-5472CAN-04-1443 21 64 65



Bibliography 83


0014-5793(96)00363-8 29 30


dxdoiorg101038ng0797-293 23








S0076-6879(05)07037-0 30 69


0008-5472CAN-05-0115 22

84 Bibliography



onc1204614 33


S0969-2126(99)80086-0 37 39

Acknowledgments





Curriculum vitae


Personal data


88 Curriculum vitae

Education
















Poster Abstracts









Oral presentations




(Armin Robubi)

Appendix A






supplmodelpdb


scriptstxt

script

__MACOSX_scriptstxt



010

2030

0

051

15

α =

1 β

= 8

Activation X(t)

Tim

e t (

min

utes

)0

1020

30020406080

α =

80

β =

8

Activation X(t)

Tim

e t (

min

utes

)0

1020

30050100

150

200

α =

1000

β

= 8

Activation X(t)

Tim

e t (

min

utes

)0

1020

30050100

150

200α

= 50

000

β =

8

Activation X(t)

Tim

e t (

min

utes

)

010

2030

0

051

α =

1 β

= 7

0

Activation X(t)

Tim

e t (

min

utes

)0

1020

30051015

α =

80

β =

70

Activation X(t)

Tim

e t (

min

utes

)0

1020

30050100α

= 10

00

β =

70

Activation X(t)

Tim

e t (

min

utes

)0

1020

30050100

150

200α

= 50

000

β =

70

Activation X(t)

Tim

e t (

min

utes

)

RA

SR

AF

ME

KE

RK

010

2030

0

051

152

α =

10

β =

80

Activation X(t)

Tim

e t (

min

utes

)0

1020

300

02

04

06

081

α =

10

β =

800

Activation X(t)

Tim

e t (

min

utes

)


Erklarung





(Armin Robubi)

Title page

Dedication

Quotation

Abstract

Zusammenfassung

Introduction






Kinase domain



DiRas3



Cell culture









Gel filtration

Bioinformatics

Molecular modeling


Results


Homology modeling



Other compounds

Dynamic modeling

DiRas3



Discussion


Dynamic modeling

DiRas3

Bibliography

Acknowledgments

Curriculum vitae


Poster Abstracts

Oral presentations


Erklaumlrung

Zusammenfassung








Contents

Abstract V

Zusammenfassung VII

1 Introduction 19









18 DiRas3 30



22 Cell culture 33













X Contents







Bibliography 71

Acknowledgments 85

Curriculum vitae 87



Erklarung 95

List of Figures









XII List of Figures



List of Tables






XVI List of Tables


List of Tables XVII




Chapter 1

Introduction












1983 2007

Time



2006RAF germline






2000BAY 43-9006 [5]

2005BAY 43-9006







braf


craf1






















Grb2

RTK

Sos

RasGDP

SHC

RasGTPGAPs

RAFRAF

Plasma membrane

Sig

na

l

MEK

ERK

MEK

ERK

PP

PP










151 Kinase domain




$

amp

(

)+-

0120134567863097+-

0+014+-lt13764=6713







hibitor



O

OH

NH

HN

N

CH3

H3C

CH3

O

S

F

O

NHN

N

H3C

I

Br

Br

O

HO

NH

H

ZM 336372 SB 203580 GW 5074









18 DiRas3



Signal Response





18 DiRas3 31



Chapter 2




22 Cell culture


























29 Gel filtration


210 Bioinformatics







Chapter 3

Results


hibitor





HN

HN

OCl

CF3

O

BAY 43-9006

N

HN

CH3

O

HN

HN

OCl

CF3

NH

O

O

1









COOHHO

NH2

HOCOOH

Br

COOK

O

COOH

O

NH2

NH

O

O

HN

HN

OCl

CF3

NH

O

O

1

2 3 4

5 6

a b

c d

e

4

6















($)

+++- ++ ++- + +- - + - +++

+

0+

+

1+

++

+230-45++24678

230-45++94678

9amplt=gt24678

9amplt=gt94678

$amp(amp)+

-$amp))amp0




amp()+

+ + + -+ + + ++

+

+

+

0+

++

+

+

+

++)12

-3)12

+)12

-3)12

)12

+3-)12

+3)12

+3+-)12

+3+)12

+3++-)12

$amp()$+-$

amp$$01












314 Other compounds
















32 Dynamic modeling



NH2

NH

O

HN

HN

OCl

CF3

NH

O

7

9 8

a

b8

NH2

H2N

NH2

HO

NHBoc

HO

NH2

O

O

HN

O

O

HN

OCl

CF3

NHBoc

O

O

11

10

12

13 14

13c d

e

f14



Conc 1 7 10 BAY ZM GW 1 7 10

3 nM 97 100 119 140 107 88 111 104 8610 nM 95 105 117 137 99 83 100 96 9333 nM 79 91 99 81 74 58 108 90 91

100 nM 81 96 104 4 51 44 87 91 95333 nM 63 86 117 4 17 33 87 93 97


100 microM 6 19 14 4 3 2 25 48 23


dXi

dt= αiXiminus1

(1minus Xi

Ci

)minus βiXi (31)




Grb2

RTK

Sos

RasGDP

SHC

RasGTPGAPs

RAFRAF

Plasma membrane

1

1

Sig

na

l

MEK

ERK

MEK

ERK

PP

2

PP3

2

3





0 10 20 30 40 50 60 70 80 90 100minus02

0

02

04

06

08

1


Inte

nsity

X(t)

Time t

RASRAFMEKERK




0 5 10 15 20 25 300

20

40

60

80

100

120

140

160BminusRAF

Activ

atio

n X(

t)

Time t (minutes)

RASRAFMEKERK

A

0 5 10 15 20 25 300

05

1

15CminusRAF

Activ

atio

n X(

t)Time t (minutes)

RASRAFMEKERK

B

0 05 1 15 20

50

100

150

BminusRAF

Activ

atio

n X(

t)

Time t (minutes)

RASRAFMEKERK

C

0 02 04 06 08 1 12 14 16 18 20

02

04

06

08

1

12

14

CminusRAF

Activ

atio

n X(

t)

Time t (minutes)

RASRAFMEKERK

D







$amp

$(

)

+

-+

(

0+1

2(

3$

45657$845-57$8



His

-B-R

afH

is-C

-Raf

-wt

GS

T-C

-Raf

-RL

GS

T-C

-Raf

-375

WG

ST-

C-R

af-3

403

41D

DG

ST-

C-R

af-w

t

P-ERK













33 DiRas3



33 DiRas3 59







33 DiRas3 61



Chapter 4

Discussion










Grb2

RTK

Sos

RasGDP

SHC

RasGTPGAPs

RAFRAF

Plasma membrane

Sig

na

l

MEK

ERK

MEK

ERK

PP

PP

Inhibitor

X

Grb2

RTK

Sos

RasGDP

SHC

RasGTPGAPs

RAFRAF

Plasma membrane

Sig

na

l

MEK

ERK

MEK

ERK

PP

PP

B

A





42 Dynamic modeling












43 DiRas3


43 DiRas3 69



Grb2

RTK

Sos

RasGDP

SHC

RasGTPGAPs

RAFRAF

Plasma membrane

Sig

na

l

MEK

ERK

MEK

ERK

PP

PP

DiRas3






Bibliography



101038ng1641 22




S0014-5793(00)02037-8 67



MCB23144778-47872003 23

72 Bibliography


org101038sjonc1202174 24






1471-4159200402530x 28 45



Bibliography 73



science1326789 22





1038383178a0 45





74 Bibliography



S1359-6349(05)81088-2 21





org101038sjonc1202603 28 45 64 65



S1097-2765(02)00528-2 29 49 52 55 66


Bibliography 75



200411105 24 64 65 66


1159000048259 30




101038372746a0 37 39




76 Bibliography









81636 29 30




1471-2407-4-24 66



Bibliography 77


1038358417a0 22




1381612023393125 20 21




dxdoiorg101038383181a0 45




78 Bibliography


abstractuid=PII0092867495904018 29 30




org101093emboj2081952 23




28654431374 22


1038nsb770 37 39 45


Bibliography 79




cbic200400280 67




dxdoiorg101016S0960-9822(02)00548-1 23






M203010200 33

80 Bibliography






org1011581078-0432CCR-04-0698 30


org101128MCB2662262-22722006 45 46 64


Bibliography 81












82 Bibliography



S0092-8674(04)00215-6 25 27 39 40 45





11580008-5472CAN-04-1443 21 64 65



Bibliography 83


0014-5793(96)00363-8 29 30


dxdoiorg101038ng0797-293 23








S0076-6879(05)07037-0 30 69


0008-5472CAN-05-0115 22

84 Bibliography



onc1204614 33


S0969-2126(99)80086-0 37 39

Acknowledgments





Curriculum vitae


Personal data


88 Curriculum vitae

Education
















Poster Abstracts









Oral presentations




(Armin Robubi)

Appendix A






supplmodelpdb


scriptstxt

script

__MACOSX_scriptstxt



010

2030

0

051

15

α =

1 β

= 8

Activation X(t)

Tim

e t (

min

utes

)0

1020

30020406080

α =

80

β =

8

Activation X(t)

Tim

e t (

min

utes

)0

1020

30050100

150

200

α =

1000

β

= 8

Activation X(t)

Tim

e t (

min

utes

)0

1020

30050100

150

200α

= 50

000

β =

8

Activation X(t)

Tim

e t (

min

utes

)

010

2030

0

051

α =

1 β

= 7

0

Activation X(t)

Tim

e t (

min

utes

)0

1020

30051015

α =

80

β =

70

Activation X(t)

Tim

e t (

min

utes

)0

1020

30050100α

= 10

00

β =

70

Activation X(t)

Tim

e t (

min

utes

)0

1020

30050100

150

200α

= 50

000

β =

70

Activation X(t)

Tim

e t (

min

utes

)

RA

SR

AF

ME

KE

RK

010

2030

0

051

152

α =

10

β =

80

Activation X(t)

Tim

e t (

min

utes

)0

1020

300

02

04

06

081

α =

10

β =

800

Activation X(t)

Tim

e t (

min

utes

)


Erklarung





(Armin Robubi)

Title page

Dedication

Quotation

Abstract

Zusammenfassung

Introduction






Kinase domain



DiRas3



Cell culture









Gel filtration

Bioinformatics

Molecular modeling


Results


Homology modeling



Other compounds

Dynamic modeling

DiRas3



Discussion


Dynamic modeling

DiRas3

Bibliography

Acknowledgments

Curriculum vitae


Poster Abstracts

Oral presentations


Erklaumlrung

raf kinases: pathway, modulation and modeling · 2013. 12. 10. · signalweges fest; ein e ekt, der...

Documents

supplmodelpdb

scriptstxt

__MACOSX_scriptstxt

supplmodelpdb

scriptstxt

__MACOSX_scriptstxt

supplmodelpdb

scriptstxt

__MACOSX_scriptstxt

supplmodelpdb

scriptstxt

__MACOSX_scriptstxt

supplmodelpdb

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__MACOSX_scriptstxt

supplmodelpdb

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supplmodelpdb

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supplmodelpdb

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