2016 Regiomeeting

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Scientific Program 2016 Regiomeeting

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Scientific Program 1

Abstracts

Keynote lecture Gwyndaf Evans Wednesday, September 28th, 19:45 - 20:45 6

Session 1 Vincent Olieric 20:45 - 21:00 7

Jörg Standfuss 21:00 - 21:15 8

Chia-Ying Huang 21:15 - 21:30 9

David F. Sargent 21:30 - 21:45 10

Session 2 Tiankun Zhou Thursday, September 29th, 09:00 - 09:15 11

Claude Sauter 09:15 - 09:30 12

Dominik A. Herbst 09:30 - 09:45 13

Fabian Renschler 09:45 - 10:00 14

Franziska M. Heydenreich 10:00 - 10:15 15

Dmitry Veprintsev 10:15 - 10:30 16

Magnus Jäckl 10:30 - 10:45 17

Session 3 Nikolaus Dietz 11:15 - 11:30 18

Felix M. Büttner 11:30 - 11:45 19

Christian Pichlo 11:45 - 12:00 20

Miki H. Feldmüller 12:00 - 12:15 21

Melanie H. Dietrich 12:15 - 12:30 22

Keynote lecture Werner Kühlbrandt 20:00 - 21:00 23

Session 4 Jennifer Fleming 21:00 - 21:15 24

Andrea E. Prota 21:15 - 21:30 25

Session 5 Michael Hennig Friday, September 30th, 08:45 - 09:00 26

Severine Freisz 09:00 - 09:15 27

Marcus Müller 09:15 - 09:30 28

Tobias Pflüger 09:30 - 09:45 29

Session 6 Yassmine Chebaro 10:30 - 10:45 30

Philip Rößler 10:45 - 11:00 31

Eric Ennifar 11:00 - 11:15 32

Marie-Laure Durand Diebold 11:15 - 11:30 33

Christophe Romier 11:30 - 11:45 34

Anna Belorusova 11:45 - 12:00 35

Fulvia Bono 12:00 - 12:15 36

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Scientific Program – 2016 Regiomeeting

Day 1: Wednesday, September 28th

15:00 - 17:00 Arrival, Registration & Check-in

17:00 - 18:00 Welcome Drinks

18:00 - 19:30 Dinner

Session 1 – Methods in Crystallography

Chair: Dr. Eric Ennifar

19:30 - 19:45 Organizer's Welcome – Prof. Thilo Stehle, University of Tübingen

19:45 - 20:45 Keynote Lecture

Gwyndaf Evans, Diamond Light Source Ltd., page 6

Developments at the interface of measurement and analysis in biocrystallography

20:45 - 21:00 Vincent Olieric, Paul Scherrer Institute, page 7

Evolving Data Collection Strategies for Experimental Phasing: from Single Crystal toSerial Crystallography

21:00 - 21:15 Jörg Standfuss, Paul Scherrer Institute, page 8

From Molecular Snapshots to Molecular Movies using Free Electron Lasers

21:15 - 21:30 Chia-Ying Huang, Paul Scherrer Institute, page 9

IMISX Structure Determination of Membrane (and Soluble) Protein Using X-RayCrystallography and Lipidic Mesophases: In Situ Diffraction Data Collection

21:30 - 21:45 David F. Sargent, ETH Zürich, page 10

Update on automated crystal harvesting with the RodBot

21:45 - Open Discussion & Socializing

Day 2: Thursday, September 29th

07:30 - 08:45 Breakfast

Session 2 – SAXS and Signaling

Chair:

09:00 - 09:15 Tiankun Zhou, University of Konstanz, page 11

Structural characterization of the complex formed by the antiapoptotic CARP protein andthe elastic N2A spring from the titin myofilament

09:15 - 09:30 Claude Sauter, University of Strasbourg, page 12

Looking at protein-only RNase P in interaction with tRNA using an integrative structuralapproach, page

09:30 - 09:45 Dominik A. Herbst, University of Basel, page 13

The Architecture of Fully Reducing Polyketide Synthases

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09:45 - 10:00 Fabian Renschler, Max Planck Institute for Developmental Biology, page 14

Specificity and Promiscuity of the Bazooka PDZ-domains

10:00 - 10:15 Franziska M. Heydenreich, Paul Scherrer Institute, page 15

The inner workings of a GPCR: Molecular basis for biased G protein activation and beta arrestin recruitment

10:15 - 10:30 Dmitry Veprintsev, Paul Scherrer Institute, page 16

Allostery in GPCR signalling

10:30 - 10:45 Magnus Jäckl, Max Planck Institute for Developmental Biology, page 17

Insights into the mechanism of HECT-mediated Ub transfer

10:45 - 11:15 Coffee Break

Session 3 – Infection and Disease - part I

Chair: Prof. Ulrich Baumann

11:15 - 11:30 Nikolaus Dietz, University of Basel, page 18

Molecular basis for Rho-family GTPase discrimination by a bacterial virulence factor

11:30 - 11:45 Felix M. Büttner, University of Tübingen, page 19

Modulation of the bacterial cell wall by N‐acetylmuramoyl‐L‐alanine amidases

11:45 - 12:00 Christian Pichlo, University of Cologne , page 20

Structural Insights into the Mechanism and Specificity of Proline-Proline Endopeptidase-1from Clostridium difficile

12:00 - 12:15 Miki H. Feldmüller, University of Cologne, page 21

Production, biochemical characterization and structure of a putative metallo-protease fromVibrio Cholerae

12:15 - 12:30 Melanie H. Dietrich, University of Tübingen, page 22

The Tail Domain of Reovirus Attachment Fiber Protein σ1

12:30 - 13:30 Picnic / Lunch Boxes

13:30 - 18:00 Excursion / Social Activity

18:00 - 20:00: Barbecue

20:00 - 21:00 Keynote Lecture

Werner Kühlbrandt, Max Planck Institute of Biophysics, page 23

High-resolution cryo-EM of protein complexes

Session 4 – Infection and Disease - part II

Chair: Dr. Christophe Romier

21:00 - 21:15 Jennifer Fleming, University of Konstanz, page 24

Investigations into the supra-assembly of the myofilament titin into filaments

21:15 - 21:30 Andrea E. Prota, Paul Scherrer Institute, page 25

Pironetin Binds Covalently to α-Cys316 and Perturbs a Major Loop and Helix of α-Tubulinto Inhibit Microtubule Formation

21:30 - Open Discussion & Socializing

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Day 3: Friday, September 30th

07:30 - 08:45 Breakfast

Session 5 – Company Talks

Chair:

08:45 - 09:00 Michael Hennig, CEO leadXpro AG, page 26

Advances in Structure Determination of Membrane Protein Targets using Serial Crystallography

09:00 - 09:15 Severine Freisz, Bruker AXS GmbH, page 27

Pushing back frontiers: Advanced in-house crystallography using the next generation D8VENTURE

09:15 - 09:30 Marcus Müller, DECTRIS Ltd., page 28

Pushing the limits of crystallography with EIGER

09:30 - 09:45 Tobias Pflüger, NanoTemper Technologies GmbH, page 29

2016: A Space Odyssey to Greek Mythology

09:45 - 10:30 Coffee Break & Group Photo

Session 6 – Complementary Methods and DNA / RNA Interaction

Chair:

10:30 - 10:45 Yassmine Chebaro, CNRS, page 30

Allosteric regulation by phosphorylation in nuclear receptor proteins: molecular dynamics study of the Retinoic Acid Receptor alpha and gamma

10:45 - 11:00 Philip Rößler, Max Planck Institute for Developmental Biology, page 31

Application of Methionine Scanning to High-Molecular Weight Complexes

11:00 - 11:15 Eric Ennifar, CNRS / University of Strasbourg, page 32

Thermodynamics of the Ribosome Translation Machinery

11:15 - 11:30 Marie-Laure Durand Diebold, Max Planck Institute for Biochemistry, page 33

The architecture of the Smc protein

11:30 - 11:45 Christophe Romier, IGBMC, page 34

Structural basis for the deposition and eviction of H2A.Z/H2B from the nucleosome byhuman YL1 and ANP32E histone chaperones

11:45 - 12:00 Anna Belorusova, IGBMC, page 35

Structural and biophysical studies of the vitamin D nuclear receptor complex with thecoactivator MED1

12:00 - 12:15 Fulvia Bono, Max Planck Institute for Developmental Biology, page 36

The bicoid mRNA localization factor Exuperantia is an RNA-binding pseudonuclease

12:15 - 12:25 Concluding Remarks

12:25 - 14:00 Lunch

14:00 Departure

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Notes on

Excursion / Social Activity

Brewery Tour

The brewery “Herbsthäuser” is located in Bad Mergentheim. A bus will pick you up at the bus stop in

front of the monastery. The bus leaves at 14:00. There will be a brewery visitor tour including a beer

tasting and some fingerfood.

Climbing Crag

The climbing crag “Waldkletterpark Hohenlohe” is located in Langenburg. Climbing will take place

irrespectively of the weather. Be prepared for bad weather. There will be a bus waiting for you at the

bus stop in front of the monastery. Departure is at 14:00.

Hiking

There will be two hiking tours with different lengths. Meeting point is at the fountain of the inner

courtyard of the monastery at 14:00.

Schöntal-Storchenturm-Rossach-Schöntal-Rundweg

The length of this tour is 7.5 km (140 height meters) with an estimated walking time of 2 h.

Schöntal-Halsberg-Westernhausen-Bieringen-Rundweg

The length of this tour is 12 km (160 height meters) with an estimated walking time of 3 h.

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Abstracts of the 2016 Regiomeeting

Session 1 – Methods in Crystallography

Session 2 – SAXS and Signaling

Session 3 – Infection and Disease – part I

Session 4 – Infection and Disease – part II

Session 5 – Company Talks

Session 6 – Complementary Methods and RNA / DNA Interactions

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Developments at the interface of measurement and analysis inbiocrystallography

Gwyndaf Evans1

1 Diamond Light Source Ltd., Harwell Science & Innovation Campus, OX11 0DE Didcot, Oxfordshire, United Kingdom.

E-mail: gwyndaf.evans@diamond.ac.uk

Since the start of user operations at Diamond Light Source in 2007 significant investment has been

made into the Macromolecular Crystallography (MX) beamlines and the software to support it. During

these years, changes in X-ray sources, beamlines and detectors have driven evolution in data

collection methodology: what was previously impossible is now challenging (Figure 1a) and what

was challenging is now routine.

Behind decades of success in the field of MX lies a goldmine of data analysis software from many

groups and individuals around the world. The recent developments outlined above however have

created a clear need for new programs that can keep pace with a rapidly evolving field that now

includes room temperature in situ data collection, free-electron lasers, serial crystallography and

electron crystallography or MicroED.

This presentation will highlight the developments at Diamond that have contributed to these changes

and introduce software developments (specifically DIALS) that are keeping pace with them (Figure

2b). The talk will conclude with a view as to what is expected in the coming years from synchrotron

beamline developments at Diamond.

(a) (b)Figure 1 (a) View of 1 μm3 crystals of CPV17 polyhedrin used for multi-crystal data collection from Diamond beamline I24. (b) Multi-experiment model used by the DIALS software for multi-crystal or serial crystallography data analysis of synchrotron and XFEL data sets.

KeywordsMacromolecular Crystallography, Data Analysis, Synchrotron, X-ray beamline

References(1) Ginn HM, et al. (2015). Nat Commun 6, 6435 .(2) Gildea RJ, et al. (2014). Acta Cryst. D70, 2652-2666. (3) Nannenga BL, Shi D, Leslie AGW, & Gonen T (2014). Nat Meth 11, 927-930. (4) Waterman DG, et al. (2016). Acta Cryst. D72, 558-575.

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Evolving Data Collection Strategies for Experimental Phasing: from Single Crystal to Serial Crystallography

Vincent Olieric1

1 Swiss Light Source, Paul Scherrer Institute, Villigen, Switzerland

E-mail: vincent.olieric@psi.ch

Because many structures cannot be solved by molecular replacement, the demand for efficient de

novo crystallographic phasing methods is still high. Phasing by SAD (single-wavelength anomalous

diffraction) is nowadays the method of choice owing to its experimental and operational simplicity.

However, this method has very high requirements for measurement accuracy and, therefore, requires

careful data collection strategies to minimise the measurement error of the anomalous signal.

Recently, advances in instrumentation and data collection protocols have improved the quality

of the anomalous data. I will review those developed at the Swiss Light Source, which are particularly

powerful for native SAD phasing. In addition, I will present experimental phasing with serial

crystallography for micron-sized LCP-grown crystals of membrane proteins.

Keywords

Native SAD phasing, anomalous signal, data-collection strategy, membrane protein

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From Molecular Snapshots to Molecular Movies using Free-ElectronLasers

Jörg Standfuss1

1 Paul Scherrer Institute, Villigen, Switzerland

E-mail: joerg.standfuss@psi.ch

In 2017 the Swiss Free Electron Laser (SwissFEL) will start its operation at the Paul Scherrer

Institute. Serial femtosecond crystallography (SFX) using such X-ray free-electron lasers (XFELs) is

a powerful method to determine radiation damage free high-resolution structures and to study protein

dynamics at room temperature and with true time resolution.

One of the current bottlenecks in XFEL science is that most facilities are still under construction

and, even when they will be finished, access will likely remain scarce. In this presentation I will

describe how we have adapted high viscosity injector systems to carry out serial millisecond

crystallography (SMX) at synchrotron sources (1), where beamtime is more abundant. Based on

these results we improved density and homogeneity of crystal preparations for efficient time-resolved

data collection at XFEL sources (2). Structural intermediates of the light-driven proton pump

bacteriorhodopsin (bR) obtained with pump probe delays in the pico- to millisecond range

demonstrate the feasibility of using sample efficient high viscosity injectors to characterize the

molecular dynamics of membrane proteins in a native like environment.

Keywords

Time-resolved serial femtosecond crystallography (TR-SFX), Free-electron lasers, membrane

protein, bacteriorhodopsin, proton pump

References

(1) Nogly P, et al. (2015) Lipidic cubic phase serial millisecond crystallography using synchrotron radiation. IUCrJ 2(2):168–176.

(2) Nogly P, et al. (2016) Lipidic cubic phase injector is a viable crystal delivery system for time-resolved se-rial crystallography. Nat Commun 7:12314.

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Structure Determination of Membrane (and Soluble) Protein Using X-RayCrystallography and Lipidic Mesophases: In Situ Diffraction Data

Collection

Chia-Ying Huang1,2, N. Howe2, V. Olieric1, R. Warshamanage1, P. Ma2,3, E. Panepucci1, X. Liu4, B.Kobilka4,5, K. Diederichs6, M. Wang1, M. Caffrey2

1 Swiss Light Source, Paul Scherrer Institute, Villigen, Switzerland, 2 Membrane Structural and FunctionalBiology Group, Schools of Medicine and Biochemistry and Immunology, Trinity College, Ireland, 3 Laboratory ofStructure and Function of Biological Membranes, Center for Structural Biology and Bioinformat ics, UniversitéLibre de Bruxelles, Belgium, 4 School of Medicine, Tsinghua University, China, 5 Department of Molecular andCellular Physiology, Stanford University School of Medicine, Stanford, USA, 6 Fachbereich Biologie, UniversitätKonstanz, Germany

E-mail: cyhuang107@gmail.com

The lipid cubic phase continues to grow in popularity as a medium in which to generate crystals of

membrane and soluble proteins for high-resolution X-ray structure determination. To date, the PDB

includes 346 records with 113 unique structures attributed to the lipid cubic phase method. However,

it is challenging to harvest crystals from lipid cubic phase. Here, we present a novel in meso in situ

serial crystallography (IMISX) method that employs a thin plastic window for in situ data collection.

The new approach has been used to generate high-resolution crystal structures of a G protein-

coupled receptor, α-helical and β- barrel transporters, and an enzyme at room (IMISX) and/or

cryogenic (IMISXcyro) temperatures. The DA+ at the PX beamlines in the Swiss Light Source (SLS)

is used for semi-automated and high-throughput crystal picking for the data collection of serial

crystallography. IMISX can apply for both MR and phasing by bromine and native sulfur SAD

methods. The method works with inexpensive materials and is compatible with high-throughput in situ

serial data collection at synchrotron beamlines.

Keywords

Experimental phasing, G protein-coupled receptor, in situ, lipid cubic phase, serial crystallography

References

(1) Caffrey, M., Cherezov, V. (2009) Nature Protocols. 4:706-731.

(2) Caffrey, M. (2015) Acta Cryst. F71, 3-18.

(3) Huang, C.-Y. et al. (2015) Acta Cryst. D71, 1238-1256.

(4) Huang, C.-Y. et al. (2016) Acta Cryst. D72, 93-112.

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Update on automated crystal harvesting with the RodBot

David F. Sargent1,2, L. Somm1, Y. Fang1, B. Zeydan1, B. J. Nelson1

1 Institute of Robotics and Intelligent Systems, ETH Zürich, 2 Institute of Molecular Biology & Biophysics, ETHZürich

E-mail: sargent@mol.biol.ethz.ch

Many approaches have been presented for mechanically-assisted crystal harvesting, but this step

remains as the main gap in the otherwise highly automated process of structure determination by X-

ray crystallography. We recently introduced a rod-shaped magnetic microrobot (the “RodBot”) to

assist in the harvesting process (1). Driven by rotating magnetic fields to roll on a substrate, RodBots

induce fluid flows that can gently lift crystals off the surface and trap them in a cylindrical vortex that

travels with the RodBot. The whole operation of crystal selection and harvesting is remotely and

gently carried out without the operator jitter or application of excessive stress that lead to high late-

stage failure rates in manual crystal harvesting. Guidance is provided by the driving magnetic field,

and can involve either manual input with a joystick or fully automated algorithms with feedback

control. In this talk I will review our progress toward the complete automation of the process involving

recognition of both the crystal and the RodBot in low intensity uv light and the development of

algorithms to select, pickup and deliver the crystal to the loop.

Keywords

Crystal harvesting, RodBot, automation

References

(1) H. W. Tung, D. F. Sargent and B. J. Nelson, Protein crystal harvesting using the RodBot: a wireless mo-bile microrobot. J. Appl. Cryst. (2014). 47, 692–700.

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Structural characterization of the complex formed by the antiapoptoticCARP protein and the elastic N2A spring from the titin myofilament

Tiankun Zhou1, J. Fleming1, B. Franke1, O. Mayans1

1 Department of Biology, University of Konstanz, D-78457 Konstanz, Germany

E-mail: t iankun. z hou@uni-konstanz.de

The cardiac ankyrin repeat protein, CARP, is induced in the heart upon disease, mechanical or toxic

stress. It increases the resistance of cardiomyocytes to apoptosis, protecting the myocardium against

damage. Stress-induced CARP targets primarily the N2A spring region of the titin filament in the

sarcomere, but the functional and molecular bases of this interaction are unclear. We investigate the

CARP/titin-N2A complex using X-ray crystallography, small-angle X-ray scattering (SAXS) and

biophysical approaches (CD, NMR). Results to date (1) have shown that CARP’s binding site in titin

spans the dual domain UN2A-Ig81. Unexpectedly, the unique sequence UN2A is not intrinsically

unstructured as previously believed, but it has a thermally-stable α-helical fold of acutely elongated

shape. Such helical domains can behave as constant-force springs, acting as mechanical buffers in

the sarcomere. In brief, our current data portray CARP/titin-N2A as a structured node, where CARP

appears to protect the N2A spring against phosphorylation by PKA (which reduces its passive force),

thereby preserving the mechanical resilience of this element.

Keywords

Recombinant proteins, CD, NMR, X-ray crystallography, SAXS

References

(1) Zhou T et al. FEBS Lett. (2016). doi: 10.1002/1873-3468.12362. [Epub ahead of print]

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Looking at protein-only RNase P in interaction with tRNA using anintegrative structural approach

F. Pinker1, 2, C. Schelcher2, P. Fernandez-Millan1, A. Gobert2, C. Birck3, A. Thureau4, P. Roblin4, P.Giegé2, Claude Sauter1

1Institut de Biologie Moléculaire et Cellulaire, CNRS, Université de Strasbourg, 2Institut de Biologie Moléculairedes Plantes, CNRS, Université de Strasbourg, 3Institut de Génétique et de Biologie Moléculaire et Cellulaire,CNRS, Université de Strasbourg, 4Synchrotron SOLEIL, Gif-sur-Yvette.

E-mail: c.sauter@ibmc-cnrs.unistra.fr

The RNase P activity is ubiquitous and consists of the 5’ maturation of pre-tRNAs. For a long time it

has been thought that all RNase P were ribozymes. However, a novel kind of RNase P composed of

proteins only, called PRORP for “Protein-only RNase P” was first discovered in human mitochondria,

then described in Arabidopsis thaliana (1,2). The latter possesses three PRORP homologs: PRORP1

located in mitochondria and chloroplasts, PRORP2 and PRORP3 in the nucleus.

We used an integrative approach to provide a structural and functional description of Arabidopsis

PRORP enzymes (3). The affinity constant between a minimal tRNA substrate and a catalytically

inactive PRORP2 enzyme determined by microscale thermophoresis (MST), ultracentrifugation and

calorimetry (ITC) is in the 0.6-1 uM range. A combination of mutagenesis and affinity measurements

helped define the respective importance of individual pentatricopeptide repeats (PPR) of PRORP2 for

RNA binding. A comparison of the crystal structure of PRORP2 and of solution structures of the

enzyme and its complex with a pre-tRNA obtained by SAXS indicated that PRORP2 undergoes

structural changes to accommodate its substrate. A dedicated SAXS setup was implemented to

stabilize the complex during analysis. Altogether this work reveals the structural diversity and

plasticity of protein-only RNase P enzymes.

Keywords

tRNA-maturation, RNase-P, PRORP, SAXS, biophysics

References

(1) Holzmann et al. RNase P without RNA: identification and functional reconstitution of the human mitochon-drial tRNA processing enzyme. Cell (2008), 135, 462-74.

(2) Gobert et al., A single Arabidopsis organellar protein has RNase P activity. NSMB (2010), 17, 740-4.

(3) Gobert et al., Structural insights into protein-only RNase P complexed with tRNA. Nat Commun (2013), 4,1353.

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The Architecture of Fully Reducing Polyketide Synthases

Dominik A. Herbst1, R. P. Jakob, F. Zähringer, T. Maier

1 Biozentrum, University of Basel, Klingelbergstr. 70, CH-4056 Basel, Switzerland

E-mail: dominik.herbst@unibas.ch

Polyketides are a diverse family of bioactive microbial secondary metabolites and amongst the most

successful compound classes in drug discovery. They are assembled via stepwise precursor

elongation by giant polyketide synthases (PKSs)(1). PKSs combine all enzymatic domains for a

single-step of precursor elongation and modification in one module, which features a structural and

functional separation into a product condensing and a modifying region.

The product of each PKS module is encoded by its substrate specificity and the variable domain

composition of the modifying region. The maximum extend of product modification is observed in

reducing PKS with a molecular organization that directly encodes the product.

Here we report a hybrid model(2) of a reducing mycocerosic acid synthase-like PKS (MAS-PKS)(3),

based on overlapping crystal structures of its condensing and modifying regions. A comparison of

experimentally observed MAS-PKS conformations provides a visualization of structural dynamics and

conformational coupling in PKSs. The modifying region of MAS-PKS adopts a unique dimeric linker-

based organization devoid of stable interactions, which is in agreement with evolutional domain

shuffling. Comparative small angle X-ray scattering demonstrates that this architecture is common to

other PKS. Our comprehensive model of PKS architecture will contribute to the functional dissection

and targeted re-engineering of PKSs for enabling combinatorial biosynthesis.

Keywords

Polyketide, PKS, antibiotics, X-ray, SAXS

References

(1) Hertweck, C. (2009) Angew. Chem. Int. Ed. Engl. 48, 4688-4716.

(2) Herbst, D.A., et al. (2016) Nature 531, 533-537.

(3) Etienne, G., et al. (2009) J Bacteriol. 191, 2613-21.

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Specificity and Promiscuity of the Bazooka PDZ-domains

Fabian Renschler1, S. Brükner1, B. J. Schroeder1, P. Salomon1,2, M. C. Schütz-Stoffregen1, S.Wiesner1

1 MPI for Developmental Biology, Spemannstr. 35, 72076 Tübingen, Germany2 present address: ImmunoGen Inc., Waltham, United States

E-mail: fabian.renschler@tuebingen.mpg.de

PDZ (Postsynaptic density-95/ Disc-large/ Zonula occludens) domains are small protein-protein

interaction domains which recognize the four to eight C-terminal amino acids of their ligands, also

called PDZ binding motif (PBM). However, PDZ domains are known to be promiscuous and are

therefore frequently able to recognize several different C-termini. Despite the efforts undertaken in

the last years the question of how PDZ promiscuity still allows PDZ specificity has not been solved

completely. To this end, I choose the dmPar-3 protein Bazooka (Baz) as a model for PDZ specificity

and promiscuity. Baz is the central scaffolding protein of the highly conserved PAR complex, a central

determinant of apical-basal cell polarity. In this context, several ligands have been proposed to bind

to at least one Baz PDZ domain in the literature.

Here, I present a combination of NMR spectroscopy and X-ray crystallography which enabled me to

dissect the specificities of the individual Baz PDZ domains towards know ligands and to reveal the

structural basis of the promiscuity and selectivity of the Baz PDZ domains. I was able to determine

the Baz PDZ binding profiles against Inscuteable (Insc), α-catenin, atypical protein kinase C, dmPar-

6, Echinoid and Shotgun (Shg) using 2D 1H,15N-HSQC experiments and to solve the X-ray

structures of Baz PDZ1 in complex with the Shg- and dmPar6-PBM as well as of Baz PDZ2 in

complex with the Insc-PBM.

Keywords

PDZ domain specificity, Bazooka/Par-3, cell polarity

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The inner workings of a GPCR: Molecular basis for biased G proteinactivation and β-arrestin recruitment

Franziska M. Heydenreich1, T. Flock2, B. Plouffe3, X. Deupi1, M. Babu2, M. Bouvier3, D. B.Veprintsev1

1 Laboratory of Biomolecular Research, Paul Scherrer Institute, 5232 Villigen, Switzerland and Department ofBiology, ETH Zurich, 8093 Zurich, Switzerland, 2 MRC Laboratory of Molecular Biology, Francis Crick Avenue,Cambridge CB2 0QH, United Kingdom, 3 Department of Biochemistry and Institute for Research in Immunologyand Cancer, University of Montreal, Montreal, Quebec, Canada

E-mail: franziska.heydenreich@psi.ch

G protein coupled receptors (GPCRs) are pharmacologically important membrane proteins involved

in the transmission of signals into the cell. While they can be activated by a diverse set of ligands

including small molecules, hormones, neurotransmitters or photons, GPCRs signal through only 16 G

proteins and 2 β-arrestins. Insights into how and why GPCRs select specific G proteins opens up

new opportunities for drug design and the possibility of drugs with fewer side effects. Using alanine

scanning mutagenesis on a GPCR, coupled with pluridimensional signalling profiling, we determined

a molecular map of residues involved in the activation of G proteins from different subfamilies and

recruitment of β-arrestins at single amino acid resolution. While some residues were important for

both G protein activation and beta-arrestin recruitment, others specifically affected either of the

signalling pathways. Clustering of the residues involved in those signalling pathways allowed us to

connect ligand binding pocket and G protein/arrestin binding region by several distinct allosteric

paths (1,2), which were specific for the activation of G proteins, G protein subtypes and the

recruitment of β-arrestins. Our data gives us insights into the molecular basis of G protein activation,

selection of G protein subtype and allows us to better understand the conformational changes

needed for β-arrestin recruitment, desensitization and internalization of receptors.

Keywords

G protein coupled receptors, G proteins, signaling, functional mapping, mutagenesis

References

(1) Heydenreich F, Brueckner F, Tsai C, Schertler GFX, Veprintsev DB, Grzesiek S. (2016). Nature 530, 237–241.

(2) Venkatakrishnan AJ, Deupi X, Lebon G, Heydenreich FM, Flock T, Miljus T, Balaji S, Bouvier M, Veprint -sev DB Tate CG, Schertler GFX, Babu MM. (2016). Nature, 536, 484–487.

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Allostery in GPCR signalling

Dmitry Veprintsev1,2

1 Paul Scherrer Institute, Villigen, Switzerland, 2 Department of Biology, ETH Zürich, 8093 Zürich, Switzerland

E-mail: dmitry.veprintsev@psi.ch

The signaling events in G protein coupled receptors (GPCRs) propagate through protein by

concerted local conformational changes, forming allosteric pathways. Using a combination of NMR

and mutagenesis as well as structural bioinformatics, we identified several independent allosteric

activation pathways of the β1-adrenergic receptor that connect the ligand binding pocket with the

activity of the receptor. In order to identify the allosteric networks in the G protein, we generated

comprehensive single amino acid resolution maps of the residues stabilising the human Gαi1 subunit

in nucleotide- and receptor-bound states. We generated these maps by measuring the effects of

alanine mutations on the stability of Gαi1 and of the rhodopsin Gαi1 complex. We identified

stabilization clusters in the GTPase and helical domains responsible for structural integrity of the

protein, the conformational changes associated with activation, as well as changes in the dynamics

of individual amino acids. The proposed methods can be readily applied to identify and study

allosteric pathways in other proteins.

Keywords

G protein coupled receptors, G proteins, NMR, mutagenesis, signalling

References

(1) Sun D., Flock T., Deupi X., Maeda S., Matkovic M., Mendieta S., Mayer D., Dawson R.J., Schertler G.F.,Babu M.M., Veprintsev D.B. (2015). NSMB 22, 686-94.

(2) Flock T, Ravarani CN, Sun D, Venkatakrishnan AJ, Kayikci M, Tate CG, Veprintsev DB, Babu MM. (2015)Universal allosteric mechanism for Gα activation by GPCRs. Nature 524, 173-9.

(3) Isogai S, Deupi X, Opitz C, Heydenreich FM, Tsai CJ, Brueckner F, Schertler GFX, Veprintsev DB, Grze-siek S (2016) Protein backbone NMR reveals efficacy-dependent allosteric signaling networks in the β1-adrenergic receptor. Nature 530, 237-41.

(4) Venkatakrishnan AJ, Deupi X, Lebon G, Heydenreich FM, Flock T, Miljus T, Balaji S, Bouvier M, Veprint -sev DB, Tate CG, Schertler GFX & Babu MM (2016) Diverse activation pathways in class A GPCRs con-verge near the G protein-coupling region. Nature 536, 484-7.

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Insights into the mechanism of HECT-mediated Ub transfer

Magnus Jäckl1, K. Hyz1, T. Strohäker1, S. Wiesner1

1 MPI for Developmental Biology, Spemannstr. 35, 72076 Tübingen, Germany

E-mail: magnus.jaeckl@tuebingen.mpg.de

The small protein Ubiquitin (Ub) can be attached to proteins as post-translational modification. This

modification plays a role in a wide array of cellular processes, e.g. as a signal for protein degradation.

Three enzymes are required for the ubiquitinylation reaction, a Ub-activating (E1), a conjugating (E2)

and a ligating (E3) enzyme. These enzymes contain strictly conserved Cys residues that form

thioester intermediates with the Ub C-terminus during Ub transfer. Among the ubiquitylation enzymes,

E3s play a crucial role as they confer substrate specificity and determine Ub chain length and linkage

type. Although the basic ubiquitylation machinery is known, many aspects of the catalytic

mechanisms are still elusive. Here, we have characterized a HECT-type E3 thioester by NMR

spectroscopy and X-ray crystallography. We show that the HECT thioester can adopt an open and a

closed conformation. In the closed conformation the Ub C-terminus forms an additional beta-strand

with the C-terminal lobe of the HECT domain presumably representing the state adopted directly after

E2-E3 transthiolation. In the open conformation, the thioester-linked Ub interacts non-covalently with

a Ub binding surface located in the N-terminal lobe of the HECT domain. Our data thus provides

important mechanistic insights into Ub transfer from the E2 to the E3 and finally the substrate.

Keywords

Ubiquitin HECT ubiquitinylation reaction, X-ray, NMR

References

(1) E. Maspero, E. Valentini, S. Mari, V. Cecatiello, P. Soffientini, S. Pasqualato, S. Polo. Nat.Struct.Mol.Biol.(2013). 20, 696-701.

(2) H. B. Kamadurai, Y. Qiu, A. Deng, J. S. Harrison, C. Macdonald, M. Actis, P. Rodrigues, D. J. Miller, J.Souphron, S. M. Lewis, I. Kurinov, N. Fujii, M. Hammel, R. Piper, B. Kuhlman, B.A Schulman, Elife (2013).doi: 10.7554/eLife.00828.

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Molecular basis for Rho-family GTPase discrimination by a bacterialvirulence factor

Nikolaus Dietz1, A. Harms1,#, I. Sorg1, G. Mas2, A. Goepfert2†,, S. Hiller2, T. Schirmer2, C. Dehio1

1 Focal Area Infection Biology Biozentrum, Universität Basel, Klingelbergstr. 70, CH-4056 Basel, Switzerland2 Focal Area Structural Biology and Biophysics, Universität Basel, Klingelbergstr. 70, CH-4056 Basel,Switzerland † Present address: Novartis Institutes for Biomedical Research, CPC/ Structural Biophysics, Basel,Switzerland #Present address: Center of Excellence for Bacterial Stress Response and Persistence (BASP),Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, DK-2200 Copenhagen N, Denmark

E-mail: ni kolaus.schmitz@unibas.ch

Ras-homology (Rho)-family GTPases are conserved molecular switches controlling fundamental

cellular activities. As such they are targeted by numerous bacterial virulence factors, which have

been intensively investigated regarding their molecular modes of action and discrete target spectra.

Surprisingly, structural elements enabling selective targeting of Rho-family GTPase subsets have

remained largely unknown. Here, we show that the bacterial FIC domain AMP-transferase Bep1 from

Bartonella rochalimae exclusively targets Rac-subfamily members. The exquisite target selectivity is

based on electrostatic interactions with subfamily-specific residues in the Rho-insert helix and the

nucleotide-binding motif. Residue substitution at the identified positions in RhoA and Cdc42 convert

these GTPases into Bep1 targets. Employing a combination of biochemistry, crystallography, nuclear

magnetic resonance (NMR) spectroscopy and mutational analysis, we identify the structural

determinants of this remarkably narrow target selectivity. Our findings further provide a rationale for

altering Bep1 target selectivity for the creation of new tools with surgical precision for dissecting Rho-

family GTPase activities.

Keywords

Crystallography, NMR, Infection biology

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2016 Regiomeeting

Modulation of the bacterial cell wall by N‐acetylmuramoyl‐L‐alanineamidases

Felix M. Büttner1, K. Faulhaber, K. Forchhammer2, I. Maldener2, T. Stehle1,3

1 IFIB, University of Tübingen, Hoppe-Seyler-Straße 4, 72076 Tübingen, Germany, 2 Interfaculty Institute forMicrobiology and Infection Medicine, Department of Organismic Interactions, University of Tübingen, Auf derMorgenstelle 28, 72076 Tübingen, Germany, 3 Department of Pediatrics, Vanderbilt University School ofMedicine, Nashville, Tennessee, United States of America

E-mail: felix.buettner@uni-tuebingen.de

The bacterial cell wall is a highly dynamic structure that undergoes constant change in order to fulfill

its various tasks, which range from physical protection against exterior stress and maintaining

homoeostasis to immune evasion. A major component of the bacterial cell wall is the peptidoglycan

network (PGN). The PGN is a net-like structure that is composed of a carbohydrate backbone linked

to a peptide stem containing non-proteinogenic amino acids. It harbors various proteins and anchors

further components of the cell wall. The composition of the peptide stems and their type of cross-

linkage determines whether the PGN is a very dense network or a rather loose mesh. N-

acetylmuramoyl-L-alanine amidases cleave the amide bond between the carbohydrate backbone and

the peptide stem. They represent a class of PGN-modulating enzymes that ensure its plasticity and

sometimes serve distinctive functions.

AmiC2 of the filamentous cyanobacterium Nostoc punctiforme fulfills such a unique task in order to

enable communication of neighboring cells within a filament. In contrast to cell-splitting amidases,

AmiC2 drills holes into the septal disk that separates neighboring cells, thus generating a nanopore

array used for nutrient exchange and communication. AmiC2 was located in the maturating septum

and we solved the structure of the catalytic domain of this enzyme, AmiC2-cat. In comparison with

the homologous enzyme AmiC E. coli, a regulatory α-helix is missing, and AmiC2-cat exhibits high

activity, which can be abolished by mutation of a catalytic glutamate. Ongoing research is focused on

the mechanism that governs activity and specificity of this unusual amidase. In particular, we study

the separate and / or cooperative influence of the additional domains of the AmiC2 holo-enzyme on

catalysis and specificity. A comparative approach of amidases may hint toward the mode of substrate

engagement by the enzyme.

Keywords

N-acetylmuramoyl-L-alanine amidase, AmiC2, bacterial cell wall, peptidoglycan, nanopore array

References

(1) Büttner et al. 2014, JBC, doi:10.1074/jbc.M114.557306

(2) Büttner et al. 2015, IJMM, doi:10.1016/j.ijmm.2014.12.018

(3) Büttner et al. 2016, FEBS, doi:10.1111/febs.13673

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Structural Insights into the Mechanism and Specificity of Proline-ProlineEndopeptidase-1 from Clostridium difficile

Christian Pichlo 1, U. Baumann1, M. Schacherl1

1 Institute of Biochemistry, University of Cologne, Otto-Fischer Str. 14, 50674 Cologne, Germany

E-mail: pichloc@uni-koeln.de

Clostridium difficile is a gram-positive hospital pathogen responsible for severe gastrointestinal

infections such as pseudomembranous colitis. Proline-proline endopeptidase-1 (PPEP-1) is a

secreted metalloprotease from C. difficile showing a unique specificity for Pro-Pro peptide bonds (1).

Its endogenous substrates are two surface proteins and one of them was identified as a collagen-

binding adhesion protein. In this line, deletion of PPEP-1 leads to an increased collagen affinity of C.

difficile (2). Therefore PPEP-1 can be seen as an important factor in colonization and dissemination

of C. difficile.

We solved crystal structures of PPEP-1 in its unbound as well as peptide-bound form shedding light

on the substrate binding mode and the strict specificity for Pro-Pro peptide bonds (3,4). Analysis of

the proteolytic activity of PPEP-1 mutants revealed that the residues K101, W103 and E184 of

PPEP-1 are crucial for substrate recognition, underlining the importance of a flexible loop, located

directly above the active site cleft, in defining substrate specificity of PPEP-1 beyond the residues

flanking the scissile bond.

Keywords

Pathogen, endopeptidase, metalloprotein, substrate specificity, peptide co-crystallization

References

(1) Hensbergen P.J., Klychnikov O.I., Bakker D., van Winden V.J., Ras N., Kemp A.C., Cordfunke R.A., Dra-gan I., Deelder A.M., Kuijper E.J., Corver J., Drijfhout J.W., van Leeuwin H.C., Mol. Cell. Pro-teomics (2014).

(2) Hensbergen P.J., Klychnikov O.I., Bakker D., Dragan I., Kelly M.L., Minton N.P., Corver J., Kuijper E.J.,Drijfhout J.W., van Leeuwen H.C., FEBS. Lett. (2015).

(3) Schacherl M., Pichlo C., Neundorf I., Baumann U., Structure (2015).

(4) Pichlo C., Montada A.A.M., Schacherl M., Baumann U., J. Vis. Exp. In Press

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2016 Regiomeeting

Production, biochemical characterization and structure of a putativemetalloprotease from Vibrio Cholerae

C. Pichlo1, Miki H. Feldmüller1, M. Schacherl1, U. Baumann1

1 Institute of Biochemistry, University of Cologne, Otto-Fischer Str. 14, 50674 Cologne, Germany

E-mail: mfeldmue@smail.uni-koeln.de

Vibrio cholerae is a bacterial species including several non-pathogenic aquatic bacteria strains as

well as pathogenic strains causing severe epidemic to pandemic gastrointestinal infections called

Cholera with symptoms such as vomiting and numerous voluminous watery stools resulting in

hypovolemic shocks or acidosis (1). All pathogenic Vibrio cholerae strains, which caused epidemic or

pandemic outbreaks of Cholera, possess a pathogenic island – a mobile genetic element carrying

genes of virulence factors improving the viability, colonialization or dissemination of Vibrio cholerae in

the host organism (2). We identified the gene of a putative metalloprotease called VcZmp1 on the

pathogenicity island of Vibrio cholerae, VcZmp1 shares approximately 20 % sequence identity with

the protease domain of PPEP-1 from Clostridium difficile and Lethal factor (LF) from Bacillus

anthracis. Both, PPEP-1 and LF have a central role during the infection of C. difficle and B. anthracis

in humans (3, 4). First genetic results already showed that deletion of the VcZmp1 gene modulates

the pathogenicity of different Vibrio cholerae strains (5, 6). Here we present the production of

recombinant VcZmp1 as well as a first biochemical characterization of the protein. Furthermore we

show the crystal structure of the protein at a resolution of 2.1 Å, confirming that VcZmp1 has a similar

fold as PPEP-1.

Keywords

Pathogen, virulence factor, metalloprotein

References

(1) Finkelstein, R. A. in Medical Microbiology (Samuel Baron, 1996).

(2) Karaolis, D. K., Johnson, J. a, Bailey, C. C., Boedeker, E. C., Kaper, J. B., & Reeves, P. R. , Proc. Natl.Acad. Sci. U. S. A. (1998).

(3) Hensbergen P.J., Klychnikov O.I., Bakker D., Dragan I., Kelly M.L., Minton N.P., Corver J., Kuijper E.J.,Drijfhout J.W., van Leeuwen H.C., FEBS. Lett. (2015).

(4) Smith, H. & Keppie, Nature (1954).

(5) Zhang, D., Xu, Z., Sun, W. & Karaolis, D. K. R., Infect. Immun. (2003).

(6) Zhang, D., Rajanna, C., Sun, W. & Karaolis, D. K. R., FEMS Microbiol. Lett. (2003).

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2016 Regiomeeting

Tail Domain of Reovirus Attachment Fiber Protein σ1

Melanie H. Dietrich1, A. Thor1, R. Ebenhoch1, T. S. Dermody2, T. Stehle1

1 Interfaculty Institute of Biochemistry, University of Tübingen, Tübingen, Germany, 2 Department of Pediatrics,University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA

E-mail: melanie.dietrich @uni-tuebingen.de

Attachment of mammalian orthoreoviruses (reoviruses) to target cells is mediated by the outer-capsid

protein σ1. The σ1 protein is a filamentous trimer with three distinct domains: the tail, the body, and

the head. Following receptor binding, reovirus virions enter cells by endocytosis. Acid-dependent

proteolysis yields ISVPs, the first disassembly intermediate. EM reconstructions of reovirus virions

and ISVPs indicate that σ1 assumes a more compact conformation on virions, while it extends as an

elongated structure from ISVPs suggesting a structural rearrangement in σ1 during virion-to-ISVP

conversion. EM images of σ1 isolated from virions show flexibility at a region near the N-terminus, at

the midpoint of the molecule coinciding with the junction of the tail and body domains, and a region

near the head domain. To investigate regions of predicted flexibility, we crystallized parts of the σ1

protein of two reovirus serotypes, T1 and T3. Our 1.4 Å resolution structure of the T1 σ1 tail domain

shows an uninterrupted α-helical coiled coil. The coiled coil harbors two chloride ion binding sites

inside its core, and has a stutter sequence located close to the transition to the body domain. This

discontinuity of the heptad repeat is compensated by a partial unwinding of the coiled coil and the α-

helix. Our 2.25 Å resolution structure of T3 σ1 comprises the tail and a portion of the body domain

and shows a stutter at the same position as in T1 σ1. The structure reveals a seamless transition

from the tail to the body domain, with direct interactions between the body and the end of the coiled

coil. This finding is in contrast to the predicted higher flexibility of σ1 within this region and requires a

reconsideration of the current model. Our structural investigations enabled us to formulate a full-

length model of the elongated σ1 protein and provide a platform for future studies to define the

flexibility of this protein.

Keywords

Virus attachment protein, coiled coil

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2016 Regiomeeting

High-resolution cryo-EM of protein complexes

Werner Kühlbrandt1

1 Max-Planck-Institut für Biophysik, Max-von-Laue-Str. 3, 60438 Frankfurt am Main, Germany.

E-mail: werner.kuehlbrandt@biophys.mpg.de

With the arrival of a new generation of electron detectors and image processing software, cryo-EM of

biological macromolecules has entered a new era (Kühlbrandt, 2014). 3D structures of a quality that

rivals or even exceeds protein crystallography can now be obtained. After determining the 3.3 Å

structure of the hydrogen transferase Frh by single-particle cryo-EM (Allegretti et al., 2014), we are

using the same technique to elucidate the structures of mitochondrial ATP synthase dimers from

Polytomella (Allegretti et al., 2015), and from the yeast Yarrowia lipolytica (Hahn et al., 2016) at 6-7

Å, which resembles the mammalian complex. Both structures show the elusive subunit a of the Fo

stator as consisting of six structurally conserved alpha helices, four of which form a long, membrane-

intrinsic bundle perpendicular to the alpha helices of the c-ring rotor. Moreover, the yeast dimer

structure reveals the structure and position of small hydrophobic subunits at the dimer interface,

explaining how ATP synthase dimers in yeast and mammalian mitochondria are held together.

Applying the new detector technology to electron cryo-tomography, we obtained maps of ATP

synthase dimers in the inner mitochondrial membrane of yeast, Polytomella and Paramecium,

revealing extensive, but different ribbons of ATP synthase dimers in these three organisms. Our

results provide new insights into how mitochondrial ATP synthases are arranged in the membrane,

and how this might help to optimize ATP production by rotary catalysis.

Keywords

Electron cryo-microscopy (cryo-EM), hydrogen transferase, membrane protein structure, ATP

synthase, mitochondria

References

(1) Allegretti, M., Mills, D.J., McMullan, G., Kühlbrandt, W. and Vonck, J. (2014). Atomic model of the F420-re-ducing [NiFe] hydrogenase by electron cryo-microscopy using a direct electron detector. ELife 3, e01963.

(2) Allegretti, M., Klusch, N., Mills, D.J., Vonck, J., Kühlbrandt, W. & Davies, K.M. (2015). Horizontal mem-brane-intrinsic α-helices in the stator a-subunit of an F-type ATP synthase. Nature 521, 237-240.

(3) Hahn A et al, (2016). Structure of the ATP synthase dimer from yeast mitochondria. Mol. Cell, Mol Cell,doi: 10.1016/j.molcel.2016.05.037.

(4) Kühlbrandt W (2014). The resolution revolution. Science 343,1443-1444.

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2016 Regiomeeting

Investigations into the supra-assembly of the myofilament titin intofilaments

Jennifer Fleming1, B. Franke1, K. Diederichs and O. Mayans

1 Department of Biology, Universität Konstanz, 78457 Konstanz, Germany.

E-mail: jennifer.fleming@uni-konstanz.de

Titin is a myofilament crucial to the development, structure and mechanics of human muscle and is a

causative factor in multiple myopathies (1). Titin consists of >300 Ig and FnIII domains linked in

series that extent over 1.2 um length, spanning both the I- and A-bands in the sarcomere (2). At the

I/A junction, titin molecules assemble laterally to form tubules called “end-filaments”, which are

oligomers of six parallel titins (3). These seem to terminate the assembly of myosin motor filaments in

the sarcomere and to bridge symmetry transitions in the lattices of the I- and A-bands. No molecular

knowledge of these formations exists to date. To reconstruct end-filaments in vitro, elucidate the

molecular basis of their assembly and estimate the impact that existing mutations on titin have in

their stability, we have implemented a medium-throughput approach, where multiple constructs

spanning 3-12 Ig domains are produced and studied using native-PAGE, MALS, SAXS and/or X-ray

crystallography. Crystal structures of a dimeric sub-assembly state of a 3Ig fragment (phased using

S-SAD) and a first SAXS model of the tubular formation of a 6Ig segment are now available. A human

mutation linked to dilated cardiomyopathy can be mapped to these structures. Data are currently

under analysis.

Keywords

Titin, SAXS, S-SAD, heart

References

(1) Gigli M, Begay RL, Morea G, Graw SL, Sinagra G, Taylor MR, Granzier H, Mestroni L. (2016). A Review ofthe Giant Protein Titin in Clinical Molecular Diagnostics of Cardiomyopathies. Front Cardiovasc Med. 3,21. doi: 10.3389/fcvm.2016.00021.

(2) Zacharchenko T, von Castelmur E, Rigden DJ, Mayans O. (2015). Structural advances on titin: towards anatomic understanding of multi-domain functions in myofilament mechanics and scaffolding. Biochem SocTrans. 43, 850-5. doi: 10.1042/BST20150084.

(3) Ahmed Houmeida Baron A, Keen J, Khan GN, Knight PJ, Stafford WF 3rd, Thirumurugan K, ThompsonB, Tskhovrebova L, Trinick J. (2008). Evidence for the Oligomeric State of ‘Elastic’ Titin in Muscle Sarcom-eres. J Mol Biol. 384, 299-312. doi: 10.1016/j.jmb.2008.09.030.

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2016 Regiomeeting

Pironetin Binds Covalently to α-Cys316 and Perturbs a Major Loop andHelix of α-Tubulin to Inhibit Microtubule Formation

Andrea E. Prota1, J. Setter2, A. B. Waight3, K. Bargsten1,3, J. Murga4, J. F. Díaz5, M. O. Steinmetz1

1 Paul Scherrer Institut, Villigen PSI, Switzerland, 2 Seattle Genetics, Inc., Bothell, USA, 3 University of Zurich, Switzerland, 4 Univ. Jaume I, Castellón, Spain, 5 CIB-CSIC, Madrid, Spain.

E-mail: andrea.prota@psi.ch

Microtubule-targeting agents are among the most powerful drugs used in chemotherapy to treat

cancer patients. Pironetin is a natural product that displays promising anticancer properties by

binding to and potently inhibiting tubulin assembly into microtubules; however, its molecular

mechanism of action remained obscure. Here, we solved the crystal structure of the tubulin-pironetin

complex and found that the compound covalently binds to Cys316 of α-tubulin. The structure further

revealed that pironetin perturbs the T7 loop and helix H8 of α-tubulin. Since both these elements are

essential for establishing longitudinal tubulin contacts in microtubules, this result explains how

pironetin inhibits the formation of microtubules. Together, our data define the molecular details of the

pironetin binding site on α-tubulin and thus offer a promising basis for the rational design of pironetin

variants with improved activity profiles. They further extend our knowledge on strategies evolved by

natural products to target and perturb the microtubule cytoskeleton.

Keywords

Microtubule-targeting agents, anticancer drug, protein-ligand interactions

References

(1) Prota, A.E., et al. (2016). J Mol Biol. 428, 2981-8.

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2016 Regiomeeting

Advances in Structure Determination of Membrane Protein Targets usingSerial Crystallography

Michael Hennig1

1 leadXpro AG, PARK innovAARE, CH-5234 Villigen, Switzerland.

E-mail: michael.hennig@leadXpro.com

Today, structure based drug discovery is well implemented in the the drug discovery engine of many

pharmaceutical companies. Whereas soluble proteins are managed within the project timelines and

portfolio changes in pharmaceutical industry, transmembrane proteins still represent a significant

challenge. GPCR’s, ion channels and transporters represent important protein drug targets and make

up about 40% of the druggable genome. Unfortunately, many targets have not been yielded drug

molecules due to challenges in discovery of efficient and safe molecules that reach the tissue of

consideration. We aim to combine expertise in drug discovery, excellence in membrane protein

science and use of cutting edge X-ray data collection at synchrotron and X-ray FEL sources enable

lead generation of challenging targets and provide an innovative route to generate and optimize lead

molecules. High quality solubilized and purified membrane proteins can be used to apply biophysical

methods to investigate ligand interaction including X-ray and single particle cryo electron microscopy

to investigate structural changes upon ligand binding. Serial crystallography opens new opportunities

for data collection in combination with synchrotron as well as X-ray free electron laser. Recent

developments and future perspectives of this method will be presented.

Keywords

Membrane protein structures, Free electron laser, Serial crystallography, Drug Discovery

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2016 Regiomeeting

Pushing back frontiers: Advanced in-house crystallography using thenext generation D8 VENTURE

Severine Freisz1, V. Smith1, M. Rappas2, G. Cseke2, J. C. Errey2, A. S. Doré2

1 Bruker AXS GmbH, Oestliche Rheinbrueckenstrasse 49, 76187 Karlsruhe, Germany, 2 Heptares Therapeutics Ltd, BioPark, Welwyn Garden City, UK.

E-mail: severine.freisz@bruker.com

Structure-based drug design (SBDD) organizations working on soluble protein targets continue to

utilize and rely on in-house x-ray sources to generate structural information due to their convenience.

However, membrane protein structural biology has remained almost exclusively reliant on

synchrotron light sources. Diffraction data collection on-site can provide fast turnaround times to

medicinal chemists, enabling key decisions to be made quickly and efficiently in real time.

Over 40% of prescription medications target G protein-coupled receptors (GPCR) a superfamily of

protein receptors which are notoriously difficult to crystallize due to their instability when removed

from the cell membrane, so remaining intractable to most SBDD platforms. The challenges are huge

and yet so are the potential rewards. Heptares proprietary StaR® technology generates

thermostabilized receptors containing a small number of point mutations, homogenous and in a

natural pharmacologically relevant conformation (agonist or antagonist) that matches the drug

product profile. These can then be readily crystallised in both classical vapour diffusion using harsh

short-chain detergents and lipidic cubic phase (LCP) to drive SBDD even with weak early stage

compounds / fragments.

Together, Heptares and Bruker have performed feasibility studies to asses if in-house sources have

reached a level that would have a role in SBDD pipelines specifically dedicated to GPCRs. We find

that Heptares StaR technology alongside Bruker’s state-of-the-art instrumentation enables high

resolution structures to be obtained in-house in a realistic timeframe using the D8 VENTURE. The D8

VENTURE x-ray diffractometer consists of state-of-the art technology; the METALJET source is the

only source available that can deliver small, high intensity x-ray beams and is coupled with the newly

launched PHOTON II CPAD detector.

Here, we present the results obtained using the combination of both technologies yielding a 2.8Å

dataset for the human Orexin-1 StaR in under 120 minutes on the D8 VENTURE. To the best of our

knowledge the structure represents the first atomic resolution GPCR structure to be determined

without the use of synchrotron radiation. Human Orexin-1 has been strongly implicated in the

treatment of cocaine addiction with potential broader applications in substance addictions (nicotine,

alcohol) and compulsive disorders (binge eating, gambling).

Keywords GPCR, Orexin, METALJET

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2016 Regiomeeting

Pushing the limits of crystallography with EIGER

A. Förster1 and Marcus Müller1

1 DECTRIS Ltd., Täfernhof 1, 5405 Baden-Dättwil, Switzerland

E-mail: marketing@dectris.com

With the recently introduced EIGER, hybrid photon counting (HPC) enters a new dimension of spatial

and temporal resolution and expands the field of X-ray experimentation. A pixel size of 75 µm and

continuous read-out with auto-summation increases the quality of the collected data. Frame rates of

up to 750 Hz enable new ways of doing X-ray crystallography like accurate positioning of

microcrystals by diffraction and synchrotron serial crystallography. EIGER R 1M and 4M are mega-

pixel HPC detectors for the laboratory that are based on the same technology as the most powerful

detectors available at select synchrotron beamlines. The absence of any detector noise in

combination with an image bit depth of 32 bit and high spatial resolution turn them into versatile

platforms for hitherto unthinkable laboratory application. A short outline of the differences between

EIGER and PILATUS3 will highlight key aspects of the new detector technology and show that higher

data quality that can be achieved with EIGER (1). Examples from several beamlines will illustrate

ways of setting up serial crystallography (2,3) and grid scanning approaches (4), especially with an

eye on increasing signal to noise from microcrystals. Data obtained from two protein arginine

methyltransferases and a prokaryotic ribosome demonstrate the power of the home source where, in

addition, the long wavelength ensures that significant native anomalous signal is almost always

measured, with benefits for experimental phasing, molecular replacement and metal ion

identification. Concerted upgrades to radiation sources, optics, software and detector instrumentation

take crystallography to the next level, beyond the previous state of the art set by fine phi slicing on

PILATUS (5).

Keywords

X-ray detectors, HPC detectors, serial crystallography, EIGER

References

(1) A. Casanas et al., Acta Cryst. D72, (2016), in the press

(2) P. Roedig et al., Sci Rep. 5 (2015) 10451.

(3) N. Coquelle et al., Acta Cryst. D71, (2015), 1184-96.

(4) U. Zander et al., Acta Cryst. D71, (2015), 2328-43.

(5) M. Mueller, M. Wang, C. Schulze-Briese. Acta Cryst. D68, (2012), 42-56.

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2016 Regiomeeting

2016: A Space Odyssey to Greek Mythology

Tobias Pflüger1

1 NanoTemper Technologies GmbH, Flößergasse 4, 81369 München, Germany

E-mail: tobias.pflueger@nanotemper.de

NanoTemper Technologies develops highly innovative instrument and software solutions for

biomolecular analytics with focus on affinity and stability. Our collaborative approach, science-driven

insights, our high-quality, proprietary instruments and analytical technologies focus on helping

researchers to make the greatest impact - with maximum speed, efficiency and precision.

This scientific presentation will give an overview about experimental setup, common and

exceptional applications and most recent developments and publications linked to biocrystallography.

Keywords

MST, thermophoresis, drug discovery and development, nanoDSF, protein stability, protein

engineering, membrane proteins, protein aggregation, label-free

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Allosteric regulation by phosphorylation in nuclear receptor proteins:molecular dynamics study of the Retinoic Acid Receptor alpha and

gamma

Yassmine Chebaro1, S. Sirigu1, I. Amal1, R. Lutzing2, R. H. Stote1, C. Rochette-Egly2, N. Rochel1, A.Dejaegere1

1 Department of Integrative Structural Biology, Institut de Génétique et de Biologie Moléculaire et Cellulaire(IGBMC), Institut National de la Santé et de la Recherche Médicale (INSERM) U964, Centre National de laRecherche Scientifique (CNRS) UMR 7104, Université de Strasbourg, 67404 Illkirch, France. 2 Department ofFunctional Genomics and Cancer, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC),Institut National de la Santé et de la Recherche Médicale (INSERM) U964, Centre National de la RechercheScientifique (CNRS) UMR 7104, Université de Strasbourg, 67404 Illkirch, France, 3 Present address:Synchrotron SOLEIL, L’Orme des Merisiers, Saint-Aubin, BP 48, 91192 Gif-sur-Yvette, CEDEX France.

E-mail: chebaro@igbmc.fr

Retinoic acid (RA) is a ligand of the retinoid acid receptors (RAR α, β and γ) mainly consisting of a N-

terminal domain (NTD), a central DNA-binding domain and a ligand-binding domain (LBD). RA-

binding induces the activation of MAPKs pathways in RARs(1,2). In RARα, MSK1 phosphorylates the

LBD(3) which increases binding of cyclin H, 40 Å away from the phosphorylation site. This leads to

the phosphorylation of the N-terminal domain of the receptor and activation of transcription. For the γ

subtype, the LBD and DBD are also phosphorylated via a similar cascade (4). Describing the

interplay between phosphorylation and the subsequent changes in structure and dynamics helps in

better understanding the allosteric mechanisms occurring. We performed molecular dynamics

simulations of the unphosphorylated and phosphorylated LBD-RARα and several mutants of the

LBD-RARγ. Although the overall structure of the receptors remains unchanged, the simulations show

that in the case of RARα the allosteric communication occurs through a subtle pathway, changing the

electrostatics network and the relative orientation of helices(5). Allosteric communication also takes

place in RARγ, changing for instance the conformational dynamics of a salt bridge in the vicinity of

the cyclin docking site. The molecular details afforded by these simulations allow us to understand

the allosteric communication related to phosphorylation in nuclear receptors and identification of key

residues in this process.

Keywords

Molecular dynamics simulations, allostery, phosphorylation, nuclear receptors

References

(1) Al Tanoury Z. et al (2014) J Cell Sci. 127, 2095-2105.(2)  Rochette­Egly C. (2015) Biochim Biophys Acta 1851, 66­75.(3)  Bruck N. et al.(2009) EMBO J 28, 34­47.(4)  Bastien J. et al. (2000) J Biol Chem 275, 21896­21904.(5)  Chebaro Y et al. (2013) PloS Comput Biol 9, e1003012.

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Application of Methionine Scanning to High-Molecular Weight Complexes

Philip Rößler1, M. Schütz-Stoffregen1, S. Wiesner1

1 MPI for Developmental Biology, Spemannstr. 35, 72076 Tübingen, Germany

E-mail: philip.roessler@tuebingen.mpg.de

NMR spectroscopy is a powerful tool to study biomolecular interactions in solution at atomic

resolution. Since NMR spectroscopy is limited to small proteins, protein complexes of high-molecular

mass are often not accessible via standard NMR methods. Methionine scanning, a technique relying

on chemical shift perturbations of newly introduced methionine residues in NMR titration

experiments, is a promising way to overcome this challenge.

Here I present that methionine scanning and additional methyl group labelling can be used to

characterise the binding interaction of a 710 kDa protein complex. The model system used is the

interaction between the 11S activator (7x25 kDa) from T. brucei and the α-subunit (7x26 kDa) of the

archeal proteasome that forms a double ring and thus binds two 11S activator moieties. Residues of

the activation loop and the C-terminal tail of the 11S activator which are known to be located at the

interface were mutated to methionine/cysteine and methyl-labelled. In NMR titration experiments, I

was able to characterise important residues in the interface. Additionally, the mutation V230I has

been identified as a reporter for the binding interaction which does not influence functionality in

kinetic assays. We thus conclude that methionine scanning in combination with additional methyl

labeling strategies is a versatile tool to study high-molecular weight complexes.

Keywords

NMR, Methionine scanning, methyl group labelling, proteasome, 11S activator

References

(1) M. C. Stoffregen, M. M. Schwer, F. A. Renschler and S. Wiesner. Methionine scanning as an NMR tool fordetecting and analyzing biomolecular interaction surfaces. Structure (2012). 20, 573-581.

(2) R. Sprangers and L. E. Kay. Quantitative dynamics and binding studies of the 20S proteasome by NMR.Nature (2007). 445, 618-622.

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2016 Regiomeeting

Thermodynamics of the Ribosome Translation Machinery

Eric Ennifar1

1 CNRS / University of Strasbourg, Strasbourg, France

E-mail: e.ennifar@unistra.fr

Translation initiation in bacteria is under the control of initiation factors IF1, IF2 and IF3. The 30S

Initiation Complex (30SIC) is formed upon binding of all partners to the 30S ribosome. The 50S

ribosomal subunit then binds to this initiation complex, leading to the elongation complex.

In the past two decades, high-resolution structural data was collected on the ribosome, but

thermodynamic data are still lacking. We have used Isothermal Titration Calorimetry (ITC) to dissect

the translation initiation in E coli. To that goal, we have developed new methods extending the

possibilities of ITC: (1) incremental ITC, which allow us to study step by step successive chemical

reactions, and (2) kinITC, to obtain kinetic information.

Using these news approaches, we explored of the thermodynamic landscape leading to the 30SIC

through the change in the order of addition of the partners. We identified a preferred assembly

pathway leading to the 30SIC formation based on thermodynamic and kinetic data, and we observed

by ITC the binding of the 50S on the 30S initiation complex leading to the formation of the 70S

Initiation Complex. Using this knowledge, we obtained the first high-resolution structure of the 30S

bound to IF3 by cryoEM. Lastly, thermodynamics of ribosome-targeting antibiotics was investigated in

the frame of the full ribosome, providing new clues about molecular forces involved into bacterial

translation inhibition.

Keywords

Thermodynamics, ITC Microcalorimetry, Ribosome, Antibiotics

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2016 Regiomeeting

The architecture of the Smc protein

Marie-Laure Durand Diebold1, L. Ruiz Avila 1, J. Basquin2, A. Durand1, F. Buermann1, S. Gruber

Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany. 1 Group of ChromosomeOrganization and Dynamics, 2 Department of Structural Cell Biology

Email: diebold@biochem.mpg.de

Structural Maintenance of Chromosome (SMC) complexes are conserved key players in the faithful

segregation of DNA during cell division from bacteria to humans. They share a conserved

organization formed by a dimer of Smc proteins associated to accessory proteins. Smc are elongated

proteins with globular domains at both ends: a hinge domain where the protein folds back on itself

and is responsible for dimerization and a head domain that carries an ATP binding and hydrolysis

activity. These two domains are held apart by a long (45nm) intramolecular and antiparallel coiled coil

whose role and detailed structure is so far unknown. The recruitment of Smc complex in Bacillus

subtilis to chromosomal DNA requires large structural changes in the coiled coil domain, the complex

switching from a ring when loaded onto the DNA to a rod when relocalizing along the chromosome

upon ATP hydrolysis. In order to understand these conformational changes, structural studies of the

coiled coil domain has been undertaken. Intramolecular crosslinking experiments allowed to identify

the register of the coiled coil helices and revealed the existence of two protruding segments located

close to the hinge and head domains. Interestingly the head-proximal domain has been shown to be

required for the recruitment of the complex to the parS sites. The structure of several fragments of

coiled coil has been solved allowing for the building of a model of a long coiled coil fragment

associated to the Smc head.

Keywords

Smc, chromosome organization and dynamic, coiled coil

References

(1) A. Minnen, F. Bürmann, L. Wilhelm, A. Anchimiuk, M. L. Diebold-Durand, S. Gruber (2016). Control ofSmc Coiled Coil Architecture by the ATPase Heads Facilitates Targeting to Chromosomal ParB/parS andRelease onto Flanking DNA. Cell Reports 14, 2003-2016.

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2016 Regiomeeting

Structural basis for the deposition and eviction of H2A.Z/H2B from thenucleosome by human YL1 and ANP32E histone chaperones

M. Marek1, M. L. Diebold1, E. Ennifar2 and Christophe Romier1

1 IGBMC, 1 rue Laurent Fries, B.P. 10142, 67404 Illkirch Cedex, France, 2 IBMC, Strasbourg, France

E-mail: romier@igbmc.fr

Histone variant H2A.Z has multiple functional roles in eukaryotes. Its mode of deposition on and

removal from the chromatin has long remained elusive. Human histone chaperones YL1 and

ANP32E respectively deposit and evict H2A.Z/H2B from the nucleosome. The X-ray structures of

YL1 and ANP32E in complex with H2A.Z/H2B reveal that recognition of this variant histone pair by

these chaperones induces a doubling in size of H2A.Z αC helix (1,2). Yet, the specificity of

H2A.Z/H2B recognition by these two chaperones is divergent and relies on common and specific

determinants. Whereas the absence of a single glycine in H2A.Z compared to H2A is sufficient to

convey recognition specificity by ANP32E, specific recognition of H2A.Z by YL1 involves a larger

interface. Analyses by isothermal titration calorimetry reveal that four mutations in H2A are sufficient

to enable recognition of the mutant H2A/H2B pair by YL1 to the same extent as H2A.Z/H2B, with

each mutated residue contributing differently to recognition. These data shed light on the

deposition/removal mechanisms of H2A.Z/H2B that involve the large ATP-dependent chromatin

remodeling complexes p400/TIP60 and SRCAP.

Keywords

Epigenetics, histone variant, histone chaperone, chromatin remodeler

References

(1) A. Obri, K. Ouararhni, C. Papin, M. L. Diebold, K. Padmanabhan, M. Marek, I. Stoll, L. Roy, S. Dimitrov, C.Romier, A. Hamiche (2014). Nature, 505, 648-653.

(2) C. M. Latrick, M. Marek, K. Ouararhni, C. Papin, I. Stoll, M. Ignatyeva, A. Obri, E. Ennifar, S. Dimitrov, C.Romier, A. Hamiche (2016). Nat Struct Mol Biol, 23, 309-16.

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2016 Regiomeeting

Structural and biophysical studies of the vitamin D nuclear receptorcomplex with the coactivator MED1

Anna Belor u sova1, B. Kieffer1, Y. Nomine1, N. Potier2, D. Moras1, N. Rochel1

1 Department of Integrative Structural Biology, Institut de Génétique et de Biologie Moléculaire et Cellulaire(IGBMC), Institut National de la Santé et de la Recherche Médicale (INSERM) U964, Centre National de laRecherche Scientifique (CNRS) UMR 7104, Université de Strasbourg, 67404 Illkirch, France,Author Affiliation, 2

Institut de Chimie LC3, CNRS, UMR 7177, 67008 Strasbourg, France.

E-mail: beloruso@igbmc.fr

The vitamin D nuclear receptor (VDR) is a transcription factor binding with high affinity its natural

ligand, the 1α,25-dihydroxyvitamin D3, or calcitriol. Together with its heterodimeric partner retinoid X

nuclear receptor (RXR), VDR modulates expression of calcitriol-regulated genes by selective

recruitment of coregulators of transcription. Nuclear receptor coregulators are, in turn, important

targets in epigenetic-oriented drug discovery. Considering the essential role of nuclear receptors and

coregulators in transcriptional control and their apparent druggability, investigation of their complexes

is highly required. Yet, the amount of available structural data for such complexes is far from being

sufficient. The primary focus of our ongoing research is on revealing the architecture of the complex

between the full-length VDR-RXR heterodimer bound to its cognate DNA response element and a

large part of the coactivator MED1, a subunit of the Mediator complex linking nuclear receptors to the

basal transcription machinery. Structural studies in solution and biophysical analysis revealed

important details of the receptor-coactivator interaction, providing new mechanistic insights into how

VDR selectively recruits the coactivator to the genomic loci.

Keywords

Transcriptional control, vitamin D nuclear receptor, MED1 coactivator, integrated structural biology

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2016 Regiomeeting

The bicoid mRNA localization factor Exuperantia is an RNA-bindingpseudonuclease

Fulvia Bono1, D. Lazzaretti1, K. Veith1, K. Kramer2,3, C. Basquin4, H. Urlaub2,3, U. Irion1

1 MPI for Developmental Biology, Spemannstr. 35, 72076 Tübingen, Germany, 2 Bioanalytical MassSpectrometry, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany, 3 Bioanalytics, Institute forClinical Chemistry, University Medical Center Göttingen, Göttingen, Germany, 4 Max Planck Institute ofBiochemistry, Martinsried, Germany

E-mail: fulvia.bono@tuebingen.mpg.de

Anterior patterning in Drosophila is mediated by the localization of bicoid (bcd) mRNA at the anterior

pole of the oocyte. This process requires the protein Exuperantia (Exu). We determined the crystal

structure of Exu at 2.6-Å resolution. The structure reveals a dimeric assembly with each monomer

consisting of a 3'-5' exonuclease (EXO)-like domain and a sterile alpha motif (SAM)-like domain. The

catalytic site is degenerate and inactive. Instead, the EXO-like domain mediates dimerization and

RNA binding. We show that Exu binds RNA directly in vitro, that the SAM-like domain is required for

RNA binding activity and that Exu binds a structured element present in the bcd 3′ untranslated

region with high affinity. Using structure-guided mutagenesis, we show that Exu dimerization is

essential for bcd localization. Therefore, Exu is a non-canonical RNA-binding protein with EXO-SAM-

like domain architecture that interacts with its target RNA as a homodimer (1). Our work further

suggests that Exu homodimerization creates a single structural platform that interacts with the mRNA

localization machinery.

Keywords

RNA regulation, pseudoenzyme, Drosophila

References

(1) Lazzaretti et al. (2016), NSMB 23, 705–713.

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Participants

Prof. Jan Pieter Abrahams Universität BaselBiozentrum, C-CINAMattenstrasse 264058 Basel, Schweizjp.abrahams@unibas.ch

Dr. Jerome BasquinMax-Planck-Institute of BiochemistryAm Klopferspitz 1882252 Martinsried, Germanybasquin@biochem.mpg.de

Dr. Shibom BasuPaul Scherrer InstituteSwiss Light SourceWSLA/2225232 Villigen, Schweizshibom.basu@psi.ch

Prof. Ulrich BaumannInstitute of Biochemistry, University of CologneOtto-Fischer-Strasse 12-14D-50674 Cologne, Germanyubaumann@uni-koeln.de

Dr. Anna BelorusovaCERBM GIE CERBM1 Rue Laurent Fries67404 Illkirch, Francebeloruso@igbmc.fr

Dr. Karin BetzFachbereich Chemie AG MarxUniversitätsstr. 1078464 Konstanzkarin.betz@uni-konstanz.de

Dr. Isabelle BillasIGBMCCentre for Integrative BiologyDepartment of Integrated Structural BiologyUMR7104 CNRS-UDS, INSERM U9641, rue Laurent Fries, BP 10142 67404 Illkirch, Francebillas@igbmc.fr

Dr. Bärbel BlaumUniversität TübingenInterfakultäres Institut für BiochemieHoppe-Seyler-Str. 472076 Tübingen, Germanybaerbel.blaum@uni-tuebingen.de

Dr. Fulvia BonoMax-Planck-Institut für EntwicklungsbiologieSpemannstr. 3572076 Tübingen, Germanyfulvia.bono@tuebingen.mpg.de

Michael B. BraunUniversität TübingenInterfakultäres Institut für BiochemieHoppe-Seyler-Str. 472076 Tübingen, Germanym.braun@uni-tuebingen.de

Dr. Michael BuchUniversität TübingenInterfakultäres Institut für BiochemieHoppe-Seyler-Str. 472076 Tübingen, Germanymichael.buch@uni-tuebingen.de

Dr. Dominique BurnoufStructure and Dynamics of Biomolecular MachinesUPR 9002 CNRSIBMC15 rue René Descartes67084 Strasbourg, Franced.burnouf@ibmc-cnrs.unistra.fr

Dr. Felix BüttnerUniversität TübingenInterfakultäres Institut für BiochemieHoppe-Seyler-Str. 472076 Tübingen, Germanyfelix.buettner@uni-tuebingen.de

Dr. Yassmine ChebaroCERBM GIE CERBM1 Rue Laurent Fries67404 Illkirch, Francechebaro@igbmc.fr

Vanessa Dacleu Siewevanessa.dacleu-siewe@etu.unistra.fr

Melanie DietrichUniversität TübingenInterfakultäres Institut für BiochemieHoppe-Seyler-Str. 472076 Tübingen, Germanymelanie.dietrich @uni-tuebingen.d e

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2016 Regiomeeting

Dr. Nikolaus DietzUniversität BaselBiozentrumKlingelbergstr. 704056 Basel, Schweiznikolaus.schmitz@unibas.ch

Violetta DimperUniversität zu KölnOtto-Fischer-Str. 12-1450674 Köln, Deutschlandvioletta.dimper@gmx.de

Dr. Alexandre DurandMax-Planck-Institut für BiochemieAm Klopferspitz 1882152 Planegg, Deutschlanddurand@biochem.mpg.de

Dr. Marie-Laure Durand DieboldMax-Planck-Institut für BiochemieAm Klopferspitz 1882152 Planegg, Deutschlanddiebold@biochem.mpg.de

Dr. Eric EnnifarCNRS UPR 900215 rue Rene Descartes67084 Strasbourg, Francee.ennifar@unistra.fr

Dr. Gwyndaf EvansDiamond Light Source LtdDiamond HouseHarwell Science & Innovation CampusOX11 0DE Didcot, Oxfordshire, UKgwyndaf.evans@diamond.ac.uk

Miki Hannah FeldmüllerUniversität zu KölnInstitut für BiochemieOtto-Fischer-Str. 12-1450674 Köln, Deutschlandmfeldmue@smail.uni-koeln.de

Anika FippelUniversität FreiburgInstitut für Biochemie und MolekularbiologieStefan-Meier-Str. 1779104 Freiburg, Deutschlandanika.fippel@biochemie.uni-freiburg.de

Dr. Jennifer FlemmingUniversität KonstanzFachbereich BiologieUniversitätsstraße 10 78457 Konstanz, Deutschlandj ennifer.fleming@uni.kn

Dr. Severine FreiszBruker AXS GmbHOestliche Rheinbrueckenstrasse 4976187 Karlsruhe, DeutschlandSeverine.Freisz@bruker.com

Dr. Arnaud GoepfertNovartis Institutes for Biomedical Research CPC/ Structural BiophysicsWSJ-182.3.101.16 Novartis Campus4002 Basel, Schweizarnaud.goepfert@novartis.com

Dr. Yinglan GuoUniversität TübingenInterfakultäres Institut für BiochemieHoppe-Seyler-Str. 472076 Tübingen, Germanyyinglan.guo@uni-tuebingen.de

Irmgard Hähnlein-SchickUniversität TübingenInterfakultäres Institut für BiochemieHoppe-Seyler-Str. 472076 Tübingen, Germanyirmgard.haehnlein-schick@uni-tuebingen.de

Christina HarprechtUniversität TübingenInterfakultäres Institut für BiochemieHoppe-Seyler-Str. 472076 Tübingen, Germanychristina.harprecht@uni-tuebingen.de

Prof. Michael HennigleadXpro AGPARK INNOVAARE5234 Villigen, Schweizmic h ael.hennig@leadXpro.com

Dominik HerbstUniversität BaselBiozentrumKlingelbergstrasse 50/704056 Basel, Schweizdominik.herbst@unibas.ch

Franziska HeydenreichPaul Scherrer InstitutLaboratory of Biomolecular ResearchOFLC-1055232 Villigen, Schweizfranziska.heydenreich@psi.ch

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2016 Regiomeeting

Dr. Chia-Ying Huang Paul Scherrer InstitutSwiss Light SourceWSLA/2145232 Villigen, Schweizcyhuang107@gmail.com

Prof. Carola Hunte Universität FreiburgInstitut für Biochemie und MolekularbiologieStefan-Meier-Str.1779104 Freiburg, Deutschlandcarola.hunte@biochemie.uni-freiburg.de

Magnus JaecklMax-Planck-Institut für EntwicklungsbiologieSpemannstr. 3572076 Tuebingen, Deutschlandmagnus.jaeckl@tuebingen.mpg.de

Dr. Wei-Chun KaoUniversität FreiburgInstitut für Biochemie und MolekularbiologieStefan-Meier-Str. 1779104 Freiburg im Breisgau, Deutschlandwei-chun.kao@biochemie.uni-freiburg.de

Prof. Werner KühlbrandtMax-Planck-Institut für BiophysikMax-von-Laue-Str. 360438 Frankfurt am Main, Deutschlandwerner.kuehlbrandt@biophys.mpg.de

Antonio Manuel LiaciUniversität TübingenInterfakultäres Institut für BiochemieHoppe-Seyler-Str. 472076 Tübingen, Germanyantonio-manuel.liaci@uni-tuebingen.de

Dr. Bernard LorberIBMC-CNRS, University of Strasbourg15 rue René Descartes67000 Strasbourg, Franceb.lorber@ibmc-cnrs.unistra.fr

Marija MarkovicUniversität KonstanzFachbereich BiologieUniversitätsstraße 1078464 Konstanz, Deutschlandmarija.markovic@uni-konstanz.de

Dr. Alastair McEwenGIE CERBM1 rue Laurent FriesB.P. 1014267404 Illkirch, Francealastair@igbmc.fr

Dr. Kareem Mohideeen AbdulCERBM GIE CERBM1 rue Laurent FriesB.P. 1014267404 Illkirch, Francemohideen@igbmc.fr

Dr. Marcus MuellerDECTRIS Ltd.Taefernweg 15405 Baden-Daettwil, Schweizmarketing@dectris.com

Erik NöldekeUniversität TübingenInterfakultäres Institut für BiochemieHoppe-Seyler-Str. 472076 Tübingen, Germanyerik.noeldeke@ifib.uni-tuebingen.de

Sinan OecalUniversität zu KölnInstitut für BiochemieOtto-Fischer-Str. 12-1450674 Köln, Deutschlandsoecal@uni-koeln.de

Dr. Vincent Olieric Paul Scherrer InstitutSwiss Light SourceWSLA/2185232 Villigen, Schweizvincent.olieric@psi.ch

Moritz PfleidererUniversität TübingenInterfakultäres Institut für BiochemieHoppe-Seyler-Str. 472076 Tübingen, Deutschlandmo.pfleiderer@gmail.com

Dr. Tobias PflügerNanoTemper Technologies GmbHFlößergasse 481369 München, Deutschlandt obias. p flueger@nanotemper.de

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2016 Regiomeeting

Christian PichloUniversität zu KölnInstitut für BiochemieOtto-Fischer-Str. 12-1450674 Köln, Deutschlandpichloc@uni-koeln.de

Dr. Andrea ProtaPaul Scherrer InstitutLaboratory of Biomolecular ResearchOFLC 1115232 Villigen, Schweizandrea.prota@psi.ch

Fabian RenschlerMax-Planck-Institut für EntwicklungsbiologieSpemannstr. 3572076 Tuebingen, Deutschlandfabian.renschler@tuebingen.mpg.de

Philip RößlerMax-Planck-Institut für EntwicklungsbiologieSpemannstr. 3572076 Tuebingen, Deutschlandphilip.roessler@tuebingen.mpg.de

Christophe RomierGIE CERBM1 rue Laurent FriesB.P. 1014267404 Illkirch, Franceromier@igbmc.fr

Nils RustmeierUniversität TübingenInterfakultäres Institut für BiochemieHoppe-Seyler-Str. 472076 Tübingen, Germanynils.rustmeier@yahoo.de

Dr. David SargentETH ZurichInstitutof Molecular Biology and BiophysicsOtto-Stern-Weg 58092 Zürich, Schweizsargent@mol.biol.ethz.ch

Claude SauterUPR 9002 - CNRS - IBMC15 rue R. DescartesF-67084 Strasbourg, Francec.sauter@ibmc-cnrs.unistra.fr

Dr. Joerg StandfussPaul Scherrer InstitutOFLB-0075232 Villigen, Switzerlandjoerg.standfuss@psi.ch

Prof. Thilo StehleUniversität TübingenInterfakultäres Institut für BiochemieHoppe-Seyler-Str. 472076 Tübingen, Germanythilo.stehle@uni-tuebingen.de

Elena StörkUniversität TübingenInterfakultäres Institut für BiochemieHoppe-Seyler-Str. 472076 Tübingen, Germanyelenastoerk@gmx.de

Carsten StollmaierMax-Planck-Institut für EntwicklungsbiologieSpemannstr. 3572076 Tuebingen, Deutschlandcarsten.stollmaier@tuebingen.mpg.de

Joana Tavares MacedoUniversität TübingenInterfakultäres Institut für BiochemieHoppe-Seyler-Str. 472076 Tübingen, Germanyjoana.tavares-macedo@uni-tuebingen.de

Dr. Anne Théobald-DietrichUPR 9002 du CNRSInstitut de Biologie Moléculaire et Cellulaire15 Rue René Descartes67084 Strasbourga.theobald@ibmc-cnrs.unistra.fr

Matthias UthoffUniversität zu KölnInstitut für BiochemieOtto-Fischer-Str. 12-1450674 Köln, Deutschlandmatthias.uthoff@uni-koeln.de

Dr. Dimitry VeprintsevPaul Scherrer InstitutOFLC 103Laboratory of Biomolecular research5232 Villigen, Switzerlanddmitry.veprintsev@psi.ch

Matthias WälchliUniversity BaselBiozentrumKlingelbergstr. 50/70CH-4056 Basel, Schweizmatthias.waelchli@stud.unibas.ch

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Dr. Tobias WeinertPaul Scherrer InstitutOFLB 0075232 Villigen, Switzerlandtobias.weinert@psi.ch

Tiankun ZhouFachbereich Biologie PF 656Universitätsstraße 10 78464 Konstanzt iankun. z hou@uni-konstanz.de

Dr. Georg ZocherUniversität TübingenInterfakultäres Institut für BiochemieHoppe-Seyler-Str. 472076 Tübingen, Germanygeorg.zocher@uni-tuebingen.d e

41