18 - protein domains and signal trans duct ion

8/8/2019 18 - Protein Domains and Signal Trans Duct Ion

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P ro te in d o m a i n s a n dsignal tran sd u ctio n

S t r u c t u r a l l y c o n s e r v ed p r o t e i n m o d u l e sM a n y o f t h e p r o t e i n s i n v o l v e d i n c e l lu l a r s ig n a l l in g p o s s e s s a m u l t i -d o m a i n a r c h i t e c t u r e ( T a b le 1 8 .1 ) . P r o t e i n d o m a i n s a r e r e g i o n s w i t h i n ap r o t e i n m o l e c u l e t h a t e x h i b i t s t r u c t u r a l h o m o l o g y . 1 M o r e s t ri ct ly , t h e y a r ec a l l e d ' s t r u c t u r a l d o m a i n s ' , b e c a u s e i t i s t h e i r t e r t i a r y s t r u c t u r e s t h a t a r eT a b l e 1 8 . I E x a m p l e s o f p r o t e i n s w i t h s t r u c t u r a l l y c o n s e r v e d d o m a i n sP r o t e in C h a p t e r S t r u c t u reRasGAP 4Sos I IPLCSzPLC71 5,Protein kinase B 13Grb2 I IN c k I Ip85 subuni t o f 13PI3-kinase13LAdrenergic 4receptor k inaseBtk 17Vav

D b lShcSrc

i ~ - I ~ - I- ~- 1 - F~ - K - i l - ~- F ~ - ~ ~ -FR ~-~ -~ - F R ~ - ~ -- polyPro-polyPro-polyPro-12B -EF- E ~ - ~ - ~ - I ~ -[ ~ - EF- IPI-PLC-X - ~ - i-s-~I - I -~-~ - I ~ 1I~ 1 - ~ -

- i -s-~I - I~1- I ~ 1 - i T ~ I - I ~. ... I ~ - - - B ~

18

- ~ - ~ - I ~

I ~ - ~ - r ~

1 2 ~ - - [ 6 - ~ 1 - F ~ - I U 1 - i ~ - ~ -I - B - ~17 ... . F ~ -F~/ -i i - - ~ - - B - ~ Ii i , 1 2 - - ] - s ~ ] - r ~ - ~

BTK, Btk moti f~ C I , homology wi th th e DAG b ind ing domain o f PKC; C2, homology w i th the Ca2+ binding dom ain o f PKC;DH , Dbl hom ology; EF, EF-hand pair, Ca2+ binding; PH, pleckstrin ho mo logy; PI-PLC -X orY, ph ospho inosit ide specif ic PLC;pkinase, protein kinase dom ain; PTB/PID, phosp hotyrosine binding/interaction dom ain; Ras-GAP, Ras GTPase activatingprotein; Ras-GEF, Ras guanine nucleotide dissociation inhibitor; RGS, regulator of G protein signall ing dom ain; SH, Srchomology.


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Nhomologous and this may extend over a wide range of proteins. Theiramino acid sequences tend to be poorly conserved. Each of the manytypes of domain is based on a compact, stable structure possessing ahydrophobic core. They consist typically of stretches of 40-100 aminoacids and are encod ed by discrete exons. A particular protein may possessseveral kinds of domains that have been brought together during evolu-tion by the shuffling of exons. Because the tertiary structures of domainsare compact, with N- and C-termini adjace nt and exposed, they can insertas 'plug-in' modules into more extended structures.

Domains originate from a set of primordial globular structures that hadthe qualities of rigidity and stability. Where sequence homology exists, itpreserves the basic properties; where the sequence is variable, it can allowvariations of function, such as the specification of the target, localizationor mode of activation. Thus, differences in sequence may reflect adapta-tions to meet special requirements. A particular protein may acquire adomain by gene fusion, allowing it to incorporate into its structure a sub-unit that was previously independent and that has been recruited for aparticular purpose. Such insertion can occur most readily at loops in theparen t structure, interrupting its linear sequence. By the same token,the linear sequence of a structural domain may itself be interrupted.For example, the chains that form one of the PH domains of PLC7 areseparated by an insert of almost 350 residues containing three Srchomology domains (Table 18.1).

m Identif ication of doma insSince the level of sequenc e homolog y amo ng domains of the same type islimited and, bec ause of insertions, their identification from sequencedata is difficult. Indeed, in the absence of structural information, theirrecognition has relied upon computer programs that make multiple com-parisons of sequences and structures. As these methods have becomemore refined, domains that were not previously perceived have becomeevident in man y proteins.

m Dom ain funct ionWhile some globular proteins possess several different types of domains,or even multiple copies of a particular domain, there are others that pos-sess only one. Many have no recognizable doma ins at all. It is co mmo n forsignalling proteins to contain multiple domains. However, although ourability to detect the presence of domains has developed rapidly, ourunderstanding of their functions is still in its infancy. Some domains havebuilt-in enzyme functions (such as kinase and DH domains); othersrecognize specific peptide sequences (such as SH2 and SH3 domains);others (such as PH domains) recognize particular lipid head groups andyet others can bind phospholipids in a Ca2+-dependent fashion (C2domains). The non-catalytic interactions can enable the incorporation ofsignalling molecules into complex structures at specific membrane sites,


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Protein Dom ains and Signal Transd uct ion

a ll a t the tou ch o f a swi tch in i t i a ted by the b ind ing o f an ac t iva t ing l igandto i ts receptor .

I n th i s c h a p t e r t h e b a s i c p r i n c i p le s o f p r o t e i n d o m a i n s a n d t h e i r ro l e s ins i g n al l in g m e c h a n i s m s a r e o u t li n e d . T h e d i s c u s s i o n is li m i t e d t o s e l e c t e de x a m p l e s o f d o m a i n s a n d m o d u l e s t h a t h a v e r e c e iv e d m e n t i o n e l s ew h e r ein th i s book . Al tho ugh no t s t r ic t ly a dom ain , the EF -han d Ca 2+ b ind ingmo t i f is a lso d i scu ssed .

m Dom ains t ha t bind o l igopept ide m ot i f sm SH2 domainsThe SH2 domain i s a r eg ion , separa te and d i s t inc t f rom the ca ta ly t i cd o m a i n i n t h e n o n - r e c e p t o r p r o t e i n t y r o s i n e k i n a s e p p 6 0 src ( r e f er e n c e 2 ;see Chap te r 12) , hence SH2, S rc homology reg ion 2 . SH2 domains a rep r e s e n t i n a l l n o n - r e c e p t o r P T K s , g e n e r a l l y l o c a t e d i m m e d i a t e l y N -t e r m i n a l t o th e k i n a s e d o m a i n . T h e y a r e al s o p r e s e n t i n a l a rg e n u m b e r o fo t h e r p r o t e i n s . T h e y c o n s i s t o f a b o u t 1 00 r e s i d u e s a n d p r o v i d e h i g h - a ff i n -i ty b i n d i n g s i t e s fo r p h o s p h o r y l a t e d t y r o s i n e r e s i d u e s ( Kd -- 50- 500 nmo l/1)(F igure 18 .1 ). The te r t i a ry s t r uc tu re cons i s t s typ ica l ly o f a cen t ra l an t ipa r -a l le l [3-sheet f lanked on ei ther s ide by two {x-hel ices . The target phospho-t y r o s in e i s p r e s e n t i n a f o u r - r e s i d u e m o t i f a n d t h is b i n d s b y s t r a d d l i n g t h eedge o f the [3 -sheet . Res idue 1 i s the p ho sp ho tyr os i ne and res idue 4 ( i.e .pY+3) i s usua l ly hydr oph obic . Each o f these i s he ld wi t h in a pocke t , o neon e i the r s ide o f the [3- shee t. F ive c lasses o f SH2 dom ain have be en re -cogn ized , d i s t ingu ished by the i r b ind ing spec i f i c i t i e s . These a re de te r -m i n e d b y t h e r e s i d u e s t h a t f o r m t h e b i n d i n g m o t i f. F o r e x a m p l e , t h eS H 2 d o m a i n o f t h e S r c f a m i l y k i n a s e s b i n d s o p t i m a l l y t o t h e s e q u e n c e

Figu re 18. I SH2 domains.Two orthogonal views of the structure of an SH2 domain(from the Src kinase Lck) with a bound pho sphotyro sylpeptide ligand (EPQPYEEIPIYL).The backbone of the ligand (11 residues) is shown in green.The binding mot if is a centralpYEEI.The phosphotyrosine and isoleucine residues are shown at left and rightrespec tively in ea ch view. (Data source: I Icj.pdb~3.)


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T h e s l i m e m o u l dDictyostel ium discoideum c a nb e r e g a r d e d a s a r e a lt r a n s i t io n a l s p e c ie s . F o r m o s to f t h e t i m e i t e x i s t s a s s i n g l ec e l ls , b u t u n d e r s t r e s s f u lc o n d i t i o n s s u c h a ss t a r v a t i o n , t h e s e c e l l sa g g r e g a t e a s a s o - c a l l e d s l u gw h i c h c a n m i g r a t e a s ac o h e r e n t o r g a n i s m . T h e c e l l sd i f f e re n t i a t e f o r m i n g a s ta l ka n d a n i n d e p e n d e n t f r u i t i n gb o d y , a n d t h e s e d i s t r i b u t es p o r e s .

'S i ii i: :~i:::: ::!:i::~ : ~i~..iiiiT i~ , i : i ~ i~.!~ i~ d i ~ i. :~ i~::::::::::::::::::::::i i ~ :~ : ~~i~;

pYEEI ( see F igu re 11 .7, page 264) . The a f f in i ty o f SH2 do m ai ns fo r no n-p h o s p h o r y l a t e d t y ro s i ne s o r f or p h o s p h o s e r i n e s o r p h o s p h o t h r e o n i n e s isneg l ig ib le .

S H 2 d o m a i n - c o n t a i n i n g p r o t e i n s m a y p o s s e s s c a t al y t ic a ct iv it y, s u c h a sS rc ( C h a p t e r 1 2 a n d b e l o w ) a n d P L C 7 ( C h a p t e r 5 ). A l t e r n a t i v e l y t h e y m a yp o s s e s s n o i d e n t i f i a b l e c a t a l y t i c a c t i v it i e s o f t h e i r o w n , s u c h a s G r b 2( C h a p t e r 1 1 ) , w h i c h a l s o p o s s e s s e s t w o S H 3 d o m a i n s ( T a b l e 1 8 . 1 ) . T h e s eb i n d a m o t i f t h a t i s q u i t e d i f f e r en t f r o m t h a t r e c o g n i z e d b y S H 2 d o m a i n s( s e e b e l o w ) . T h u s , i t c a n a c t a s a n a d a p t e r m o l e c u l e , d i r e c t i n g a t y r o s i n ep h o s p h o r y l a t i o n s i g na l i nt o a R as p a t h w a y s i g n al .

S H 2 d o m a i n s h a v e b e e n i d e n t i f i e d i n a n im a l s , b u t n o t s o fa r i n t h e o t h e rm a i n k i n g d o m s o f e u k a r y o t i c o r g a n i s m s , s u c h a s p l a n t s a n d f un g i. I n e v o -l u t i o n a r y t e r m s , t h e m o s t a n c i e n t f o r m a p p e a r s i n t h e S T A T p r o t e i n( C h a p t e r 1 2) o f t h e s l i m e m o u l d Dictyostelium discoideurn. T h i s s u g g e s t sa r o l e f o r t h e S H 2 d o m a i n i n t h e e v o l u t i o n o f m u l t i c e l l u l a r a n i m a l s . I nr e s p o n s e t o a n a c t i v a ti n g s i gn a l, S T A T m o l e c u l e s d i m e r i z e t h r o u g h am u t u a l i n t e r a c t i o n i n vo l vi n g a n S H 2 d o m a i n o n e a c h a n d a p h o s p h o ty r o -s i n e o n t h e o t h e r . T h e d i m e r i c f o r m t h e n e n t e r s t h e n u c l e u s a n d b i n d s t oD N A t o d i r e c t t r a n s c r i p t i o n , e s s e n t i a l l y a s i t d o e s i n h i g h e r a n i m a l s .

m P T B / P I D d o m a i n sA n o t h e r t y p e o f d o m a i n t h a t r e c o g n i z e s p h o s p h o t y r o s i n e r e s i d u e s is t h eP T B ( p h o s p h o _ t yr o s i n e b i n d i n g ) d o m a i n ( a l t er n a t i v e ly : p h o s p h o t y r o s i n ei n t e r a c t i o n d o m a i n , P ID ) . H o w e v e r, t hi s h a s c o m p l e t e l y d i f f e re n t m o l e c u -l a r a r c h i t e c t u r e f r o m t h e S H 2 d o m a i n . A l so , i n c o n t r a s t w i t h S H 2 d o m a i n s ,t h e s p e c i fi c it y o f i n t e r a c t i o n i s d e t e r m i n e d b y th e s e q u e n c e o f a m i n oa c i d s i m m e d i a t e l y o n t h e N - t e r m i n a l s i de o f t h e p h o s p h o r y l a t e d t y r o s i n er e s i d u e ( N P X pY m o t i f ) . 3,4 C u r i o us l y , P T B / P I D d o m a i n s a r e s t r u c t u r a l l yve ry s imi la r to PH domains , wi th a 13 -ba r re l s t ruc tu re and a long cz -he l ixt h a t p a c k s a g a i n s t o n e e n d ( c o m p a r e F i g u r e 1 8 .2 w i t h F i g u r e 1 8 .6 ). H o w -e v e r , P H d o m a i n s h a v e q u i t e d i f f e r e n t t a r g e t s ( s e e b e l o w ) , w h i c h b i n de i t h e r t o t h e l o o p s t h a t j o i n t h e f ~ - c h a i n s o r t o t h e m a j o r h e l i x . I n P T Bd o m a i n s , b y c o n tr a s t , t h e t a r g e t p h o s p h o t y r o s i n e b i n d s t o o n e s i de o f t h e[3-barrel (Figure 18.3).

I I S H 3 d o m a i n sS H 3 d o m a i n s c o n s i s t o f 5 5 - 7 5 a m i n o a c i d s t h a t f o r m a t w i s t e d [ 3 -b a rr els t r u c t u r e ( F i g u r e 1 8 . 4 ) . T h i s s t a b l e c o r e i s c o n s e r v e d , b u t t h e l o o p s t h a tj o i n t h e [ 3 -s t ra n d s a re v a r i a b le . T h e m o t i f o n t h e t a r g e t p r o t e i n s , t o w h i c hS H 3 d o m a i n s b i n d , c o n s i s t s o f a p r o l i n e - r i c h s t r e t c h o f 8 - 1 0 r e s i d u e s .S u c h s e q u e n c e s f o r m e x t e n d e d l e f t - h a n d e d h e l i c e s h a v i n g t h r e e r e s i d u e sp e r t u r n ( c a l le d a t y p e I I p o l y p r o l i n e h e l i x ). T h e b i n d i n g s i t e is l i n e d w i t hh y d r o p h o b i c a r o m a t i c a m i n o a c id s . 5 T h e p l a n a r s i d e - c h a i n s f o r m r i d g e st h a t f i t i n t o t h e g r o o v e s o f t h e p o l y p r o l i n e h e l ix , r a t h e r l i k e th e t h r e a d s o ft w o a d j a c e n t s c r e w s t h a t f i t t o g e t h e r ( F i g u r e 1 8 . 5 ) . A b o u t t h r e e t u r n s a r ei n v o l v ed i n b i n d i n g a n d t h e c o n s e n s u s m o t i f is e i t h e r R X L P PL P XX o r


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P r o t e i n D o m a i n s a n d S ig na l T r a n s d u c t i o n

F i g u r e 1 8 . 2 P T B / P I D d o m a i n s t r u c t u r e . S te re o sco p ic d ia g ra m o f th e s t ru c tu re o fth e P T B d o ma in o f S h c.T h e co n se rve d se co n d a ry s t ru c tu re th a t fo rms th e co re is sh o wnin co lo u r ( s -h e l i x , ma ge n ta; ~ -s t ru c tu re , ye l lo w) .T h e h e l i x c lo se s o f f o n e e n d o f atwis ted ~-barre l . (Data source : Ishc .pdb l4. )

How to view stereoimages of molecularstructuresThere is much moreinformation in a stereoscopicimage than a conventional flatprojection. Almost everyonecan view stereo pictures withunaided eyes, but it does takea while t o get used to it.Practice is the key, unless youare unfortunate enough tohave one very w eak eye.There are two ways ofseeing a three-dim ensionalimage by observing a stereopair.You may either crossyour eyes, so that the left eyeviews the right-hand imageand vice versa, or you mayallow y our eyes to diverge, sothat each eye looks at theimage in front of it. Rasmol-generated images are, bydefault, for convergent(cross-eyed) viewing andCHIM E images are fordivergent viewing. To viewthe images in this book (andmost printed images), youshould view the left imagewith the left eye and the rightimage with the right eye. Ifthe two do not readily fuseinto a single three-dimensional image, tr y thefollowing:9 First touch your nose tothe page between and belowthe stereo pair. The twoimages will now besuperimposed, but thepicture will be very blurred(although you may noticesome three-dimensionality atthis stage).9 No w move the pageslowly away from you, buttake care not to rotate it.Concentrate on the three-dimensional aspect and waitfor y our eyes to bring it intofocus.Note: i f you attempt to v iewa divergent pair using theconvergent strategy (crossedeyes) the image will be three-dimensional but inverted in aconfusing way.

F i g u r e 1 8 . 3 P T B d o m a i n w i t h b o u nd p h o s p h o t y r o s i n e - c o n t a i n in g m o t i f . T h eta rg e t m o t i f o f th e P T B d o ma in i s d e p ic te d in g re e n a n d th e p h o sp h o ty ro s in e is co lo u re dred. (Data sourc e: I shc.pdbl4.)


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Figure 18.5 Molecularsurface of an SH3domain bound to aproline-rich peptide.The SH3 domain of Fyndepicted as a molecularsurface (using CHIME).The target peptide isshown in spacefill formatusing the same colou rscheme as Figure 18.4.(Da ta source : l azg.pdb~5.)

Figure 18.4 Stereoscopic vie w of a left-handed proline-rich helix binding tothe surface of an SH3 domain. Structure, obtained by NMR, of the SH3 domain ofthe Src kinase Fyn, comp lexed w ith a synthetic peptide c orrespon ding to residues 9 I- I 04of the p85-subunit of PI3-kinase (PPRPLPVAPGSSLT).The peptide, in ball and stick fo rmat,is col ou red to sho w Pro pink; Arg, Lys blue; Leu,Va l green; Ala, Gly grey; Ser, Thr, orange.(Dat a source: l azg.pdblS.)XXXPPLPXR, giving rise to two classes of SH3 domain-binding proteins.Those bearing the first sequence will align their motifs in the oppositedirection to proteins with the second sequence. However, the binding inboth cases is usually weak and these interactions are promiscuous. Suchunreliability may account for the prese nce of two or more SH3 domains inadaptor proteins such as Grb2 (Chapter 11) and Nck (Table 18.1). In thecase of Grb2, these bind to two neighbouring proline-rich regions on thetarget, thereby increasing the affinity and specificity of binding. The tan-dem arrangement of SH2 and SH3 domains found in a variety of sig-nalling proteins may provide a conformational mechanism for regulatingSH3-d epend ent interactions thr ough tyrosine phosphorylat ion. 6

m D o m a i n s t h a t b i n d p r o t e i n s a n d l i p i d sI I PH domainsPH (pleckstrin homology) domains are a relatively recent addition to theinventory of known structural domains. Consisting of some 100 aminoacid residues, sequence matches for them have been found in over 600proteins, though detailed structures are known for only a few of these.Their function is less clear than that of the SH2 and SH3 domains and itvaries among different PH domains. Many have been shown to mediateprote in-p rote in or protein- lipid interactions. They were first identified asinternal repeats in pleckstrin, the major substrate of protein kinase C inplatelets. 7,8 Pleckstrin itself, rather unusually, possesses two PH domains,one at either end of the molecule.

The sequences which make up the various PH domains are much more


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P rotein Domains and Signal T ransduction

variable than those of SH2 and SH3 domains. Like all domains, the fewconserved residues ten d to be immerse d in the interior, where they main-tain the core structure. Multiple se quence alignments of PH domainsfrom diffe rent pro teins reveal this lack of similarity, as illustrated in Table18.2.

The conserved three- dimensio nal structures of PH domain s are char-acterized by a [3-barrel structure, consisting of seven antiparalle113-strandswith a C-terminal s-helix packed against one end of the barrel. This formsa rigid flame. The process of evolution has adapted this structure, partic-ularly in the interchain loop regions, to form PH domains with differentbinding specificities. It is noteworthy that the same fold forms the core ofthe PTB/PID domains (compare Figure 18.6 with Figure 18.2).

The assignme nt of function to PH domains is not straightforward. Forsignalling proteins, it is useful to identify specific ligands. Some are listedin Table 18.3. A numb er of prote ins cont ain PH domains t hat can bind tothe [37-subunits of G-proteins. The best known of these is [3ARK, thekinase that catalyses phosphorylation of [3-adrenergic receptors, butalthough it is known that the [37-subunits bind to the C-terminal region ofthe PH domai n, the structural details of the intera ction are unclear. Incontra st to [3ARK, the homo logous prot ein rhodopsin kinase, which hasno PH domain, possesses a farnesyl group at its C-terminus and thishydrophobic modification enables it to associate with membranes. Thissuggests that association of a PH domain with a membrane-tethered[37-subunit also functions to attach the host molecule at a membrane site.

PH domains may also interact with membrane polyphosphoinositides.This occurs through binding of the 4- and 5-phosphate groups of thephospholipid headgroup to residues in a cleft between the loops con-necting the [3-strands. For instance, this is a property of the PH domain ofPLC8 whi ch binds to PI(4,5)P 2 (or IP 3) (Figure 18.6). This high-aff inityinteraction may direct the phospholipase to membrane regions contain-ing PI (4,5)P2, which is also the substr ate for the catalytic dom ain. Anotherexample is provided by the Btk/Tec family of non-receptor PTKs, exceptthat in this case the PH doma in is selective for PI(3,4,5)P 3, the pro duc tof PI3-kinase. It seems that the main function of these PH domains is totarget proteins to specific membrane locations determined by the pres-ence of individual polyph osphoi nosit ides. The p resence of IP 3, whichmight prevent this form of interaction, provides yet anothe r possible linkto signal trans duc tion processes. The PH do mai n of [3ARK can bind toboth PI(4,5)P 2 and [37-subunits to ensu re its tran sloca tion to a me mbra nesite. A link with the regulation of heterotrimeric G-proteins and the acti-vation status of 7TM receptors is then provided by the finding that thedissocia tion of the G~/[3ARK com ple x is regu late d by the level of PIP 2.Finally, although we have good clues about the functions of a fewimportant PH domains, we know little about the remainder. PH domainsmay use either or both of the two basic mechanisms described above formembrane translocation, but some use neither and much remains to beclarified.


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Protein Dom ains and Signal Trans duction

Figu re !8.6 PH do ma in wit h bou nd IP 3. Stereoscopic diagram of the structure ofthe PH domain of PLCSI complexed with IP3.The secondary structure forming theconserved core is indicated in colour (s-hel ix, magenta; 13-structure, yellow). As for PTBdomains, the major helix closes off one end of a twisted 13-barrel. IP3 is depicted as a balland stick model in Rasmol cpk colours (red, oxygen; yellow, phosphorus). (Data source:I mai.pdb~6.)

Ta bl e 18.3 Some physiological ligands of PH domainsHos t protein Ligands Protein function13-Adrenergic rec epto r l[37-subu nits, I(4,5)P2kinasesPhospholipase C61 PI(4,5)P 2, IP3AI


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,~ ~' ~ ,,~ S ii !i{ n~a~.ii T~~qs~.~~,s di ~is


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Protein Doma ins and Signal T ransduction

Figure 18.8 C2 do ma in str uct ure . Stereo image showing secondary structure withCa 2 in green. (Data source: l a25.pdb~8.)m i c r o m o l a r l e ve ls . It r e p e a t s a t h e m e t h a t w e h a v e e n c o u n t e r e d b e fo r e:t he r ec r u i t m en t o f a s i gna ll i ng m o l ec u l e t h r oug h o ne o f i ts dom a i ns t o as p e ci fi c m e m b r a n e l o c a ti o n .

In gene ra l, C 2 dom a i ns do no t neces s a r i l y exh i b it a ll t he se cha rac t e r i s -t ic s . T he re a r e va r i a t i ons i n t he s t o i ch i o m e t ry o f b i nd i ng (nu m b er o f s it es )an d in the a f f in i ty for Ca 2 Indeed , so me do n ot b ind Ca 2 but c an assoc i -a t e w i t h o t he r p ro t e i n s , sugges t i ng a pos s i b le a l t e rna t i ve func t i on .m P r o t e i n k i n a s e d o m a i n sm Protein kinases share a common domainP ro t e i n k i na se s ca t a l y se t he t r an s fe r o f a ph os ph a t e g roup f rom A TP t o ahyd roxy l r e s i due on a n am i n o ac i d s i de -cha i n . T he re a r e tw o p r i nc i pa lc l as se s : s e r i ne / t h reo n i n e k i na se s an d t y ro s i ne k ina se s . In bo t h , t he ca t -a l y ti c ac t iv i ty is con f i ned t o a s truc t u ra l l y con se rv ed do m a i n ca l l ed a p ro -t e i n k i na se dom a i n . T he bas i c a r ch i t ec t u re o f k i na se d om a i n s i s t yp i f iedby the ca t a l y t ic subu n i t o f t he cA M P -de pen den t p ro t e i n k i na s e A (P KA )and th i s i s i l l us t ra t ed in F igure 18 .9 . The pept ide cha in i s fo lded to formt w o l obes tha t a r e i n c l o se appos i t i on , t he c l ef t be t w e en t he m h ous i ng t heca ta ly t i c s it e . The re i s a l so an N- ter mi na l a -he l i ca l ch a in ( the A he l ix) t ha tb i nds t o the su r f ace o f bo t h l obes . I t i s som e t i m es r e f e r r ed t o a s t he l inke r.T h e l o b e s a r e c o n n e c t e d t o e a c h o t h e r b y a s i ng l e p o l y p e p t i d e c h a i nw h i ch ac t s a s a h i nge .

T he N - t e rm i na l l obe i s t he sm a l l e r o f t he t w o , pos s e s s i ng abo u t 100am i no ac i d s . A t t he N - t e rm i nus i t i s m yr i s t oy l a t ed , bu t t he re i s no ev i -dence t ha t t he m yr i s t oy l g roup i s f r ee t o a s soc i a t e w i t h m em branes .In s t ead , i t o ccup i e s a pocke t and p rov i des s t ruc t u ra l s t ab i l i t y , he l p i ng t o


12/18

S i ~.~i iis S=~ i :Ti[ :~'a, iis 5


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Protein Dom ains and Signal Transd uct ion

both lobes. Two Mg 2+ ions are also bound, but for clarity these are no tshown in the following figures. Next, the rec ognition and binding of a con-sensus motif on the target pro tein mus t take place. For PKA, the se quenceof the motif is RXXT/Sh (where h indicates a hydrophobic side-chain).The target hydroxyl group on serine or threonine must be accuratelyaligned with the terminal phosp hate of ATP and ultimately the ADP thatis formed must be exchanged for new ATP.

All this requires the coordinated interaction of amino acid side-chainsand peptide bonds in specific structures on both lobes. These are shownin bold below and indicated in Figure 18.10. The residues on the smalllobe that form contacts with the nucleotide and align it, include the mainchain nitrogens of a glycine-rich loop, between [3-strands 1 and 2, and alysine (K72) on ]3-strand 3 that inte racts with a gluta mat e (D91) on theC-helix. In the large lobe, a 24 amino acid segment controls activation. Itforms the loop following [3-strand 8, termed the activation loop, and alsopart of the ensuing helix. On the activation loop is a crucial threonine(T197) that must itself be in a phosphorylated state for activation to takeplace. Once thought to be a stable post-translational modification, phos-phorylat ion of T197 is now considered to be a regulatory event for PKA.Also following [3-strand 8 are residues that cont ribu te to the binding of thetwo Mg 2+ ions (not shown in the figure) and, finally, following [3-strand 6 isthe catalytic loop that contains a glutamate (D166) which is thought toinitiate the phosphate transfer reaction.

Fig ure 18. I 0 C a ta ly t i c c o r e o f P K A . Some of the important structures and residuesthat for m the catalytic site. (Orien tation as in Figure 18.9 (bott om), bu t the frame wor k isfaded and the linker and substrate are not shown). Key residues include K72 on 13-strand3, E91 in the C -helix, T I9 7 in the act ivation loop and R165 in the catalytic loop (allcoloure d cyan). DI 66 in the catalytic loop is coloure d red.The binding of ATP is indicatedby the grey, spacefilled molecu le. For exp erim enta l reasons this is an ATP analogue,adenylyl imid odiph ospha te. (Data source: I cdk.pdb~9.)


14/18

S{gna{ Tr ans duc t{o n

Effec tive ca ta ly t ic ac t iv i ty dep end s on th e com ple t ion of the ac t ive s ite,and th is requ ires the b ind ing of ATP and Mg 2 the c losure o f the c lef tb e twe e n th e two lo b e s a n d p h o s p h o ry la t io n o fT 1 9 7 . T h e p o si t io n o f th eac t iva t ion segment i s c r i t ica l fo r the correc t a l ignment o f the ca ta ly t icre sid u es , a n d th e c o n ta c t s ma d e b y th e p h o s p h o th re o n in e a re p a r t ic u la r lyimportan t . Also , ATP b ind ing is ve ry sens i t ive to the pos i t ion o f theC-he l ix . Thus , bo th the ac t iva t ion segment and the C-he l ix can ac t a sregula tors of kinas e activity .Bind ing of subs t ra te in the correc t o r ie n ta t io n is a lso c r i tica l, and th is isi l lus tra ted in Figure 18.11. In pan el (a) the m ole cul ar sur face of PKA in the

Figure 18. I I Molec ular surfaces of P KA showing AT P and substrate binding:(a) Stereoscopic diagram of the calculated molecular surface of P KA (Van der Waalsradii).T he linker and small lobe are coloured blue; the large lobe is viole t.T he bound AT P(analogue) is just visible .T I97 is coloured green. (b) T he surface has been renderedtransparent to reveal the nucleotide.The 'substrate' peptide is indicated in red (residues5-24 of a protein called P KC inhibitor, PKI). (Data source: I cdk.pdb~9.)


15/18

P rotein Domains and Signal T ransduction

c l o s e d c o n f o r m a t i o n i s s h o w n , w i t h A TP b o u n d d e e p i n th e c l ef t. I n p a n e l( b ) t h e s u r f a c e i s r e n d e r e d t r a n s p a r e n t t o r e v e a l t h e n u c l e o t i d e a n d ap s e u d o s u b s t r a t e p e p t i d e i s s h o w n i n t h e s u b s tr a t e b i n d i n g s it e.

P h o s p h o r y l a t i o n o f T 1 9 7 is i m p o r t a n t b e c a u s e i t n e u t r a l iz e s t h e p o s i -t iv e c h a r g e s o n a n u m b e r o f n e a r b y c a t i o n i c r e s i d u e s , i n c l u d i n g a n a r gi -n i n e ( R 16 5 ) a d j a c e n t t o D 1 6 6. It a l s o i n t e r a c t s w i t h r e s i d u e s o n t h e s m a l ll o b e , h e l p i n g t o s e a l t h e c l e f t . T 1 9 7 i s p r e s e n t i n t h e s e q u e n c e R T W T L .T h i s m a t c h e s t h e PK A t a rg e t c o n s e n s u s s e q u e n c e a n d s o in d i c a t e s t h a tk i n a s e a c t i v i t y i s r e g u l a t e d b y a u t o p h o s p h o r y l a t i o n . A n u m b e r o f o t h e rk i n a s e s a r e al s o p h o s p h o r y l a t e d a t p o s i t i o n s e q u i v a l e n t t o T 19 7 o nP K A . T h e s e i n c l u d e i s o f o r m s o f P K C , E R K , M E K 1, t h e c y c l i n - d e p e n d e n tk i n a s e s , C D K 2 a n d C D K 7 , a n d S r c f a m i l y p r o t e i n t y r o s i n e k i n a s e s . H o w -e ve r, a l th o u g h a p h o s p h o r y l a t i o n i n th e a c t i v a ti o n l o o p m a y b e c o n d i -t i o n a l fo r k i n a s e a c t i v a t i o n , a u t o p h o s p h o r y l a t i o n a t t h i s s i t e d o e s n o to c c u r i n e v e r y c a s e . F o r e x a m p l e , i n P K C , E RK a n d t h e c y c l i n - d e p e n d e n tk i n a s e s , th e s e q u e n c e o f t h e m o t i f i n t h e a c t iv a t i o n lo o p d o e s n o t m a t c ht h e c o n s e n s u s s e q u e n c e r e c o g n i z e d b y t h e k in a s e . T h i s i m p l i e s a r e q u i re -m e n t f o r a n u p s t r e a m k i n a s e ( fo r e x a m p l e , P D K 1 f o r P K C , s e e C h a p t e r 9 ) .

O t h e r p r o t e in k i n a s e s a r e n o t r e g u l a te d b y a p h o s p h o r y l a t i o n ( s e e T a b le1 8 .4 ) . I n s t e a d , t h e p o s i t i v e l y c h a r g e d a r g i n i n e r e s i d u e ( e q u i v a l e n t t o R 1 6 5i n P K A ) m a y b e n e u t r a l i s e d b y a n a c i d i c ( g l u t a m a t e) r e s i d u e ( p h o s p h o r -y l a s e k i n a s e ) , o r s u b s t i t u t e d b y a n o n - p o l a r r e s i d u e ( m y o s i n l i g h t c h a i nk i n a s e ) .

:,, : . . .

T able 18.4 R egulation of kinase activity by phosphorylation of the activation loopPhosp horyla ted in the ac t iva t ions e g m e n t

Not phosphory l a t ed in the activations e g m e n t

Cyclic AMP- PKAdependent kinaseCyclin-dependentkinaseMAP kinaseMAP kinase kinaseRafl kinaseCa2+/calmodulinkinaseProtein kinase CInsulin-stimulatedkinaseGlycogen synthase GSK3kinaseInsulin recep tor IRKkinasePDGF receptorc-Src family

p34 cdc2 c d c 2 Pho sph ory las e inaseCDK2 CDK7MAPK/ERK2 Casein kinase IMEKI EGF recep torRafl C-te rmin al Src kinaseC a M K I C a 2 + / c a l m o d u l i ninasePK C ~, 1311ISPK

PDGFRSrc,Yes, Fyn, Fgr,Lyn, Lck, BIk

Myosin light chain kinase

EGFRCskCaMKIIMLCK


16/18

__. l l ! I l ll l

For historical reasons, henumbering of the residues ofhuman c-Src corresponds tothe sequence of chickenc-Src.Thus 86-536 actuallydenotes residues 83-533.

S ig n ~ T~-an~,~ductior~

l The regu lator y dom ains of Src cont ro l prote in k inase act iv i tyT h e n o n - r e c e p t o r p r o t e i n t y r o s in e k i n a s e c -S r c p r o v i d e s a n e x c e l le n te x a m p l e o f t h e r e g u l a t i o n o f k i n a s e a c t iv i ty b y s t r u c t u r a l d o m a i n s t h a t c a nt a k e p a r t in p r o t e i n - p r o t e i n ( or d o m a i n - d o m a i n ) i n t e r a c ti o n s . ( T h e S rcf a m i l y o f p r o t e i n t y r o s i n e k i n a s e s a r e d e s c r i b e d o n p a g e 2 9 4 .

T h e t h r e e - d i m e n s i o n a l s t r u c t u r e o f h u m a n c -S r c (r e s id u e s 8 6- 8 36 ) isdep ic ted in F igure 18 .12. The l inker cha in and the two lob es o f the k inased o m a i n a r e e v id e n t . N - t e r m i n a l t o t h e s e s t r u c t u r e s t h e r e a r e th e t w o S rch o m o l o g y d o m a i n s ( SH 2 a n d S H3 ) a n d a t t h e C - t e r m i n a l t h e r e i s a sh o r t,f le x ib l e s e g m e n t t h a t b e a r s a n i n h i b i t o r y p h o s p h o t y r o s i n e ( pY 52 7).

R e g u l a t i o n f ol lo w s a g e n e r a l p ri n c i p le . T h e o p e r a t i o n a l m a c h i n e r y t h a tenab les ca ta ly t i c ac t iv i ty i s con ta in ed w i th in the k inase d om ain i tsel f. Thec o n t r o l s a r e l o c a t e d o n a d j a c e n t d o m a i n s a n d r e g u l a t i o n d e p e n d s o nt h e i r i n t e r a c t i o n s w i t h t h e k i n a s e d o m a i n . I n t h e c a s e o f S rc a n d r e l a t e dk inases , the e lements tha t d i rec t the regu la t ion a re the SH2 and SH3domains . S rc i s he ld in an inac t ive s ta te by the C- te rmina l cha in phos -p h o t y r o s i n e . T h i s p r o v i d e s a n i n t r a m o l e c u l a r b i n d i n g s i t e f o r t h eN - t e r m i n a l S H 2 d o m a i n . T h e r e i s a l s o a n i n t e r a c t i o n b e t w e e n t h e S H 3d o m a i n a n d t h e l i n k e r c h a i n , w h i c h a d o p t s a l e f t - h a n d e d h e l i c a l c o n f o r -ma t ion ( resem bl ing a type II po lyp ro l ine he l ix , a l thou gh th e re i s on ly onepro l ine res idue) . Toge ther , these in te rac t ions fo rce the molecu le to adop ta c o m p a c t c o n f i g u r a t io n , d i s t o r t in g t h e s m a l l lo b e, r e d u c i n g a c c e ss t o t h ec lef t and caus ing an o u tw ard ro ta t ion o f the C-he l ix .

Ac t iva t ion o f k inase ac t iv i ty fo llows even ts tha t d es tab i l i ze the co mp ac tc o n f o r m a t i o n . L a c k i n g a n i s o l e u c i n e a t p Y +3 , t h e a m i n o a c i d s e q u e n c e

; i ; ~ ~. ~ ~ ~ T he st ruc ture of c -Src in its inact ive s tate . This is the compact auto-inhibited conformation of c-Src.The kinase domain is to the r ight and the l inker andlobes are coloured as in previous diagrams.The C-terminal tail region (red) containspY527, which is shown in spacefi ll format, but is mos tly hidden.The activation loop issketched as a dotted line because it is disordered in this structure. (Data source:I fmk.pdb2~


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Pro te in Do m ains and S igna l T ransd uct ion

a d j a c e n t t o t h e C - t e r m i n a l p Y 5 2 7 d o e s n o t m a t c h t h a t o f t h e m o t i f t h a tb i n d s t h e S H 2 d o m a i n o f S rc m o s t s t r o n g l y ( s e e F i g u r e 1 1 .7 , p a g e 2 6 4 a n dF i g u r e 1 8 .1 ) . C o n s e q u e n t l y , t h e b i n d i n g is l e s s t i g h t, g i v i n g t h e o p p o r t u -n i t y f o r p Y 5 2 7 t o b e c o m e d e p h o s p h o r y l a t e d . D i s p l a c e m e n t o f p Y 5 2 7 b y ah i g h e r - a f f i n i t y p h o s p h o t y r o s i n e m i g h t f a c i l i t a t e th i s . R e m o v a l o f t h ep h o s p h a t e g r o u p a l lo w s th e m o l e c u l e t o r e la x i n t o a n a c t iv e c o n f o r m a -t i on . H o w e v e r , a s in o t h e r k i n a s e s , p h o s p h o r y l a t i o n o f a k e y r e s i d u e i n t h ea c t i v a t i o n l o o p ( Y 41 6 , e q u i v a l e n t t o T 1 9 7 in PK A ) is a l s o e s s e n t i a l f o ra c t i v i t y .

A s p e c i f i c k i n a s e , C s k ( F i g u r e 1 2 .6 , p a g e 2 9 5 ) , is r e s p o n s i b l e f o r p h o s -p h o r y l a t i n g t h e C - t e r m i n a l t a i l o f S rc , i n h i b i t i n g i ts a c ti v it y . M u t a t i o n o ft y r o s i n e 5 2 7 t o p h e n y l a l a n i n e i n a v i a n c -S r c p r o d u c e s a c o n s t i t u t i v e l ya c t iv e o n c o g e n i c p r o t e i n . L i k e w i s e t h e o n c o g e n i c v i ra l f o r m , v - S rc , l a ck s aC - t e r m i n a l t a i l .

I R e f e r e n c e s1 Wet lau fe r , D .B . Nuc lea t ion , rap id fo ld ing , and g lobu la r in t racha in reg ions in p ro te ins .

P r o c . N a t l . A c a d . S c i . U S A 1973; 70: 697-701.2 Koch, C.A. , M oran, M. , Sadowski , I ., Paw son, T . The co m m on src ho m olo gy regio n 2dom ain o f cy top la s mic s igna l ing p ro te ins i s a posi t ive e f fec to r o f v - fps ty ros ine k inas efunc t ion . M o l . C e l l B i o l . 1989; 9: 4131-4 0.3 Kavanaugh , W. M. , Wi l l iams , L .T . An a l t e rna t ive to SH2 do ma ins fo r b ind ing ty ros ine -p h o s p h o r y l a t e d p r o t e i n s. S c i e n c e 1994; 266: 1862-5.4 Kav anaugh , W.M. , Turck, C.W. , Wil l iams, L .T. PTB dom ain bin din g to s ign al ing pro te inst h r o u g h a s e q u e n c e m o t i f c o n t a i n i n g p h o s p h o t y r o s i n e . S c i e n c e 1995; 268: 1177-9.5 Yu, H., Rosen, M .K., Shin, T.B. e t a l . Solu t ion s t ruc tu re o f the SH3 dom ain o f Src andiden t i f i ca t ion o f i ts l igand-b ind ing s it e . S c i e n c e 1992; 258: 1 665-8.6 Hu , K . Q., Se t t l eman , J . T and em SH2 b ind ing s i t e s med ia te the RasGAP-RhoGAPi n t er a c ti o n : a c o n f o r m a t i o n a l m e c h a n i s m f o r SH 3 d o m a i n r e g u l a t io n . E M B O ]. 1997;16: 473-83.7 Has lam, R . J ., Ko ide , H . B ., Hem ming s , B . A. P lecks t rin d om a in homology . N a t u r e 1993;363: 309-10.8 Imao ka, T ., Lynh am, J .A., Has lam , R.J . Purif ica t ion and cha racter iza t io n of the 47,000-d a l t o n p r o t e in p h o s p h o r y l a t e d d u r i n g d e g r a n u l a t i o n o f h u m a n p l a te l et s . I . B i o l . C h e m .1983; 258 :11404-14 .9 Lem mo n, M.A. , Falasco, M. , Ferg uson, K.M. , Schless inger, J . Reg ula tory recr ui tm en t ofs i gn a ll in g m o l e c u l e s t o t h e c el l m e m b r a n e b y p l e c k s tr i n - h o m o l o g y d o m a i n s . T r e n d sC e l l B i o l . 2000; 7: 237-42.10 Kre t singer , R . H. Ca lc ium -b ind ing p ro te ins . A n n u . R e v. B i o ch e m . 1976; 45: 239-66 .11 Bab u, Y.S. , Sack , J .S., Gr een ho ug h, T.J. e t a l . T h r e e - d i m e n s i o n a l s t r u c t u re o f c a lm o d u l i n .N a t u r e 1985; 315: 37-40.12 Ub ach , J . , Zh ang , X., Shao, X., Sfidhof, T.C., Rizo, J . Ca 2+ b ind in g to s ynap to tagm in : howm a n y C a2 ions b in d to the t ip o f a C2-do main? E M B O ] . 1998; 17: 3921-30.13 E ck , M. J. , Shoe l s on , S .E . , Ha r r i s on , S . C . Recogn i t ion o f a h igh-a f f in i ty phos ph o ty ros y lpep t ide by the Src hom ology-2 d om a in o f p561ck . N a t u r e 1993; 362: 87-91.14 Zho u, M.M . , Rav ichand ran, K.S. , Ole jniczak, E .E e t a l . St ruc tu re and l igand recogn i t iono f t h e p h o s p h o t y r o s i n e b i n d i n g d o m a i n o f S h c. N a t u r e 1995; 378: 584-92.15 Ren zoni, D .A., Pug h, D .J., Siligardi, G. e t a l . S t r u ct u r al a n d t h e r m o d y n a m i ccha rac te r i za t ion o f the in te rac t ion o f the SH3 dom ain f rom Fyn wi th the p ro l ine - r i chb ind ing s i te on the p85 s ub un i t o f PI3 -k inas e . B i o c h e m i s t r y 1996; 35 :15646-5 316 Fergu son, K.M. , Lem mo n, M .A., Schless inger, J ., Sigler , P.B. Struc ture of the h igh aff ini tyc o m p l e x o f i no s i to l t r i s p h o s p h a t e w i t h a p h o s p h o l i p a s e C p l ec k s t ri n h o m o l o g yd o m a i n . C e l l 1995; 83: 1037-46.


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5i[~na[ TraHsd~Jctior~

17 Chat top adhya ya, R., Meador, W.E., Means , A.R., Quiocho, EA. Calm odulin s t ructurerefined at 1.7 A resolution. ] . Mol . B io l . 1992; 228: 1 177-92.18 Sut ton, R.B., Sprang, S .R. Structure of the prote in kinase C[3 pho spho l ipid-b indin g Ca2~d o m a i n c o m p l e t e d w i th C a2+. S t r u c t u r e 1998; 6: 1395-405.19 Bossem eyer, D., Engh, R.A., Kinzel, V., Ponstingl, H., Hub er, R. Pho sph otra nsf eras e an dsubs t ra te b inding me chani sm of the cA MP-dependent pro te in k inase ca ta ly t i c subuni t

f rom porc ine hear t a s deduc ed f rom the 2 .0 A s t ruc ture of the co mplex wi th Mn 2adenyly l imidodip hospha te and inhib i tor pept ide PKI(5-24). E M B O 1 . 1993; 12(849):859.20 Xu, W., Harrison, S .C., Eck, M.J . Thr ee-d imen sion al s t ructur e of the ty ros ine kinasec-Src. N a t u r e 1997; 595(602): 385.21 Runne ls, L.W., Jenco, J., Morris, A. Scarlata, S. M em br an e bin din g of ph osp hol ipa sesC-~-1 and C-2 i s indep ende nt of phospha t idyl inos i to l 4 ,5-b i sphospha te and the a and[37 sub uni ts of G prote ins. B i o c h e m i s t r y 1996; 35: 16824-32.

18 - protein domains and signal trans duct ion

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