Madrid 17–19 September 2012 Allosteric Regulation of Cell Signalling SUMMARY

005 031 087 Detailed Session II. Poster session Programme DYNAMICS AND MODELLING OF 115 015 ALLOSTERIC SYSTEMS CNIO Frontiers Session I. Meetings 2013 ALLOSTERIC SIGNALLING 049 MECHANISMS Session III. 119 ALLOSTERIC INHIBITION Previous CNIO Frontiers Meetings 059 and CNIO Cancer Speakers’ Conferences Biographies Allosteric Regulation of Cell SignallinG DETAILED PROGRAMME MONDAY, September 17th

09:00 11:15 14:55 17:15 CONTRIBUTED TALK Welcome Address Structural Basis for Relay Allosteric control of From the molecular mechanism of allostery in I integrins lipidated protein traffic by the of regulation of AGC kinases to the 09:20 Timothy A. Springer, Harvard GTP-binding proteins Arl2 and Arl3 development of allosteric activa- Keynote Speaker: Medical School, Boston, USA Alfred Wittinghofer, MPI for Molecular tors and allosteric inhibitors INTRODUCTION TO ALLOSTERY. Physiology, Dortmund, Germany Ricardo Biondi, Frankfurt University Allosteric mechanisms of signal 11:50 Hospital, Frankfurt am Main, Germany tranduction: an introduction Surface assemblies in cell 15:30 CONTRIBUTED TALK Jean-Pierre Changeux, Collège de guidance signalling systems Crystal structure and 17:40 CONTRIBUTED TALK France, Pasteur Institute, Paris, France Yvonne Jones, Wellcome Trust Centre for mechanism of activation of TBK1 The structure of the second Human Genetics, University of Oxford, UK Daniel Panne, EMBL, Grenoble, France pair of fibronectin type III re- peats of the integrin β4 subunit 12:25 CONTRIBUTED TALK 15:55 suggests a mechanism for an Allosteric Conversation Coffee break & poster session intramolecular interaction Session I in the Human Androgen Receptor José M. de Pereda, Instituto de Biología ALLOSTERIC SIGNALLING MECHANISMS Ligand-Binding Domain Surfaces 16:15 Molecular y Celular del Cáncer, Chair Ermanno Gherardi and Daniel Lietha Eva Estébanez-Perpina, Universidad PKA: Assembly of Salamanca, Spain de Barcelona, Spain dynamic macromolecular signaling complexes 18:05 CONTRIBUTED TALK 10:20 12:50 Susan S. Taylor, Howard Hughes Coordination of Critical Hormone-mediated activation Lunch and poster session Medical Institute, University of Cytoskeletal Changes by Nck of G proteins by GPCRs: insights California, San Diego, USA Enables Endothelial Cell Po- from the crystal structure 14:20 larization and Morphogenesis of a GPCR-G protein complex Designing therapeutics that 16:50 Gonzalo M. Rivera, Texas A&M University, Roger Sunahara, University of Michigan modulate multiprotein regulatory Allosteric regulation College Station, USA Medical School, Ann Arbor, USA systems: Allo-targeting of oligomeric assembly: the Tom L. Blundell, University of Cambridge, UK role of PIP2 in activation of 18:30 10:55 focal adhesion kinase Poster Session Coffee break & beginning Daniel Lietha, CNIO, Madrid, Spain of poster session

Note Talks: 30 minutes / Short talks: 20 minutes Discussion: 5 minutes after each talk / 6 DETAILED PROGRAMME 5 minutes after each short talk Allosteric Regulation of Cell Signalling 7 TUESDAY, September 18th

Session II 10:40 14:20 17:05 CONTRIBUTED TALK DYNAMICS AND MODELLING Allostery and conforma- Proteins in flagrante: Impact of mutations on the OF ALLOSTERIC SYSTEMS tional control in signaling: excursions in silico and in proteo allosteric conformational Chair Francesco Luigi Gervasio the ubiquitin E3 ligases Dorothee Kern, Howard Hughes Medical equilibrium and Alfonso Valencia Ruth Nussinov, National Cancer Institute Institute, Brandeis University, Waltham, USA Patrick Weinkam, University of California, and Tel Aviv University, Israel San Francisco, USA 14:55 09:00 11:15 Dynamical effects in the 17:30 CONTRIBUTED TALK The Ins and Outs Controlling allosteric allosteric control of kinases Characterizing protein of EGFR Asymmetry networks in proteins Francesco Luigi Gervasio, conformation changes through Daniel Leahy, Johns Hopkins University Nikolay V. Dokholyan, University CNIO, Madrid, Spain torsional linear response School of Medicine, Baltimore, USA of North Carolina at Chapel Hill, USA and allosteric profiles 15:20 Ugo Bastolla, Centro de Biologia Molecular 09:35 11:50 Co-evolution based methods Severo Ochoa CSIC-UAM, Madrid, Spain Conformational dynamics Exploring the landscape in the prediction of allosteric in multidomain kinases, kinase- for proteins and the ribosome: channels and contact networks 17:55 CONTRIBUTED TALK phosphate complexes, and ESCRT- connecting simple models and Alfonso Valencia, CNIO, Madrid, Spain Oncogenic mutations corrupt Protein supercomplexes detailed simulations the ordered autophosphorylation Gerhard Hummer, National Institute Jose Onuchic, Center for Theoretical Biologi- 15:45 of the RET receptor tyro- of Diabetes and Digestive and Kidney cal Physics, Rice University, Houston, USA Coffee break and poster session sine kinase: structural and Diseases, NIH, Bethesda, USA molecular basis of RET-MEN2B 12:25 CONTRIBUTED TALK 16:05 Ivan Plaza Menacho, London Research 10:10 Intrinsic disorder in wild-type The innate immunity Institute, Cancer Research UK, London, UK Group picture and Coffee break EGFR kinase underlies onco- molecular machinery genicity of cancer mutations Giulio Superti-Furga, CeMM Research 18:20 Yibing Shan, D. E. Shaw Research, USA Center for Molecular Medicine of the Austrian Poster Session Academy of Sciences, Vienna, Austria 12:50 Lunch AND POSTER SESSION 16:40 Crystal structure of the 1 MDa mam- malian TRiC/CCT complex with tubulin Guillermo Montoya, CNIO, Madrid, Spain

8 DETAILED PROGRAMME Allosteric Regulation of Cell Signalling 9 WEDNESDAY, September 19th

Session III 11:05 ALLOSTERIC INHIBITION Allosteric kinase inhibition Chair Giulio Superti-Furga Jeffrey R. Peterson, Fox Chase Cancer Center, Philadelphia, USA

09:00 11:40 Structural insights into the Non-ATP competitive kinase mechanism Jak kinase activation inhibitors: potentials and limitations in myeloproliferative disease Doriano Fabbro, Novartis Pharma AG, Michael J. Eck, Dana-Farber Cancer Basel, Switzerland Institute, Harvard Medical School, USA 12:15 09:35 Regulation of HGF/SF-MET signalling Allosteric regulation of Ermanno Gherardi, MRC Laboratory of intracellular and membrane Molecular Biology, Cambridge, UK proteins by DARPins Markus G. Grütter, University 12:50 CONTRIBUTED TALK of Zurich, Switzerland Identification and characterisation of novel 10:10 allosteric regulators of PKM2 Elucidation of the Molecular using fragment based screening Mode of Action of SSR128129E, the Marc O’Reilly, Astex Pharmaceuticals, First Small Molecule allosteric Cambridge, UK Inhibitor of FGF receptor signaling Françoise Bono, Sanofi R&D, 13:15 Toulouse, France Final Remarks

10:45 Coffee break

10 DETAILED PROGRAMME KEYNOTE SPEAKER: INTRODUCTION TO allostery. Allosteric mechanisms of signal tranduction: an introduction

Jean-Pierre Changeux Collège de France, Pasteur Institute, Paris, France

Exactly 50 years ago, biochemists raised the and theoretical comparisons with the question of the mechanism of the confor- formal models of both scenarios. Also, it mational change that mediates “allosteric” has been shown that mutated receptors interactions between regulatory sites and can adopt constitutively active confirma- biologically active sites in regulatory/recep- tions in the absence of ligand. There have tor proteins. Do the different conformations also been demonstrations that the atomic involved already exist spontaneously in the resolution structures of the same protein absence of the regulatory ligands (Monod- are essentially the same whether ligand is Wyman-Changeux), such that the comple- bound or not. These and other advances mentary protein conformation would be in past decades have produced a situation selected to mediate signal transduction, or where the vast majority of the data using do particular ligands induce the receptor to different categories of regulatory proteins adopt the conformation best suited to them (including regulatory enzymes, ligand-gat- (Koshland-Nemethy-Filmer-induced fit)? ed ion channels, G protein-coupled recep- This is not just a central question for bio- tors, and nuclear receptors) support the physics, it also has enormous importance conformational selection scheme of signal for drug design. Recent advances in tech- transduction. niques have allowed detailed experimental

Allosteric Regulation of Cell Signalling 13 Allosteric Regulation of Cell SignallinG SESSION I. ALLOSTERIC SIGNALLING MECHANISMS Hormone-mediated activation of G proteins by GPCRs: insights from the crystal structure of a GPCR-G protein complex

Roger K. Sunahara University of Michigan Medical School, Ann Arbor, USA

SESSION I Recent advances in the structural biology deuterium exchange mass spectrometry, of G protein-coupled receptors have helped reveal dramatic changes in the G protein ALLOSTERIC to unravel the intricacies of ligand bind- α-subunit. The crystal structure also sug- ing. Similarly, structural and biochemical gests that G proteins may allosterically SIGNALLING MECHANISMS analyses of heterotrimeric G proteins have regulate GPCRs by stabilizing a closed con- Chair: Ermanno Gherardi and Daniel Lietha affirmed our understanding of the mecha- formation on the extracellular face of the nism underlying effector interactions and receptor. Consistent with these changes GTPase activity. We recently elucidated the radioligand binding analyses suggest that G crystal structure of a prototypic GPCR, the protein coupling slows ligand dissociation.

β2-adrenergic receptor (β2AR), in a com- These structural changes would account plex with the stimulatory G protein, Gs, for the slower observed dissociation rates trapped in its nucleotide-free state. These of agonists and likely account for their G data have helped us to understand how protein-dependent high affinity binding. hormone binding to GPCRs leads to GDP Cumulatively these data support a model release on G proteins, the principle step for the mechanism for receptor-mediated that precedes in GTP binding and G protein nucleotide exchange, G protein activation activation. The crystal structure, together and agonist binding. with data from electron microscopy and

Allosteric Regulation of Cell Signalling 17 Structural basis for relay Surface Assemblies in Cell of allostery in αI integrins Guidance Signalling Systems

Timothy A. Springer Harvard Medical School, Boston, USA E. Yvonne Jones Wellcome Trust Centre for Human Genetics, University of Oxford, UK

Integrins are complex adhesion receptors and movement of its C-terminal α7-helix The molecular mechanisms that underpin multiple interactions within assemblies of with 12 or 13 extracellular domains in their toward the βI domain metal ion-depend- cell-cell communication depend on recep- receptors. To analyse such systems we need transmembrane α- and β-subunits. Half of ent adhesion site (MIDAS). An invariant tors embedded in the plasma membrane to draw on an increasingly broad range of integrin α-subunits (αI integrins) contain Glu immediately following the α7-helix of the cell. Many of these receptors have methodologies which extend from high res- a ligand-binding αI domain that is inserted moves 26 Å from its position in the closed extracellular and cytoplasmic regions olution crystallographic studies to imaging in their β-propeller domain, whereas αI domain to directly coordinate to the linked by a single membrane spanning the behaviour of molecules in the context αI-less integrins bind ligand at the inter- βI MIDAS Mg2+. Surprisingly, the α7-helix helix. Crystallographic and biophysical of the cell. I will present some examples of face between the α-subunit β-propeller substantially unwinds, and its C-terminal analyses of the extracellular architecture the progress we are making in the study of domain and the βI domain. Large rear- portion with conserved hydrophobic resi- and recognition complexes of such recep- cell surface interaction assemblies involved rangements in integrin ectodomains trans- dues loses all contact with the αI domain tors have provided a wealth of detail on the in the balance between cell guidance and mit signals between the membrane and the and completely restructures to form an structural determinants of ligand binding adhesion, specifically drawing on examples membrane-distal, ligand-binding domains. internal ligand. This internal ligand binds affinities and specificity. However, for many from the ephrin/Eph and semaphorin/

Despite hypothesized mechanisms, how to a hydrophobic pocket formed by the αX systems it is increasingly apparent that the plexin families of cell guidance cues. allostery is relayed to integrin αI domains β-propeller domain and the specificity- mechanisms controlling signalling involve remains unknown. Here, we capture the determining and metal-binding loops of

relay mechanism in an αXβ2 integrin crystal the β2 βI domain. The MIDAS touch captured

structure. Complete activation of αI (open here in bent αXβ2 relates to headpiece open-

conformation) is relayed by deformation ing and extension in αXβ2 on cell surfaces, as shown by effect of mutations on ligand binding and exposure of conformation- specific epitopes.

18 Session I: ALLOSTERIC SIGNALLING MECHANISMS Allosteric Regulation of Cell Signalling 19 CONTRIBUTED TALK Allosteric Conversation in the Human Androgen Designing therapeutics that modulate Receptor Ligand-Binding Domain Surfaces multiprotein regulatory systems: allo-targeting

Eva Estébanez-Perpina Universidad de Barcelona, Barcelona, Spain Tom L Blundell University of Cambridge, UK

Androgen receptor (AR) is a major therapeu- conformation is indeed closely coupled to Detailed structural knowledge of the inter- I will describe databases that enable the tic target that plays pivotal roles in prostate BF-3 and provide mechanistic proof of their actions between molecules in the cell —the utilisation of structural information on cancer (PCa) and androgen insensitivity structural interconnection. BF-3 mutations structural interactome— can contribute to protein-molecule interactions. I will focus syndromes.Wepreviously proposed that may function as allosteric elicitors, prob- understanding of cellular networks. It can on the use of fragment-based approaches compounds recruited to ligand-binding ably shifting the AR LBD conformational also facilitate the selection of targets and to target protein-protein interactions in domain (LBD) surfaces could regulate AR ensemble toward conformations that alter the design of candidate therapeutics that order to make chemical tools and candidate activity in hormone-refractory PCa and AF-2 propensity to reorganize into subpock- modulate protein-protein interactions. therapeutic molecules. Topics will include discovered several surface modulators of ets that accommodate N-terminal domain Such molecules may target allosteric sites (i) the human recombinase, Rad51, inter- AR function. Surprisingly, the most effec- and coactivator peptides. The induced con- and mediate their actions through confor- actions with BRCA2, inhibitors of which tive compounds bound preferentially to formation may result in either increased or mation change or electrostatics, or directly would be helpful in modulating DNA repair a surface of unknown function [binding decreased AR activity. Activating BF-3 muta- modulate protein-protein interactions during chemo— or radiotherapy and (ii) function 3 (BF-3)] instead of the coacti- tions also favor the formation of another involved in colocation of regulatory com- the Met receptor interaction with HGF/SF, vator-binding site [activation function pocket (BF-4) in the vicinity of AF-2 and ponents; we class both as allotargeting. important in regulating metastasis. 2 (AF-2)]. DifferentBF -3 mutations have BF-3, which we also previously identified been identified in PCa or androgen insen- as a hot spot for a small compound. We sitivity syndrome patients, and they can discuss the possibility that BF-3 may be strongly affectAR activity. Further, com- a protein-docking site that binds to the parison of AR x-ray structures with and N-terminal domain and corepressors. AR without bound ligands at BF-3 and AF-2 surface sites are attractive pharmacological showed structural coupling between both targets to develop allosteric modulators pockets. Here, we combine experimental that might be alternative lead compounds evidence and molecular dynamic simula- for drug design. tions to investigate whether BF-3 mutations affect AR LBD function and dynamics pos- · Mol Endocrinol. 2012 Jul;26(7):1078- 90. Epub 2012 May 31. sibly via allosteric conversation between surface sites. Our data indicate that AF-2

20 Session I: ALLOSTERIC SIGNALLING MECHANISMS Allosteric Regulation of Cell Signalling 21 CONTRIBUTED TALK Allosteric control of lipidated protein traffic Crystal structure by the GTP-binding proteins Arl2 and Arl3 and mechanism of activation of TBK1

Alfred Wittinghofer Max-Planck-Institute for molecular Physiology, Dortmund, Germany Daniel Panne EMBL, Grenoble, France

GTP-binding (G) proteins are molecular By solving the structures of PDEδ and HRG4 Members of the IKK family of kinases play Comparison with the activated form shows switches that cycle between a GDP-bound and their complexes with Arl we have shown a central role in the innate immune system the requirements for kinase activation. We OFF and a GTP-bound ON state and are regu- that they have a β-sandwich structure with by integrating signals from pattern recog- also have analyzed the structure in solu- lated by Guanine nucleotide exchange fac- a large hydrophobic pocket. The pocket of nition receptors and by mediating regu- tion by SAXS and determined positioning tors (GEFs) and GTPase Activating Proteins PDEδ is specific for farnesylated C-terminal lation of the inflammatory, immune and of the missing C-terminal domain. The (GAPs). Arl2 and 3 are members of the Arf and of HRG4 for myristoylated proteins. apoptotic responses. The IKK kinase family TBK1 structure together with functional subfamily of the Ras superfamily of G pro- We have shown how the binding of cargo contains two canonical family members analysis indicates that dimerization is teins. Arl3 has been implicated in the func- is allosterically regulated by Arl2 and Arl3. IKKα, IKKβ and two non-canonical family required for kinase activation. However, tion of cilia, while not much is known about The specificities of interactions and the members IKKε and TBK1. TBK1 is a major as such TBK1 dimers cannot autoactivate the function of Arl2. In the GTP-bound form biological function will be discussed. target for drug development with appli- in cis, we propose that activation occurs Arl2/3 interact with a variety of proteins cations in the treatment of cancer and a when multiple called downstream effectors. These are the Keywords: Photoreceptor, Arl, PDEd, HRG4 variety of inflammatory diseases including delta subunit of phosphodiesterase PDEδ, rheumatoid arthritis and obesity-related TBK1 dimers are brought together into HRG4 (human retina gene 4) also called metabolic disorders. We have determined higher-order signaling assemblies. Unc119a, which is mutated in certain rod/ the 2.6 Å X-ray crystal structure of close to cone diseases, and various other proteins full-length TBK1 in complex with specific Polyubiquitination has been repeatedly such as BART (Binder of Arl2). inhibitors. The structure reveals a dimer associated with IKK kinase activation and with a trimodular architecture containing one hypothesis is that polyubiquitin bind- the kinase domain (KD) a Ubiquiting-like ing to TBK1-associated scaffold proteins domain (ULD) and a Scaffold dimerization triggers higher-order assembly and kinase domain (SDD). activation. The structure described here will allow development of more specific The ULD packs against a hydrophobic patch inhibitors that might find application as at the base of the KD C-lobe and is involved anti-cancer or anti-inflammatory drugs. in allosteric regulation of kinase activity. The kinase we have crystallized is in an inactive, autoinhibited conformation.

22 Session I: ALLOSTERIC SIGNALLING MECHANISMS Allosteric Regulation of Cell Signalling 23 PKA: Assembly of dynamic Allosteric regulation of oligomeric assembly: The

macromolecular signaling complexes role of PIP2 in activation of Focal Adhesion Kinase

Susan S. Taylor, P. Zhang, R. Ilouz, J. Wu, M. Keshwani, and A. Kornev Howard Hughes Medical Institute, University of California, San Diego, USA Daniel Lietha CNIO, Madrid, Spain

cAMP-dependent protein kinase (PKA), is determined by the isoform diversity of Focal Adhesion Kinase (FAK) is a non-re- activator. Indeed, PIP2 does interact with ubiquitous in every mammalian cell, reg- the R-subunits and by targeting, which is ceptor tyrosine kinase which is activated the regulatory domain of FAK, however, the ulates a plethora of biological processes. typically mediated by A Kinase Anchoring downstream of growth factor receptor mechanism of promoting FAK activation While the PKA catalytic (C) subunit is Proteins (AKAPs), which bind through an and integrin signalling. FAK is believed appears to be quite unique. We show that

the best understood protein kinase and amphipathic helix to the D/D domains of to be the key signalling component inte- PIP2 enhances FAK autophosphorylation on serves in many ways as a prototype for the the R-subunits. In this way specificPKA grating growth and adhesion signals. FAK a linker site via promoting the formation of entire superfamily, the in cells it is pack- holoenzymes are localized in close proxim- is essential during embryogenesis and FAK clusters. Supported by several lines of aged as holoenzyme with regulatory (R) ity to dedicated substrates. The functional wound healing and in pathology FAK is experiments we propose a model where in

subunits so that the activity of PKA is fully non-redundancy of the R-subunits was frequently upregulated in many types of PIP2 induced clusters FAK adopts an inter- dependent on the second messenger, cAMP. not understood in molecular terms until tumours and its upregulation correlates mediate conformation where the inhibitory There are four functionally non-redundant structures of tetrameric holoenzymes were with tumour invasion and poor patent prog- FERM-kinase interaction is partially main- R-subunits (RIα, RIβ, RIIα, and RIIβ), and solved. Although the 1º, 2º, and 3º struc- nosis. We have previously structurally and tained, but trans-autophosphorylation all have a conserved domain organization tures of all four isoforms are very similar, biochemically defined the mode of FAK is promoted. Only in a subsequent step,

with a dimerization/docking (D/D) domain the quaternary architecture of each R2C2 autoinhibition. In the autoinhibited state which involves phosphorylation of the FAK at the N-terminus followed by a flexible tetramer is remarkably different. Forma- the N-terminal regulatory FERM domain kinase domain by Src, is FAK switched to a linker that contains an inhibitor site and tion of each tetramer creates a novel 2-fold of FAK intramolecularly interacts with fully open conformation. Such a mecha- finally at the C-terminus two tandem cyclic symmetry and defines distinct isoform- the kinase domain, thereby promoting nism, where ligand binding allosterically nucleotide binding (CNB) domains. In the specific mechanisms for allosteric regula- an “off-state”. As observed for other non- induces a change in the oligomeric state, absence of cAMP each R-subunit dimer is tion. (Supported in part by grants from the receptor tyrosine kinases, it was proposed conceptually resembles ligand induced bound to two C-subunits creating and inac- National Institutes of Health (GM19301, that allosteric binding of activators to the activation of receptor tyrosine kinases, but tive tetramer. Specificity inPKA signaling GM34921, and DK54441). regulatory domain would switch FAK to has not been described previously for non- an “on-state”. Recently we identified the receptor tyrosine kinases. phosphoinositide PIP2 as a potential FAK

24 Session I: ALLOSTERIC SIGNALLING MECHANISMS Allosteric Regulation of Cell Signalling 25 CONTRIBUTED TALK CONTRIBUTED TALK From the molecular mechanism of regulation The structure of the second pair of fibronectin of AGC kinases to the development of allosteric type III repeats of the integrin β4 subunit suggests a activators and allosteric inhibitors mechanism for an intramolecular interaction

Ricardo Biondi Frankfurt University Hospital, Frankfurt am Main, Germany José M de Pereda Instituto de Biología Molecular y Celular del Cáncer, Salamanca, Spain

The group of AGC protein kinases includes PIF-pocket is also the key site that mediates The integrin α6β4 is a component of the the FnIII4 was refined against data to 1.8 more than 60 protein kinases in the human the activation by Zipper/turn-motif- and hemidesmosomes, protein complexes that Å resolution. The structure in solution of genome (e.g. PDK1 and isoforms of PKA, PKC, hydrophobic motif- phosphorylation of mediate the stable anchoring of basal epi- the FnIII-3,4 region was analyzed by SAXS. PRK, PKB/Akt, S6K, SGK, RSK, MSK, ROCK, PKB/Akt, S6K, RSK, SGK, PKC, PRK, and MSK. thelial cells to the basement membrane. The radius of gyration, calculated from GRK, amongst others). Human AGC kinases Interaction with the PIF-pocket also regu- The cytodomain of β4 is unique among the SAXS data, is 21 Å, and the maximum are involved in diverse cellular functions lates the intramolecular inhibition of atypi- the integrin family and it is responsible distance is ~63 Å. The low resolution struc- and are potential targets for the treatment cal PKCs by their N-terminal domains and for most of the intracellular interactions ture of the FnIII-3,4 modelled using the of cancer, diabetes, obesity, neurological the oligomerization that inhibits PRK2 [6]. of α6β4. The cytoplasmic region of β4 con- SAXS data and ab initio methods reveals disorders, inflammation, viral infections, We have developed reversible low-molec- tains a Calx- β domain and four fibronectin a heart-shaped structure with two lobes. etc. Most drugs to protein kinases target ular-weight compounds that, by targeting type III domains (FnIII1 to FnIII4) arranged The limited resolution of the SAXS-derived the ATP-binding site, that is conserved and the PIF-pocket site, allosterically activate in two pairs separated by a connecting seg- model hinders the unequivocal docking therefore most inhibitors to date are non- PDK1, or allosterically inhibit atypical PKCs. ment (CS). A 90-residue long C-terminal of the crystal structures of the FnIII3 and selective. Targeting non-ATP binding sites As a prototype for the group of AGC kinases tail extends after the FnIII4. An interaction the FnIII4 into the molecular envelope. can potentially achieve high degree of selec- we showed in PDK1 that small compounds between the CS and the C-tail has been Thus, we have used site direct spin labelling tivity that could allow protein kinase inhibi- binding to the PIF-pocket produce allosteric observed by yeast-2-hybrid and blot overlay (SDSL) in combination with EPR to obtain tors to be used in long term treatments. We effects that ultimately affect theATP -bind- assays, and these two regions of β4 are in inter-domain distance restraints that will identified that a particular pocket on the ing site. Together, our work shows that the close proximity in living keratinocytes. We help us to elucidate the relative orientation kinase domain, termed the PIF-pocket, is PIFpocket on AGC kinases is an allosteric have combined x-ray crystallography, small of the FnIII3 and FnIII4 in solution. Our a key mediator of the allosteric regulation regulatory site that can be targeted by small angle x-ray scattering (SAXS), mutagenesis, results have implications for the organiza- of many AGC kinases. We first described the molecules to produce pharmacological acti- and electron paramagnetic resonance spec- tion of the cytodomain of the integrin β4 regulatory properties of the PIF-pocket vators, inhibitors and substrate-selective troscopy (EPR) to characterize the FnIII- subunit and support an intramolecular on PDK1. Follow-up work verified that the inhibitors of AGC kinases. 3,4 region of β4. The crystal structure of interaction between the connecting seg- the FnIII3 was refined against data to 1.6 ment and the C-terminal tail. Å resolution, and the crystal structure of

26 Session I: ALLOSTERIC SIGNALLING MECHANISMS Allosteric Regulation of Cell Signalling 27 CONTRIBUTED TALK Coordination of Critical Cytoskeletal Changes by Nck Enables Endothelial Cell Polarization and Morphogenesis

Gonzalo Rivera Texas A&M University, College Station, USA

The establishment of cell polarity ena- In subconfluent cultures, in contrast, Nck bles directional migration of endothelial contributed to the acquisition of a migrat- cells and their organization into vascular ing phenotype such as the one observed in networks. Extracellular signals that alter tip endothelial cell. Time-lapse differential tyrosine phosphorylation drive vascular interference contrast and total internal formation by inducing reorganization of reflection fluorescence microscopy showed the actin cytoskeleton. Nck, an important that Nck couples the formation of polarized link between tyrosine phosphorylation membrane protrusions with their stabiliza- and actin dynamics, has been involved tion through the assembly and maturation in physiological responses of endothe- of cell-substrate adhesions. Measurements lial cells following stimulation by potent by atomic force microscopy showed that angiogenic stimuli including VEGF and integrin α5β1-fibronectin adhesion force integrin engagement. Nevertheless, the and cell stiffness are also modulated by role of Nck in cytoskeletal remodeling dur- Nck. Mechanistically, the loss of front- ing endothelial cell morphogenesis and back polarity in Nck-depleted cells was the underlying molecular mechanisms associated with altered spatiotemporal remain largely undetermined. Here we activation of FRET biosensors for the Rho used a combination of molecular genetics GTPases Cdc42, Rac, and RhoA, as well as and quantitative live cell microscopy to decreased phosphorylation of the regula- highlight a previously unappreciated role tory light chain of myosin II. Collectively, of Nck in the coordination of endothelial these results underscore a mechanism cell morphodynamics. Depletion/replen- whereby Nck orchestrates critical cytoskel- ishment experiments revealed an essential etal changes that enable endothelial cell role for Nck in directional migration, inva- polarization and morphogenesis. Based on sion, matrix degradation, and endothelial these findings, we postulate that Nck is an cell morphogenesis in vitro. In confluent important target for therapeutic interven- monolayers, Nck-dependent actin rear- tion in diseases associated with abnormal rangements promoted the assembly of angiogenesis. VEcadherin-dependent cell-cell contacts.

28 Session I: ALLOSTERIC SIGNALLING MECHANISMS Allosteric Regulation of Cell SignallinG SESSION II. DYNAMICS AND MODELLING OF ALLOSTERIC SYSTEMS The ins and outs of EGFR asymmetry

Daniel J. Leahy Johns Hopkins University School of Medicine, Baltimore, USA

SESSION II The epidermal growth factor receptor and head-and-neck cancers. It has long (EGFR/ErbB) family of receptor tyrosine been known that ligand binding results in DYNAMICS AND MODELLING kinases comprises EGFR, HER2/ErbB2, ErbB dimerization, stimulation of their HER3/ErbB3, and HER4/ErbB4. Each ErbB intrinsic kinase activity, and initiation OF ALLOSTERIC SYSTEMS mediates cell proliferation and differen- of intracellular signaling cascades. Early Chair: FRANCESCO LUIGI GERVASIO AND ALFONSO VALENCIA tiation events essential for normal animal structural and biophysical studies of EGFR/ development, but abnormal activity of each ErbB fragments appeared consistent with ErbB is associated with cancer. Unregulated this model and rationalized many aspects activity of EGFR and HER2 in particular are of ErbB behavior. Recent biochemical, associated with increased cancer severity, biophysical, and structural studies have and drugs targeting EGFR or HER2, including indicated that active, singly-ligated EGFR Trastuzumab (Herceptin®), Cetuximab dimers also form, and new developments in (Erbitux®), Erlotinib (Tarceva®), and our understanding of the molecular mecha- Lapatinib (Tykerb®), have proven suc- nisms governing EGFR/ErbB activation and cessful therapies for breast, colon, lung, inhibition will be presented.

Allosteric Regulation of Cell Signalling 33 Conformational dynamics in multidomain Allostery and conformational control kinases, kinase-phosphate complexes, in signaling: the ubiquitin E3 ligases and ESCRT-Protein supercomplexes

Gerhard Hummer National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, USA Ruth Nussinov National Cancer Institute and Tel Aviv University, Israel

We used simulation and experiment to multi-domain protein kinase C [6] to the Allostery is important for all cellular events. 1 Flexible cullins in cullin-RING E3 ligases study large-scale conformational motions in MAP kinases ERK2 and p38α in dynamic In E3 ubiquitin ligases, substrate binding allosterically regulate ubiquitination. Liu J, Nussinov R. J Biol Chem. 2011 multidomain proteins involved in signaling complexes with the phosphatase HePTP [7,8]. proteins, e.g. VHL-box, SOCS-box or the Nov 25;286(47):40934-42. and protein trafficking. A coarse-grained The dynamic character and the binding coop- F-box proteins, recruit protein substrates 2 Rbx1 flexible linker facilitates cullin-RING transferable energy function combined erativity of the protein assemblies emerge for ubiquitination. Ubiquitination involves ligase function before neddylation and after deneddylation. Liu J, Nussinov R. enhanced sampling methods [1] allowed as essential elements of their function. several steps. The cullin-RING E3 ligase Biophys J. 2010 Aug 4;99(3):736-44. us to simulate these large, dynamic and machinery is involved in one of these. In 3 Molecular dynamics reveal the essential partially disordered systems. To combine 1 Y. C. Kim, G. Hummer, J. Mol. Biol. this step, ubiquitin is transferred from E2 role of linker motions in the function of 375, 1416–1433 (2008). cullin-RING E3 ligases. Liu J, Nussinov R. the simulations with experiment, we devel- 2 B. Rozycki, Y. C. Kim, G. Hummer, to the substrate protein, labeling the sub- J Mol Biol. 2010 Mar 12;396(5):1508-23. oped ensemble refinement procedures [2,3] Structure 19, 109-116 (2011). strate protein for degradation. However, 4 The mechanism of ubiquitination in the cullin- that incorporate data from small-angle 3 E. Boura, B. Rozycki, D. Z. Herrick, H. S. when E3, E3-substrate and E2-ubiquitin RING E3 ligase machinery: conformational Chung. J. Vecer, W. A. Eaton, D. Cafiso, G. control of substrate orientation. Liu J, Nussinov X-ray scattering (SAXS), spin-label distance Hummer, J. H. Hurley, Proc. Natl. Acad. crystal structures are modeled together, R. PLoS Comput Biol. 2009 Oct;5(10):e1000527. measurements (EPR), single-molecule Sci. USA 108, 9437-9442 (2011). the distance between the ubiquitinated 5 Allosteric effects in the marginally stable fluorescence energy transfer (FRET), and 4 Y. C. Kim, C. Tang, G. M. Clore, G. Hummer, Proc. E2 and the substrate binding site is 50-59 von Hippel-Lindau tumor suppressor Natl. Acad. Sci. U.S.A. 105, 12855-12860 (2008). protein and allostery-based rescue mutant paramagnetic relaxation enhancement 5 E. Boura, B. Rozycki, H. S. Chung, D. Z. Herrick, B. Angstrom, raising the question how the E3 design. Liu J, Nussinov R. Proc Natl Acad (PRE) experiments as well as conventional Canagarajah, D. Cafiso, W. A. Eaton, G. Hummer, machinery bridges the distance and orients Sci USA. 2008 Jan 22;105(3):901-6. solution NMR [2-5]. This combination with J. H. Hurley, Structure 20, 874-886 (2012). the substrate for the ubiquitin transfer? 6 T. A. Leonard, B. Rozycki, L. F. Saidi, G. experiment allowed us not only to validate Hummer, J. H. Hurley, Cell 144, 55-66 (2011). Allostery is a key factor, and the talk will the simulation approach, but also to obtain 7 D. M. Francis, B. Rozycki, D. Koveal, address our recent work addressing this detailed representations of the structures G. Hummer, R. Page, W. Peti, Nature fascinating question. Chem. Biol. 7, 916-924 (2011). and motions in systems ranging from the 8 D. M. Francis, B. Rozycki, A Tortajada, supercomplexes of the ESCRT membrane- G. Hummer, W. Peti, R. Page, J. Am. protein trafficking system [2,3,5] over the Chem. Soc. 133, 17138–17141 (2011).

34 SESSION II: DYNAMICS AND MODELLING OF ALLOSTERIC SYSTEMS Allosteric Regulation of Cell Signalling 35 Controlling allosteric Exploring the landscape for networks in proteins proteins and the ribosome: connecting simple models and detailed simulations

Nikolay V. Dokholyan University of North Carolina at Chapel Hill, USA José N. Onuchic Center for Theoretical Biological Physics, Rice University, Houston, USA. *Supported by the NSF

We present a novel methodology for to control protein function allosterically. Energy landscape theory and the funnel protein complexes, not only folding but also delineating allosteric pathways in proteins. We design a novel protein domain that can concept have enormously advanced our function associated to large conformational We use this methodology to uncover the be inserted into identified allosteric site understanding of protein folding and motions. Therefore a discussion of how structural mechanisms responsible for cou- of target protein. Using a drug that binds more recently the role of global motions global motions control the mechanistic pling of distal sites on proteins and utilize to our domain, we alter the function of in protein function. Going beyond folding processes in the ribosome and molecular it for allosteric modulation of proteins. We the target protein. We successfully tested small and mid size proteins, the power of motors will be presented. In this presenta- will present examples where inference of this methodology in vitro, in living cells reduced models to study the physics of tion we will focus on how the combination allosteric networks and its rewiring allows and in zebrafish. We further demonstrate protein assembly, protein binding and of detailed atomistic simulations and sim- us to “rescue” cystic fibrosis transmem- transferability of our allosteric modulation recognition, and larger biomolecular plified structure-base models has allowed brane conductance regulator (CFTR), a pro- methodology to other systems and extend machines has also proven impressive. Since us to explore the entire landscape for the tein associated with fatal genetic disease it to become ligh-activatable. energetic frustration is sufficiently small, Ribosome. This would be impossible with cystic fibrosis. We also use our methodology native structure-based models, which cor- one of these techniques alone. The effect of respond to perfectly unfrustrated energy magnesium in controlling structure transi- landscapes, have shown to be a powerful tions will also be discussed mostly in the approach to explore larger proteins and context of riboswitches.

36 SESSION II: DYNAMICS AND MODELLING OF ALLOSTERIC SYSTEMS Allosteric Regulation of Cell Signalling 37 CONTRIBUTED TALK Intrinsic disorder in wild-type Proteins in flagrante: excursions EGFR kinase underlies oncogenicity in silico and in proteo of cancer mutations

Yibing Shan D. E. Shaw Research, New York, United States Dorothee Kern Howard Hughes Medical Institute, Brandeis University, Waltham, USA

Using molecular dynamics simulations, we these findings, our experiments indicate Understanding biological function, such I will discuss our exploration of the full find that the dimerization interface of the that the cancer mutation causes abnormally as the fascinating rate acceleration of energy landscape of enzyme catalysis wild-type EGFR kinase domain is intrinsical- high activity primarily by promoting EGFR enzymes, specificity of protein/protein through a combination of time-resolved ly disordered, and that it becomes ordered dimerization. Additionally, we elucidated interactions or the delicately controlled NMR including high-pressure NMR, crys- only upon dimerization. Our simulations the conformational pathway of the deacti- action of signaling has been a long-standing tallography, single-molecule FRET and MD suggest, moreover, that some cancer-linked vation of EGFR kinase and the mechanism challenge. Despite remarkable informa- simulation. Allosteric is in play for 3 very mutations distal to the dimerization inter- of the cross-membrane coupling of EGFR tion generated using chemical tools in different systems: an enzyme, a phosho- face facilitate EGFR dimerization by sup- embedded in membrane by modeling the the last 100 years, complemented more rylation-mediated signaling protein and pressing this local disorder. Corroborating dimers of full-length EGFR dimers. recently with structural and computational the inhibition of a kinase via protein/pro- approaches, we cannot yet identify with a tein interactions. For the latter example, complete energy inventory how ANY protein binding via an allosteric shift and not via works. The secret of enzymes lies in their an induced fit is directly demonstrated by ability to partition energetic contributions flux measurements, the only rigorous test among many atoms in a well-coordinated for selected binding. style. To unravel these secrets, proteins in action are spied on at atomic resolution to The presented data stress the point that provide a comprehensive description of highly choreographed chemical integrity enzyme catalysis in the form of an energy AND optimized conformational sampling is landscape. Since the rate of catalysis is a prerequisite for efficient enzyme catalysis. determined by the climb over a sequence The power of an intimate marriage between of energy barriers, we focus here on the NMR and other biophysical methods and critical question of transition pathways MD simulations including a variety of novel with the highest energy state being the pathway algorithms will be illustrated. transition state.

38 SESSION II: DYNAMICS AND MODELLING OF ALLOSTERIC SYSTEMS Allosteric Regulation of Cell Signalling 39 Dynamical effects in the allosteric Co-evolution based methods in the prediction control of kinases of allosteric channels and contact networks

N Dölker, J Juraszek and Francesco Luigi Gervasio CNIO, Madrid, Spain Alfonso Valencia CNIO, Madrid, Spain

Protein kinases (PK) are one of the larg- In Abl a shift of the SH2 domain from the C- In this presentation I will describe the Together with the development of methods est and most functionally diverse protein to the N-terminus of the catalytic domain current status of co-evolution based meth- my group is interested the practical use of families and are involved in most cellular has been found to be involved in activation ods that for the last 20 years have addressed this type of computational methods for the pathways. PK malfunction is related to an [1]. The allosteric mechanism, by which the prediction of protein interactions (inter analysis of the consequences of mutations important number of human diseases, such the SH2 domain induces conformational protein) and protein contacts (intra pro- in the context of cancer genome projects as cancer, diabetes and cardiovascular dis- changes at the active site, is still debated. We tein). I will emphasize the utility of methods (Vazquez et al., 2012). eases. Thus, PK represent major targets for have used elastic network models, normal developed by my group for the study of con- drug development. mode analysis, molecular dynamics simula- certed evolution between interacting pro- · Juan D, Pazos F, Valencia A (2008) High- confidence prediction of global interactomes tion and mutagenesis to gain insight into tein families (Juan et al., 2008), the detec- based on genome-wide coevolutionary networks. Historically, drug discovery programs the interplay between the SH2 domain and tion of the residues potentially responsible Proc Natl Acad Sci USA. 105(3):934-9. have been dominated by efforts to deve- the relevant motifs at the catalytic site. We of binding specificity (Rausell et al., 2010), · Rausell A, Juan D, Pazos F, Valencia A (2010) Protein interactions and ligand binding: from lop antagonists that compete for binding propose a mechanism, by which the SH2 and the potential implications of these and protein subfamilies to functional specificity. with endogenous ligands at orthosteric domain influences the dynamics of the other developments for modeling protein Proc Natl Acad Sci USA. 107(5):1995-2000 sites. However, allosteric drugs might offer crucial residues directly involved in the complexes (Wass et al., 2011). · Vazquez M, et al, in preparation. · Wass MN, Fuentes G, Pons C, Pazos F, several therapeutic advantages over tradi- catalytic process. In FgFr we use free ener- Valencia A (2011) Towards the prediction tional orthosteric ligands, including greater gy calculations, crystallography and NMR of protein interaction partners using safety and/or selectivity. approaches to shed light on the mode of physical docking. Mol Syst Biol. 7:469. action of a novel allosteric inhibitor. [2] Here, by combining state-of-the-art compu- ter simulations with spectroscopy, chemical 1 Nagar B, Hantschel O, Seeliger M, Davies JM, Weis WI, Superti-Furga G, Kuriyan and molecular biology approaches we study J Molecular Cell 2006, 21, 787-798. in great details the role of conformational 2 F. Bono et al., submitted to Cancer Cell changes in the allosteric control of two pharmaceutically relevant kinases: Abl and FGFr.

40 SESSION II: DYNAMICS AND MODELLING OF ALLOSTERIC SYSTEMS Allosteric Regulation of Cell Signalling 41 The innate immunity molecular machinery Crystal structure of the 1 MDa mammalian TRiC/CCT complex with tubulin

Giulio Superti-Furga1, Yazan M. Abbas2, Andreas Pichlmair1,3, Kumaran Kandasamy1, Maria W Górna1, Tilmann Bürckstümmer1,4, Adriana Goncalves1, Carol-Ann Eberle1, Leonhard Heinz1, Marielle Klein1, Astrid Fauster1, Adrijana Stefanovic1, 1Christoph L Baumann1, Alexey Stukalov1, Andre Müller1, Keiryn L Bennett1, Jacques Colinge1 and Bhushan Nagar2 1CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria; 2McGill University, Montreal, Canada; 3Max-Planck Institute for Bio- chemistry, Martinsried / Munich, Germany; 4Haplogen GmbH, Vienna, Austria, Vienna, Austria Guillermo Montoya CNIO, Madrid, Spain

Innate immune processes are very much affinity baits to map the cell's recognition Protein folding is assisted by molecular structure of the open conformation of this centered on the management of molecular machinery. Overall, we have obtained an chaperones. CCT (chaperonin containing nanomachine in complex with tubulin, pro- interactions. A variety of activities demand overview of host cellular networks involved TCP-1, or TRiC) is a 1-MDa oligomer that is viding information about the mechanism by it: Detection of pathogens and danger signals, in innate immunity as well as the dynamic built by two rings comprising eight different which it aids tubulin folding. The structure distinction of self- from non-self molecules, assembly, disassembly and regulation of 60-kDa subunits. This chaperonin regu- showed that the substrate interacts with signaling to initiate both transcriptional and several molecular machines, such as the IFIT, lates the folding of important proteins loops in the apical and equatorial domains non-transcriptional responses, neutraliza- TLR and NLRPs complexes as well as on the including actin, α-tubulin and β-tubulin. of CCT. The organization of the ATP-bind- tion of pathogens, resolution of infection logic that a variety of different pathogens We used an electron density map at 5.5 ing pockets suggests that the substrate is and inflammation. Using affinity proteom- use to overturn cellular processes to their Å resolution to reconstruct CCT, which stretched inside the cavity. Our data provide ics coupled to mass spectrometry, bioinfor- own advantage. New three-dimensional showed a substrate in the inner cavities the basis for understanding the function of matics, pathogen infections, RNAi and gene structures of components of these machines of both rings. Here we present the crystal this chaperonin. inactivation in mice for selected cases, we reveal new recognition mechanisms as well have step-wise mapped the cellular proteins as allosteric movements involved in the involved in these processes and monitored process. From a biochemical and systems- interactions among themselves as well as biological point of view it is already clear with perturbing proteins from invading that subtle calibration of accurate molecular pathogens. We have used viral ORFs encod- interactions is of uttermost importance for ing immune modulators to map the human the cell to safeguard its homeostasis in face cell defence network. We have also used of perilous environmental challenges such pathogen-associated molecular patterns as as pathogen infections.

42 SESSION II: DYNAMICS AND MODELLING OF ALLOSTERIC SYSTEMS Allosteric Regulation of Cell Signalling 43 CONTRIBUTED TALK CONTRIBUTED TALK Impact of mutations on the Characterizing protein conformation allosteric conformational equilibrium changes through torsional linear response and allosteric profiles

Ugo Bastolla, Helena Gomes, Javier Klett and Raul Mendez-Giraldez Patrick Weinkam University of California, San Francisco, USA Centro de Biologia Molecular Severo Ochoa, CSIC-UAM, Madrid, Spain

Allostery in a protein involves effector transitions, as defined by the effector- An intriguing property of the functional to increase the free energy barrier opposed binding at an allosteric site that changes bound and unbound protein structures. We conformation changes of proteins is that to the conformation change. We assess the structure and/or dynamics at a distant, develop a set of metrics based on simulated they are strongly correlated with thermal the null model and the deviations from it functional site. In addition to the chemical trajectories, molecular mechanics ener- motions, despite their very different scales: through a massive computational analysis equilibrium of ligand binding, allostery gy functions, and stereochemical effects. tenths of angstrom for thermal motions of conformation changes in the PDB and involves a conformational equilibrium Using a machine-learning algorithm on a versus several angstroms for conformation several detailed case studies. We argue between one protein substate that binds dataset of 10 proteins and 179 mutations, we changes. This relationship is predicted by that this approximate analysis of energy the effector and a second substate that predict both the magnitude and direction linear response theory. We develop a null barriers provides valuable information less strongly binds the effector. We run of the allosteric conformational equilib- model of non-functional protein deforma- for characterizing conformation changes, molecular dynamics simulations using rium shift by the mutation; the impact of a tions based on the linear response of the and discuss its relationship with confor- simple, smooth energy landscapes to sam- large identifiable fraction of the mutations torsional network model (TNM) that we mational selection and induced fit. This ple specific ligand-induced conformational can be predicted with an average unsigned recently introduced, an elastic network framework, based on the torsional linear error of 1 kBT. model (ENM) that adopts main-chain and response, naturally allows to predict pro- side-chain torsion angles as degrees of tein positions that are most able to produce freedom and all-atoms contacts as inter- allosteric changes, and protein positions actions. Within the harmonic approxima- that perform highly robust motions. We tion, significant deviations from this null show that active sites of proteins tend to model have the effect to either reduce or belong to this type of sites class.

44 SESSION II: DYNAMICS AND MODELLING OF ALLOSTERIC SYSTEMS Allosteric Regulation of Cell Signalling 45 CONTRIBUTED TALK Oncogenic mutations corrupt the ordered autophosphorylation of the RET receptor tyrosine kinase: structural and molecular basis of RET-MEN2B Ivan Plaza Menacho1, Barnouin K.2, Borg A. 3, Murray-Rust J.1, Knowles P.1 and McDonald N. Q.1 1Structural Biology Laboratory; 2Protein Analysis and Proteomic Laboratory and 3Protein Production Facility, London Research Institute, Cancer Research UK, London, UK

Despite the large body of genetic and bio- within the flanking regions of the kinase logical evidence suggesting the importance domain leads to a sequential open confor- of the RET receptor tyrosine kinase in early mation fully achieved when tyrosines in the development and neoplastic processes, the activation loop are phosphorylated at later precise mechanisms of RET kinase activa- time points. These results further indicate tion and signal transduction at molecular that the flanking regions ofRET core kinase and structural level remain unknown. In domain can allosterically regulate kinase this study we investigate the temporal activity, which we prove by testing N- and sequence of RET intracellular domain (ICD) terminal deletions of RET ICD by LFQMS, autophosphorylation by using label free biochemical and structural analysis. In quantitative mass spectrometry (LFQMS) order to uncover the molecular mechanism and phospho-specific antibodies. We dem- driving oncogenic MEN2B-RET activation onstrate that upon kinase domain activation and signalling we show a perturbation in the earliest sites of RET autophoshorylation the kinetics of autophosphorylation by are outside the core kinase domain. In con- oncogenic RET V804M and M918T, together trast, a distinct set of autophosphorylation with higher basal levels of autophosphor- sites are formed within the kinase domain, ylation at specific sites in the case of RET including RET “activation loop” tyrosines, M918T. These findings were further sup- only at later time points. Low angle X-ray ported with a structural analysis of onco- scattering (SAXs) data show that the radius genic RET-MEN2B M918T kinase crystal of gyration (Rg) of the non-phosphorylated structure solved at 2.1Å resolution. RET ICD is smaller than when it is phospho- rylated, consistent with a model where Other Information · RET (REarrange during Transfection) the intracellular domain of RET displays a · MEN2 (Multiple Endocrine Neoplasia type 2) closed conformation in the non-phoshpor- ylated state. Upon kinase activation, phos- phorylation of tyrosines residues situated

46 SESSION II: DYNAMICS AND MODELLING OF ALLOSTERIC SYSTEMS Allosteric Regulation of Cell SignallinG SESSION III. ALLOSTERIC INHIBITION Structural insights into the mechanism Jak kinase activation in myeloproliferative disease

Michael J. Eck Dana Farber Cancer Institute, Harvard Medical School, Boston, USA

SESSION III Jak-family tyrosine kinases drive signaling mutant reveals that the mutation induces from diverse growth factor and cytokine a conformational switch that remodels ALLOSTERIC INHIBITION receptors. Jaks contain a pseudokinase one surface of the N-terminal lobe of the Chair: Giulio Superti-Furga domain, which is thought to participate pseudokinase domain. A key element of in regulating the activity of the adjacent this switch is a helical segment that lies just tyrosine kinase domain. Mutations within N-terminal to the pseudokinase domain; the pseudokinase domain are the cause of in the intact protein this segment consti- myeloproliferative disorders and hemato- tutes a link to the adjacent SH2-like domain. logic malignancies. The V617F mutation in This linker helix is also a site of activating Jak2 leads to development of polycythemia mutations. Comparison of the two crystal- vera, and the equivalent mutation in Jak1 lographically distinct molecules in the wild (V658F) is a frequent cause of T-cell acute type pseudokinase structure shows that the lymphoblastic leukemia (T-ALL). A lack of conformation adopted by the V658F mutant structural information for the Jak pseu- is also accessible in the normal protein, dokinase domains has hampered elucida- suggesting that the conformational switch tion of the mechanism by which this muta- triggered by the V658F mutation is closely tion leads to constitutive kinase activation. related to the physiological mechanism Here we describe crystal structures of the of Jak activation in response to receptor pseudokinase domain of Jak1. Comparison engagement. of the WT structure with that of the V658F

Allosteric Regulation of Cell Signalling 51 Allosteric regulation of intracellular Elucidation of the molecular mode of action and membrane proteins by DARPins of SSR128129E, the first small molecule allosteric inhibitor of FGF receptor signaling

Markus G. Grütter University of Zürich, Switzerland Françoise Bono SanofiR &D, Toulouse, France

Repeat proteins are ubiquitous protein- 1 Kohl, A., Binz, H. K., Forrer, P., Stumpp, M. The fibroblast growth factor (FGF) / fibrob- for binding to the receptor but inhibits FGF- protein interaction molecules fundamental T., Plückthun, A. & Grütter, M. G. Designed last growth factor receptor (FGFR) signaling induced signaling linked to receptor inter- to be stable: crystal structure of a consensus to many biological processes. This feature ankyrin repeat protein. Proc. Natl. Acad. network plays an important role in cell nalization in an allosteric manner as shown is exploited in vitro, by designing ankyrin Sci. USA 100, 1700-1705 (2003). growth, survival, differentiation and ang- by crystallography studies, NMR, Fourier repeat proteins (DARPins) and combinato- 2 Binz, H. K., Amstutz, P., Kohl, A., Stumpp, iogenesis. Deregulation of FGFR signaling transform infrared spectroscopy, molecular M.T., Briand, C., Forrer, P., Grütter, M. G. & rial libraries thereof [1]. By using ribosome Plückthun, A. High affinity binders selected can lead to cancer development. Here we dynamics simulations, free energy calcula- display DARPins having high affinity (nM from designed ankyrin repeat protein libraries. report a new FGFR inhibitor, SSR128129E tions and FGFR mutagenesis. SSR is the first range) and specificity for any target protein Nature Biotechnology, 22, 575-582 (2004). (SSR), which binds to the extracellular part reported small molecule allosteric inhibitor 3 Schweizer, A., Roschitzki-Voser, H., Amstutz, can be selected [2]. Their use in structural P., Briand, C., Gulotti-Georgieva, M., of the receptor, thus inhibiting FGF-induced of FGF/FGFR signaling. biology, in modulating the activity and in Prenosil, E., Binz, HK., Capitani, G., Baici, signaling. SSR does not compete with FGF determining the role of a target protein in A., Plückthun, A. & Grütter MG. Inhibition of caspase-2 by a designed ankyrin repeat signaling events will be discussed. The meth- protein: specificity, structure, and inhibition odology as well as structural and functional mechanism. Structure 15, 625-636 (2007). data of DARPins in complex with caspases [3], 4 Kohl, A., Amstutz, P., Parizek, P., Binz, H.K., Briand, C., Capitani, G., Forrer, P., Plückthun, kinases [4], the membrane proteins AcrB [5] A. & Grütter, M.G. Allosteric inhibition and ABC-transporters [6] will be presented. of aminoglycoside phosphotransferase The examples illustrate the importance of by a designed ankyrin repeat protein. Structure 13, 1131-1141 (2005). specificity of recognition when analyzing 5 Sennhauser, G., Amstutz, P., Briand, C., the role of a particular protein in a signaling Storchenegger, O., & Grütter, M.G. Drug export pathway but also the potential of the DARPin pathway of multidrug exporter AcrB revealed by DARPin inhibitors. PloS Biol. 5, e7 (ePub), (2007). technology in structural biology and inhibi- 6 Mittal, A., Boehm, S., Grütter, MG., tor design. Bordignon, E. & Seeger, MA. Asymmetry in the homodimeric ABC transporter MsbA recognized by a DARPin. J Biol Chem. 287, 20395-20406 (2012).

52 Session III: ALLOSTERIC INHIBITION Allosteric Regulation of Cell Signalling 53 Allosteric kinase inhibition Non-ATP competitive kinase inhibitors: potentials and limitations

Jeffrey R. Peterson Fox Chase Cancer Center, Philadelphia, USA Doriano Fabbro Novartis Pharma AG, Basel, Switzerland

Protein kinases are an important class of target. These off-target effects can pro- Conformational bias, i.e. a shift in the inhibitors and the limited set of chemo- drug targets in cancer due to their drugga- duce unwanted toxicities and complicate equilibrium between active and inac- types targeting the ATP binding site —a bility and their central roles in promoting the interpretation of experiments using tive conformations is a key determinant highly crowded area— are issues in kinase cell survival and proliferation. The develop- these agents. Inhibition of kinase cata- in kinase regulation and can be brought drug discovery. In contrast less is known ment of ATP-competitive, small-molecule lytic activity by allosteric mechanisms is about by many factors including post- about targeting the protein kinases outside kinase inhibitors has been a key strategy in an alternative approach that, by targeting translational modifications, regulatory of the “classical ATP pocket”. However, a anti-cancer drug development but the evo- less-conserved binding sites, is expected to proteins, ligand binding and pathologic few well documented examples like the lutionary conservation of the ATP-binding allow more selective kinase inhibition. I will kinase deregulation. inhibitors for MEK, ABL, mTOR, AKT and IGF- pocket between different kinases generally discuss examples of allosteric kinase inhibi- 1R have shown that protein kinase can be leads to promiscuous inhibition of mul- tion from our own work and others with an Kinase inhibitors can be viewed as particu- inhibited by mechanisms that are outside tiple kinases in addition to the intended emphasis on screening approaches, mecha- lar ligands to protein kinases. As the mode of the beaten tracks. nism of action, and target selectivity. of action is linked to the binding mode, the selectivity as well as the kinetics of kinase In this lecture we will review the field of inhibitors can often be rationalized based non-ATP site directed inhibitors and we will on the target conformation. Our knowledge discuss the potentials as well as the limita- on the structural determinants of kinase tion of these approaches that should lead inhibition by small molecules binding to to improved target selectivity as well as the the ATP pocket has advanced steadily in the use of these types of inhibitors to prevent past years. Selectivity of ATP directed kinase resistance protein kinase inhibition.

54 Session III: ALLOSTERIC INHIBITION Allosteric Regulation of Cell Signalling 55 CONTRIBUTED TALK Regulation of HGF/SF-MET signalling Identification and characterisation of novel allosteric regulators of PKM2 using fragment based screening

Ermanno Gherardi MRC Centre, Cambridge and University of Pavia, Italy Marc O’Reilly Astex Pharmaceuticals, Cambridge, UK

Receptor tyrosine kinases are potent related to blood proteinase plasminogen Cancer cells exhibit several unique meta- uncharacterised amino acid binding pocket. allosteric machines capable of initiat- and expressed in a tightly regulated, stage- bolic phenotypes that are critical for cell I will present X-ray crystallographic, bio- ing intracellular signals through activa- and tissue-specific manner alongside with growth and proliferation. Specifically, they physical, enzymatic and cellular data that tion of the cytoplasmic, catalytic kinase two truncated variants of the HGF/SF tran- over-express the M2 isoform of the tightly prove that the hPKM2 amino acid binding domain upon binding of specific ligands script known as NK1 and NK2, which act as regulated enzyme pyruvate kinase (hPKM2), pocket represents a key hPKM2 reglulatory to the receptor ectodomains. Hepatocyte partial receptor agonists and antagonists. which controls glycolytic flux. This makes node that modulates hPKM2 activity via an growth factor/scatter factor (HGF/SF) and We have determined several structures PKM2 an attractive target for anti-cancer allosteric mechanism. I will also discuss the receptor tyrosine kinase MET control of the NK1 and NK2 fragments of HGF/SF therapy. A crystallographic, fragment the potential biological implications for a number of developmental processes, either alone or in complex with heparin (a based, screen identified multiple PKM2 allosteric regulation of hPKM2 activity by regeneration of several epithelial organs structural analogue of heparan sulphate ligand binding sites, including a previously natural amino acids. in adult life and distant migration of a co-receptor) and further structures of NK1, variety of cancer cells. This signalling sys- HGF/SF and anti-MET antibodies in com- tem, therefore, has attracted considerable plex with MET fragments. These studies interest both from the point of view of shed some initial light on the mechanisms the control of embryogenesis and regen- of ligand-induced MET activation as well eration in vertebrate organs and from a as the mechanism of MET inhibition by therapeutic point of view. HGF/SF is a com- engineered fragments of HGF/SF or anti- plex multidomain protein structurally MET antibodies.

56 Session III: ALLOSTERIC INHIBITION Allosteric Regulation of Cell Signalling 57 Allosteric Regulation of Cell SignallinG Speakers’ biographies Jean-Pierre Changeux Collège de France, Pasteur Institute, Paris, France

A pioneer in the field of modern neuro- allosteric membrane protein and to its role science, Jean-Pierre G. Changeux is emeri- in synaptic plasticity, nicotine addiction tus professor at the Pasteur Institute and at and higher brain functions such as cogni- the Collège de France in Paris. He received tive learning, reward mechanisms, and a doctorate in 1964 under the tutelage of consciousness. Jacques Monod of the Pasteur Institute and completed postdoctoral studies at the Changeux has written or co-written several University of California, Berkeley, and the books on neuroscience for general audi- Columbia University College of Physicians ences, including Neuronal Man; Conversa- and Surgeons before returning to the Pas- tions on Mind, Matter and Mathematics; teur Institute. What Makes Us Think; and The Physiology of Truth. As a graduate student, Changeux conduct studies on the experimental basis and theo- He is a member of the U.S. National Academy retical foundations of allosteric interactions of Sciences and the recipient of numerous between topographically distinct sites in honors including the Gairdner foundation proteins. He subsequently identifies the award, the Richard Lounsbery Prize, the first protein receptor of a neurotransmit- Wolf Prize, the Balzan Prize, the National ter—the nicotinic receptor of acetylcho- Academy of Science's Award in Neuro- line— and contributes to the understanding science. of its function in signal transduction as an

Allosteric Regulation of Cell Signalling 61 Roger Sunahara Timothy A. Springer University of Michigan Medical School, Ann Arbor, USA Harvard Medical School, Boston, USA

My graduate training with Dr. Philip See- and/or arrestin recruitment. Indeed these Dr. Timothy Springer is a 1971 Phi Beta Kap- Velcade). In 2004, he was co-recipient of man (University of Toronto) and post- approaches served to be invaluable for our pa graduate of the University of California, the prestigious Crafoord Prize given by doctoral work with Dr. Alfred G. Gilman recent elucidation of the crystal structure Berkeley, and earned a Ph.D. in molecular the Royal Swedish Academy of Sciences.

(University of Texas Southwestern) have of the β2-adrenergic receptor bound to the biology and biochemistry from Harvard Dr. Springer is one of the most highly cited provided a strong appreciation for the appli- heterotrimeric G protein, Gs. The crystal University in 1976. He is a member of the scientists in the world, with his over 540 cation of pharmacology, biochemistry and structure and functional analyses of the National Academy of Sciences and the papers cited in excess of 55,000 times. He structural biology to delineate mechanisms GPCR-G protein complex have now provided American Academy of Arts and Sciences. has held a 10-year NIH MERIT award not of action. During my postdoctoral felllow- a plausible mechanism for hormone activa- In 1993 he founded LeukoSite, a company once, but twice and is responsible for many ship we solved the crystal structure of the tion of G proteins. We continue to utilize through which two drugs were brought of our current concepts on how conforma- catalytic core of adenylyl cyclase bound these approaches to better understand to market following a merger with Mil- tional changes affect integrin structure to the stimulatory G protein, Gsα, as well the basis for receptor-G protein specifi- lennium Pharmaceuticals (Campath and and function. as activated Gsα alone. These and other city as well as the mechanism underlying data led to the elucidation of the catalytic receptor-arrestin interactions. It is our mechanism of adenlylyl cyclase. hope that by understanding the molecular mechanism and structural bases for ligand My laboratory focuses on understanding efficacy that we may engineer ligands that the mechanism by which hormone binding specifically target downstream signaling to G protein-coupled receptors leads to pathways. Such signaling-specific or biased activation of signaling cascades. We develop ligands may provide health professionals and utilize biochemical and biophysical with more selective, more potent and safer methodologies to elucidate how hormone therapeutics. binding can modulate G protein activation

62 Speakers’ biographies Allosteric Regulation of Cell Signalling 63 E. Yvonne Jones Sir Tom Blundell Wellcome Trust Centre for Human Genetics, University of Oxford, UK FRS, FmedSci, Department of Biochemistry at Cambridge, University of Cambridge, UK

E. Yvonne Jones is Joint Head of the Divi- receptors and signalling assemblies. Her Tom researches on molecular and struc- He has written extensive software for struc- sion of Structural Biology and Deputy current research aims to integrate high tural biology of growth factors, receptor tural bioinformatics and developed new Director of the Wellcome Trust Centre resolution mechanistic detail with lower activation, signal transduction and DNA approaches to structure-guided fragment- for Human Genetics at the University of resolution cellular context and is focused repair, important in cancer and other based drug discovery. In 1999 he co-founded Oxford. Over the last 20 or so years she on cell guidance cues, cell adhesion recep- diseases. He has published 500 research Astex Therapeutics, an oncology company has developed a programme of research tors and Wnt signalling. papers, including 30 in Nature. that is now public with eight drugs in clini- into the structural biology of cell surface cal trials. He was founding CEO of BBSRC 1991-1996, Chairman of Royal Commission on Envi- ronment 1998-2005, Deputy Chair of Institute of Cancer Research since 2008 and President of the UK Science Council since 2011.

64 Speakers’ biographies Allosteric Regulation of Cell Signalling 65 Alfred Wittinghofer Susan S. Taylor MPI for Molecular Physiology, Dortmund, Germany Howard Hughes Medical Institute, University of California, San Diego, USA

Academic History and Appointments Major Research Interests Taylor was born in Wisconsin and gradu- 2006 has been a Professor in the Depart- Since 2009 Emeritus (Emeritus-Group- We are interested in GTP-binding proteins, ated with a major in Chemistry from the ment of Pharmacology. In 1993 she was Leader, MPI Dortmund) their regulation by Guanine Nucleotide University of Wisconsin. She received her elected to the American Academy of Arts since 1993 Honorary Professor of Biochem- Exchange Factors and GTPase-Activating PhD in Physiological Chemistry from the and Sciences, and in 1997 she was elected istry, Faculty of Chemistry/Biochemistry, Proteins. We also study the downstream Johns Hopkins University and then did to the National Academy of Sciences, to Ruhr-University Bochum, Germany effects of these proteins, their interaction postdoctoral research first at the Medical the Institute of Medicine, and became a · Director of the Dept. Structural Biology at with effectors and their biology. Recently Research Laboratory of Molecular Biology fellow of the Howard Hughes Medical Insti- the MPI of Molecular Physiology we started looking at the function of Arl with Brian Hartley and then with Nathan tute. She is married to Palmer W. Taylor, 1992 Habilitation in Biochemistry, Uni- proteins in the function of cilia. Kaplan in the Department of Chemistry Professor of Pharmacology and Dean of versity Heidelberg, Germany at the newly founded University of Cali- the Skaggs School of Pharmacy and Phar- 1980 Research group leader, Max Planck Selected Publications: fornia, San Diego. In 1972 she joined the maceutical Sciences at the University of Institute for Medical Research, Heidelberg, C. Thomas, I. Fricke, A. Scrima, A. Berken, and Department of Chemistry as an Assist- California, San Diego. They have three chil- A. Wittinghofer. Structural evidence for a Germany common intermediate in small G protein-GEF ant Professor. In 1985 she was promoted dren, Tasha, Ashton, and Palmer Andrew 1974-1980 Scientific employee at the Max reactions. Molecular Cell 25, 141-149 (2007) to full Professor in the Department of and three grandchildren, Elian Vera, and Planck Institute for Medical Research, Hei- J.L. Bos, H. Rehmann, and A. Wittinghofer. GEFs Chemistry and Biochemistry and since Natalia and Rowan Taylor. and GAPs: Critical Elements in the Control of delberg Small G Proteins. Cell 129, 865-877 (2007) 1971-1973 Postdoctoral fellow, University S. Veltel, R. Gasper, E. Eisenacher and A. of North Carolina, USA Wittinghofer. The retinitis pigmentosa 2 (RP2) gene product is a GTPase-Activating 1971 Ph.D., German Wool Research Insti- protein (GAP) for Arl3. Nature Struct tute, Aachen, Germany Mol. Biol. 15, 373-380 (2008) 1968 Diploma in Chemistry, Technical B. Sot, C. Kötting, D. Deaconescu, Y. Suveyzdis, K. Gerwert, and A. Wittinghofer. Unravelling University of Aachen, Germany the mechanism of dual-specificity GAPs. EMBO J. 29, 1205-1214 (2010) Awards S. A. Ismail, Y.-X. Chen, A. Rusinova, A. Chandra, M. Bierbaum, L. Gremer, G. Triola, H. 2001 Louis-Jeantet Prize for Medicine Waldmann, P.I.H. Bastiaens and A. Wittinghofer. 2002 Richard-Kuhn-Medal of the Gesells- Arl2-GTP and Arl3-GTP regulate a GDI-like chaft Deutscher Chemiker (GDCh) transport system for farnesylated cargo. Nature Chemical Biology 7, 942-949 (2011) 2003 Deutscher Krebspreis 2003 der Deutschen Krebsgesellschaft e.V. 2003 Otto-Warburg-Medal of the GBM

66 Speakers’ biographies Allosteric Regulation of Cell Signalling 67 Daniel Lietha Daniel J. Leahy CNIO, Madrid, Spain Johns Hopkins University School of Medicine, Baltimore, USA

Daniel Lietha obtained his undergraduate Cancer Institute (USA), where he studied My laboratory has a longstanding interest Biochemistry, Columbia University, degrees in Chemistry from the Zürcher signalling mechanisms triggered by cell- in the molecular mechanisms governing · Assistant Professor, Johns Hopkins Uni- Hochschule für Angewandte Wissenschaf- matrix adhesion. In 2009 Daniel joined activity of cell-surface receptors in normal versity School of Medicine ten (Switzerland) and in Biotechnology the CNIO as Junior Group Leader in the and disease states. Specific projects in the · Associate Professor, Johns Hopkins Uni- from Teesside University (UK). In 2003 Structural Biology and Biocomputing Pro- lab involve X-ray structural and biophysi- versity School of Medicine he obtained his PhD in Protein Crystal- gramme. His research focuses on molecular cal characterizations of components of the 2004-present Professor, Johns Hopkins lography from Birkbeck College (UK). For mechanisms of cell signalling and adhesion, epidermal growth factor receptor (EGFR/ University School of Medicine his postdoctoral training he joined the downstream of growth factor receptor and ErbB) and Hedgehog signaling pathways. laboratory of M.J. Eck at the Dana-Faber integrin signalling. A particular expertise of my laboratory is Other Experience and Professional expression of cysteine-rich glycoproteins Memberships in eukaryotic cells in sufficient amounts 2000-2007 Ad hoc reviewer on several and suitable forms for structural and bio- NIH P01, R01, and F32 panels physical analysis. 2007 Editorial Advisory Board, Protein Science Professor of Biophysics & Biophysical 2009-2012 Member, NIH MSFC study sec- Chemistry and Professor of Oncology tion (chair 2010-2012)

Positions and Employment Honors 1982-1984 Medical Student, Stanford 1989-1992 Helen Hay Whitney Fellow University Columbia University 1984-1988 Graduate Student, Department 1994-1997 Searle Scholar Johns Hopkins of Chemistry, Stanford University University 1988 Postdoctoral Fellow, Dept. Physik,Technische Universität München, 1988-1993 Postdoctoral Fellow, Dept. of

68 Speakers’ biographies Allosteric Regulation of Cell Signalling 69 Gerhard Hummer Ruth Nussinov National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, USA National Cancer Institute and Tel Aviv University, Israel

Gerhard Hummer uses theory and simula- to the National Institutes of Health where Dr. Nussinov's interests largely focus on sequence analysis (already in 1980); the tions to study biomolecular systems. After he is currently Senior Investigator, Chief protein folding and dynamics, protein- proposition of conformational selection receiving his Ph.D. in physics (1992; Uni- of the Theoretical Biophysics Section, and protein interactions, binding mechanisms and population shift as an alternative bind- versity of Vienna, Austria and Max-Planck- Deputy Chief of the Laboratory of Chemical and regulation, amyloid conformations and ing mechanism to induced fit and the role Institute for Biophysical Chemistry, Ger- Physics, NIDDK. He is Fellow of the Ameri- toxicity, and large multi-molecular asso- of conformational ensembles in protein many), he joined the Los Alamos National can Physical Society (2005) and received ciations with the ultimate goal of under- function (1999), including protein allostery Laboratory, first as a postdoctoral fellow the Raymond and Beverly Sackler Interna- standing the protein structure-function (2004). More recently, she proposed new (1993-1996) and then as an independent tional Prize in Biophysics (2010). relationship. Among her accomplishments mechanistic concepts related to allostery researcher (1996-1999). In 1999, he moved are the dynamic programming algorithm and the roles of allosteric mechanisms and for the prediction of the secondary struc- population shift in cellular functions. ture of RNA (1978); pioneering work in DNA

70 Speakers’ biographies Allosteric Regulation of Cell Signalling 71 Nikolay V. Dokholyan José Nelson Onuchic University of North Carolina at Chapel Hill, USA Rice University, Houston, USA Dr. Onuchic did his undergraduate work at the University of São Paulo, Brazil, and Dr. Dokholyan received his PhD in Physics 150 per review articles, 15 book chapters, a JOSÉ ONUCHIC is a Professor of Physics and received his PhD from Caltech at 1987 at Boston University (advisor Prof. Gene book, entitled “Computational Modeling Astronomy, Chemistry and Biochemistry under the supervision of John J. Hopfield. Stanley). He completed postdoctoral train- of Biological Systems: From Molecules to and Cell Biology at Rice University and His thesis work was on new aspects of the ing in Biophysics at Harvard University Pathways”. He studies the physical nature is the co-Director of the NSF-sponsored theory of electron transfer reactions in (advisor Prof. Eugene Shakhnovich). Dr. of interactions between atoms, molecules, Center for Theoretical Biological Phys- biology. He then spent six month at the Dokholyan joined University of North cells, and organisms. The underlying ques- ics. Within the CTBP, Dr. Onuchic and his Institute for Theoretical Physics in Santa Carolina at Chapel Hill Department of tion throughout his research is how these research group have led the biological phys- Barbara and after that went back to Brazil at Biochemistry and Biophysics in 2002 and interactions shape the complex organiza- ics community as it attempts to devise an the University of São Paulo as an Assistant was further promoted to Associate (2008) tion, behavior, and evolution of biomol- integrated picture of a variety of model Professor for two and half years. During this and Full Professor (2011). Dr. Dokholyan ecules and organisms. To approach this biochemical and biological systems. His period he continued his work on electron is a member of numerous centers and pro- question Dr. Dokholyan's group has been research has expanded across the scales transfer theory as well as on the theory of grams and a member of Faculty 1000. He studying structure, dynamics, function, and of molecular-level interactions to cellular chemical reactions in condensed matter serves as an editor in chief for Research evolution of biological molecules. Such a systems to organized multi-cellular struc- and molecular electronics. He came to the and Reports in Biochemistry and a book broad approach is necessary to tie together tures. At Rice he will move this view towards University of California at San Diego in series editor “Series in Computational Bio- the diverse pieces of knowledge of molecu- medical applications focusing on cancer. 1990. In 1989 he was awarded the Inter- physics”. Dr. Dokholyan has published over lar properties and evolution that is to us. In protein folding, he has introduced the national Centre for Theoretical Physics concept of protein folding funnels as a Prize in honor of Werner Heisenberg in mechanism for the folding of proteins. Trieste, Italy, in 1992 he received the Beck- Convergent kinetic pathways, or folding man Young Investigator Award, and he is funnels, guide folding to a unique, stable, a fellow of the American Physical Society. native conformation. Energy landscape In 2006 he was elected a member of the theory and the funnel concept provide the National Academy of Sciences, USA, and in theoretical framework needed to pose and 2009 he was elected a fellow of the Ameri- to address the questions of protein folding can Academy of Arts and Sciences and of the and function mechanisms. He also works Brazilian Academy of Sciences. In 2011 he on the theory of chemical reactions in was awarded the Einstein Professorship by condensed matter with emphasis on bio- the Chinese Academy of Sciences (CAS) and logical electron transfer reactions. He is recently he has been elected Fellow of the now broadening his interests to stochastic Biophysical Society. In 2011, he has joined effects in genetic networks. the faculty of Rice University.

72 Speakers’ biographies Allosteric Regulation of Cell Signalling 73 Dorothee Kern Francesco L. Gervasio Howard Hughes Medical Institute, Brandeis University, Waltham, USA CNIO, Madrid, Spain

Dorothee Kern is Professor and Chair of Dr. Kern is the recipient of the Pfizer Award Francesco L. Gervasio was trained in Gervasio joined the CNIO in February Biochemistry at Brandeis University and in Enzyme Chemistry from the American physical chemistry and spectroscopy at the 2009 to lead the Computational Biophys- an Investigator of the Howard Hughes Chemical Society, the Dayhoff Award from Molecular Spectroscopy Laboratory and ics Group. Since 2011 he is member of the Medical Institute. She received her PhD the Biophysical Society and the National the European Laboratory for Non-linear Scientific Committe ofCNIO . He is an expert at the Martin Luther University in Halle, Lecturer of the Biophysical Society. Before Spectroscopy, the Universitá di Firenze. in molecular modelling and simulations Germany followed by postdoctoral stud- her professional scientific carrier, she was He received a PhD in Chemistry in 2002 and has developed effective algorithms to ies at UC Berkeley. She joined the faculty captain of the German National Basket- from the Universitá di Firenze and the study large-scale protein dynamics and to at Brandeis in 1999. ball team for many years and won the MVP International School for Advanced Stud- predict the structure-activity relationship award. ies in Trieste. The same year he joined the of drug-like molecules. He has authored Swiss National Supercomputer Centre as 57 scientific papers in international peer- a Junior Scientist. reviewed journals, which have been cited more than 1500 times so far. His h-index In 2004 he joined the group of M. Parrinello is 25. He is part of the Innovative Medi- as a Post-doctoral Fellow at the Eidgenös- cines Initiative Open PHACTS consortium sische Technische Hochschule (ETH) in (funded by EU under the 7th Framework Zurich (Switzerland). In 2006 he was Programme). promoted as Assistant Professor in com- putational chemistry at the ETH. He was appointed as Professor (2006-2009) at the Scuola Normale di Pisa (Italy).

74 Speakers’ biographies Allosteric Regulation of Cell Signalling 75 Alfonso Valencia Giulio Superti-Furga CNIO, Madrid, Spain CeMM, Research Center for Molecular Medicine of the Austrian Academy of Sciences,Vienna, Austria

Prof. Valencia is the Director of the Struc- Prof. Valencia is Fellow of the International Scientific Director ofC eMM Research Center Academy of Sciences, German Academy tural Biology and Biocomputing Pro- Society for Computational Biology, elected of Molecular Medicine of the Austrian Acad- of Sciences, EMBO, European Academy of gramme of the Spanish National Cancer member of the EMBO, founder “e-biolab” emy of Sciences. Italian. Performed stud- Cancer Sciences. Chair of the EMBL Alumni Research Centre and of Spanish Bioinfor- science and art initiative and Professor ies in molecular biology at the University Association. 2009 Advanced Investigator matics Institute. He is a biologist by train- Honoris Causa of the Danish Technical Uni- Zurich, Genentech, and IMP/Vienna. Post- ERC Grant and Knight Officer Order of Merit ing interested in the analysis of genomic versity. As Executive Editor of Bioinformat- doctoral fellow and Team Leader at EMBL. of the Republic of Italy. 2011, Prize of the information with particular emphasis in ics (OUP) he has promoted the integration of Co-founded biotech company Cellzome, City of Vienna for Natural Sciences, and the evolution of protein families and pro- computational and molecular biology. (Scientific Director). Member of: Austrian “Austria's scientist of the Year”. tein interaction networks.

76 Speakers’ biographies Allosteric Regulation of Cell Signalling 77 Guillermo Montoya Michael J. Eck CNIO, Madrid, Spain Dana-Farber Cancer Institute, Harvard Medical School, USA

Guillermo Montoya was born in Madrid in Montoya has been Head of the CNIO's Michael J. Eck MD, PhD is Professor of Bio- Dr. Eck received his Bachelors degree in 1967 and obtained his Bachelor degree in Macromolecular Crystallography Group logical Chemistry and Molecular Pharma- Electrical Engineering from Rice Univer- Biochemistry from the Universidad del País since 2002 and was acting Director of the cology at the Dana-Farber Cancer Institute sity in 1985, and his M.D. and Ph.D. from the Vasco in 1990, and his PhD in Chemistry Structural Biology and Biocomputing Pro- and Harvard Medical School. He directs University of Texas Southwestern Medical from the Universidad de Zaragoza in 1993. gramme from November 2003 to January the Laboratory of X-ray Crystallography at School in 1991. He completed postdoctoral Montoya worked at the European Molecu- 2006. His work has addressed the structure the DFCI. Dr. Eck's research centers on the training at Children's Hospital, Boston and lar Biology Laboratory (EMBL) in Heidel- of different protein complexes involved in structural biology of signaling and cancer. Harvard Medical School in 1996. He joined berg (Germany), working in I. Sinning's cell cycle and the redesign of protein-DNA His research group works to unravel the the faculty of Harvard Medical School Group, where he pioneered the study of interactions for genome modification. In mechanisms by which mutations in kinase and the Dana-Farber Cancer Institute in the structure of the signal recognition par- 2009 he was awarded the National Prizes oncogenes alter their activity and drug sen- 1996. ticle founded by an EMBO and Marie Curie of the Fundación Mutua Madrileña and sitivity and to develop novel therapeutics Fellowships. In 1998 he was awarded a Peter the Fundación Caja Rural de Granada- that specifically target these cancer-causing und Traudl Engelhorn Foundation Research Ministerio de Sanidad. proteins. Major interests include EGFR Fellowship establishing his research team. mutations in lung cancer and JAK kinase mutations in leukemia.

78 Speakers’ biographies Allosteric Regulation of Cell Signalling 79 Markus G. Grütter Françoise Bono University of Zürich, Switzerland SanofiR &D, Toulouse, France

Markus Grütter has a PhD degree in Bio- in Structural Biology and since 2009 he is Dr. Françoise Bono is currently Vice Presi- Dr. Bono has served as the project team physics (University of Basel, Switzerland) coordinating the EUprogram P-CUBE (Infra- dent of Early to Candidate Unit in sanofi leader for 5 drug discovery programs and and training in Protein Crystallography. He structure for Protein Production Platforms). Research and Development in charge of a 2 of them are already in clinical evalua- was head of a structural biology research His research group focuses on the DARPin research team of 90 scientists. Dr Bono first tion. And actively contributes to the dis- unit at Novartis. He is full professor at the technology and the structure-function joined Sanofi Research in 1989 as a Senior covery of more than 10 others. She has Department of Biochemistry of the Uni- relationship of proteins involved in apop- Scientist (Exploratory biochemistry) She published more than 50 research articles versity of Zürich and currently Director of tosis and inflammation and of membrane held group leader position in Cardiovascu- in various peer-reviewed scientific journals the Department. Since 2001 he is directing proteins. lar Thrombosis Department, (cell biology including Nature, Cell, Nature Medicine, the Swiss National Research Center (NCCR) & biochemistry) in 1999 and become Vice Cancer research, Blood, and Circulation President of the Thrombosis and Angiogen- Research, and has been granted more than esis Department in Sanofi Aventis Research 30 patents. and Development between 2005 and 2008.

80 Speakers’ biographies Allosteric Regulation of Cell Signalling 81 Jeffrey R. Peterson Doriano Fabbro Fox Chase Cancer Center, Philadelphia, USA Novartis Pharma AG, Basel, Switzerland

Dr. Peterson is a biochemist and chemi- developed novel allosteric inhibitors (tar- Doriano Fabbro received his PhD (cell biol- Dr. Fabbro, a member of various profes- cal biologist who trained at the Harvard gets include: N-WASP, Pak1 kinase, mDia1) ogy & biochemistry, Biocenter Basel) study- sional societies and several journal edito- Institute of Chemistry and Cell Biology as well as conventional active site-directed ing the mechanisms of activation of PKC. In rial boards, has contributed, among many under the direction of Drs. Marc Kirschner, inhibitors (targets: Pak isoforms, Ack, IGF1R 1991, he joined the Oncology Research of other things, to the discovery and develop- Timothy Mitchison, and Stuart Schreiber. and others) for proteins relevant to can- Ciba where he contributed to the discovery ment of kinase inhibitors for the treatment His work focuses on basic mechanisms of cer. He is currently a tenured Associate of Midostaurin and Glivec. Following the of cancer including RAD001 (Everolimus, signal transduction and their inhibition by Professor at Fox Chase Cancer Center in merger of Ciba with Sandoz to Novartis in Afinitor, launched),STI 571 (Imatinib, Glivec, small molecules with a particular focus on Philadelphia. 1996, he was promoted Executive Direc- launched), AMN107 (Nilotinib, Tasigna, protein kinase targets. His laboratory has tor in Oncology where he was responsible launched) and PKC412 (Midostaurin, Ph for the drug discovery efforts focusing on III). He has holds many patents and has ATP-dependent enzymes (Kinases and written numerous publications in the area ATPases) and other approaches. In 2006, of kinase regulation, structure, screening he was instrumental to the establishment and drug discovery. of NIBR Expertise Platform Kinases where he serves as Head of the Kinase Biology until today.

82 Speakers’ biographies Allosteric Regulation of Cell Signalling 83 Ermanno GherardI MRC Centre, Cambridge and University of Pavia, Italy

Ermanno Gherardi has a degree in medicine subsequently as a group leader on growth and a Ph.D. in molecular biology from the factors. He has recently taken up a posi- University of Cambridge, UK. He worked tion at the University of Pavia, Italy. His at the Medical Research Council (MRC) research interests are structure-function Laboratory of Molecular Biology in Cam- relationships and protein engineering of bridge, UK, between 1987 and 2011, first antibodies and growth factors. on antibodies with César Milstein and

84 Speakers’ biographies Allosteric Regulation of Cell SignallinG Poster session Structural basis for 1 the Regulation of Protein Kinase B

Deborah Balzano and Daniel Lietha CNIO, Madrid, Spain

Protein Kinase B (PKB) is a member of the kinase domain depends on the activation serine/threonine AGC protein kinase family. state of PKB. The inactive form is called PKB is a key signalling molecule downstream the closed conformation, while the mem- of growth factor induced PI3K signalling brane-associated form is called the open regulating important cellular processes conformation. Once phosphorylated, PKB is such as metabolism, growth, proliferation released from the membrane to the cytosol. and survival. Three highly homologous The aim of our work is to investigate on a isoforms of PKB exist in mammals (α, β structural level i) the modeof PKB autoin- and γ), each containing a (N)-terminal PH hibition in its basal state, ii) how the bind- domain, a kinase domain and a 21-amino ing to PIP3 and phosphorylation by PDK1 acid (C)-terminal hydrophobic motif (HF). regulates the catalytic activity of PKB, and Aberrant regulation of PKB pathway is iii) understand what triggers the release of implicated in the pathogenesis of differ- the active enzyme from the membrane. We ent diseases, including several human can- will complement the structural studies with cers and type-2 diabetes. PKB is therefore extensive biochemistry, like kinase activ- considered a good drug target and several ity assays as well as protein-protein and small molecule inhibitors have already been protein-lipid interaction studies. Further, described. A multistep process involving new insights on PKB structure and regula- PIP3 and upstream kinases, such as PDK1, tion could allow the interpretation of the activates PKB. When active, PKB is located to effects of different classes ofPKB inhibitors the inner surface of the plasma membrane. and enable the design of inhibitors with Previous work has revealed that a change in improved selectivity. the position of the PH domain relative to the

Allosteric Regulation of Cell Signalling 89 P38α MAP kinase dynamics LIGAND-DETECTED 19F NMR-BASED 2 3 in free and ligand-bound forms FRAGMENT SCREENING FOR ALLOSTERIC LIGAND DISCOVERY AGAINST SELECTED PROTEIN TARGETS Anna Berteotti1, Vittorio Limongelli2, and Michele Parrinello3,4 1 Istituto Italiano di Tecnologia, Genova, Italy; 2 Università di Napoli “Federico II”, Naples, Italy; 3 Computational Science (ICS), Università della Svizzera Ital- iana, Lugano, Switzerland; 4 Computational Sciences, Department of Chemistry and Applied Biosciences, ETH Zurich, Switzerland, Faculty of Informatics, Universita’ della Svizzera Italiana, Lugano, Switzerland Blanca López-Méndez and Ramón Campos-Olivas CNIO, Madrid, Spain

Kinases play pivotal roles in the cell cycle metadynamics simulations[1] allowed us Fragment-based drug discovery is an alter- Using 1D 19F NMR spectroscopy we were regulation and in several pathological to enhance the sampling of the DFG flip native approach to target-focused drug able to monitor the possible binding of conditions. Thus in the very recent years, for p38α MAP kinase in the apo form and discovery based on high throughput cell the compounds present in each cocktail to a selective kinase inhibition has become in two ligand-bound forms[2]. The data or biochemical assays, which has gained different protein targets by monitoring the an important goal for both the academia coming from X-ray and NMR[2,3] experi- widespread interest and application. It is increase of 19F NMR signal linewidth upon and pharmaceutical industry. In many ments are assessed and complemented based on the idea of building drugs piece protein addition. Typically 20 μM concen- kinases, a typical conformational change, shedding light on the dynamical charac- by piece, detecting first the weak binding of tration in each CF3-containing fragment, called DFG flip characterizes the move- ter of this movement. From our results low molecular weight (typically MW <150- or 50 μM for those with CF groups, are suf- ment of three well conserved amino acids the dynamical aspects of the DFG flip at 300 Da) and low complexity compounds ficient to obtain high quality spectra, and as at the beginning of the activation loop. atomistic level are disclosed and the data (fragments), that could subsequently be low as 1:50 of protein equivalents produce During this movement the Aspartate obtained here will be of precious help for chemically developed into compounds a detectable effect on fragment binders, moves away from the ATP binding site, future experiments and structure-based with lead-like properties. The approach therefore requiring very small amounts and its position is taken by the neighbor- drug design. is particularly promising to target protein- of protein. With a sample changer and a ing Phenylalanine. This transition allows protein interactions, and to unveil pro- dual H-F probe in our 700 MHz instrument, the creation of an additional binding site 1 A. Barducci et al., Phys. Rev. Lett. tein allosteric sites that may or may not screening of the complete fluorinated frag- Jan 18,100, 2, 020603 (2008). that can be targeted by some selective 2 C. Pargellis et al., Nat. Struct. be physiologically exploited. In our Unit ment library can be performed in less than 2 inhibitors. Here the use of well-tempered Biol. 9, 4, 268-72 (2002). we have developed tools to conduct NMR- days. Results obtained in the initial screen- 3 M. Vogtherr et al., Angew. Chem. Int. based screening to identify and characterize ing and the follow up of hits discovered Ed. Engl. 30,45(6),993-7 (2006). the binding of small molecules to protein against selected macromolecular targets, targets, potentially providing hits for drug in particular in the targeting of an auto- discovery research and chemical tools for inhibitory domain of a protein tyrosine biophysical and functional applications. In kinase, will be presented in this poster. particular, we put together a collection of ~370 fluorinated small molecule compounds with good solubility in aqueous buffer and assembled them into mixtures of eight each.

90 POSTER SESSION Allosteric Regulation of Cell Signalling 91 Structural Biology of TIM proteins: Towards unravelling the molecular 4 5 A family of cell surface phosphatidylserine mechanism and regulation of the pro/anti- receptors that regulate immunity oncogene proteins SHIP1 and 2

José M. Casasnovas, Angela Ballesteros, César Santiago, Meriem Echbarthi, Gerardo G. Kaplan, Gordon J. Freeman Centro Nacional de Biotecnologia, CSIC, Madrid, Spain; Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Johanne Le Coq, Luis Heredia and Daniel Lietha Cell Signalling and Adhesion Group, USA. Dana-Farber Cancer Institute, Harvard Medical School, Boston, USA Structural Biology and Biocomputing Programme, CNIO, Madrid, Spain

The T-cell immunoglobulin and mucin to bind to phosphatidylserine (PtdSer) SHIP (SH2-containing Inositol 5'-Phos- and SHIP1/2 are responsible for its degrada- domain (TIM) proteins are involved in and to mediate phagocytosis of apoptotic phatase) proteins 1 and 2 (SHIP1/2) are two tion. SHIP1/2 have been heavily implicated the regulation of immune responses. The cells. A model for TIM protein binding to important signalling enzymes homologs with several serious diseases such as can- TIM are type I membrane proteins with an PtdSer in cellular membranes was built from the inositol polyphosphate 5-phos- cer and type 2 diabetes. The paucity in the N-terminal immunoglobulin (Ig) domain based on structural and functional data. phatase family, involved in several growth predictability of their behaviour reflects followed by a heavily glycosylated mucin In this model the tip of the N-terminal Ig factor receptor-signalling pathways. our incomplete understanding of their domain in the extracellular region, a single domain of the protein penetrated into the They catalyse the dephosphorylation of exact role and the molecular mechanism transmembrane region and a cytoplasmic membrane, whereas the hydrophilic moi- the phospholipid phosphatidylinositol by which they are regulated. Therefore it is

tail with tyrosine phosphorylation motifs. ety of PtdSer entered into the MILIBS. The 3,4,5-triphosphate (PI(3,4,5)P3) to PI(3,4) key for cancer drug development and poten-

The N-terminal Ig domain is engaged in MILIBS modulate the biology of the TIM P2. PI(3,4,5)P3 is a key lipid second messen- tially for other diseases associated directly ligand binding with some contribution of proteins, such as the trafficking of TIM-1 ger which can recruit signalling proteins to or indirectly with these two enzymes that the mucin domain. The structures of the to the cell surface, TIM-TIM interactions the plasma membrane and subsequently we unravel their respective regulatory Ig domains determined by our group pro- and the conformation of the proteins on initiate numerous downstream signalling molecular mechanisms. To that effect we vided relevant insights on ligand recogni- the cell surface. Therefore, the MILIBS is a pathways responsible for the regulation of a are studying both enzymes and the roles tion by the TIM proteins. A loop disulphide critical determinant of the ligand binding plethora of cellular events such as prolifera- of their different domains at the structural linked to the CFG β-sheet of the Ig domain specificity and functional properties of the tion, growth, apoptosis, actin cytoskeletal and biochemical level. We are employing is a distinctive structural feature of the TIM TIM family. Overall, we conclude that the rearrangement, to name a few. Because kinetic and binding studies to elucidate the proteins. Moreover, the structures identi- unique structure of TIM IgV domains sug- of its central role, three enzymes tightly regulation mechanisms of both enzymes

fied a unique ligand-binding pocket with gests that the TIM molecules evolved as a regulate the levels of PI(3,4,5P)P3: PI3K is and have recently solved the structure of a metal ion to which ligands coordinate family of pattern recognition receptors for responsible for its generation while PTEN, SHIP2 phosphatase domain in conjunction (Metal Ion-dependent Ligand Binding Site, PtdSer that determine whether apoptotic a well-characterized tumour suppressor, with its C2 domain (2.2 Å). MILIBS). The TIM proteins use the MILIBS cell recognition leads to immune activa- tion or tolerance, depending on the TIM molecule engaged and the cell type it is expressed on.

92 POSTER SESSION Allosteric Regulation of Cell Signalling 93 The domain interplay in protein kinases: Structural insight into Mu transposition: MuB is a 6 7 a study on the allosteric effect of the SH2 AAA+ ATPase that forms helical filaments on DNA

domain on kinase activation Marija Dramićanin1, Naoko Mizuno2, Michiyo Mizuuchi3, Yong-Woon Han3, Wei Yang3, Alasdair Steven1, Kiyoshi Mizuuchi3, Santiago Ramón-Maiques1 1Structural Biology and Biocomputing Programme, Spanish National Cancer Research Centre (CNIO), Madrid, Spain; 2Laboratory of Structural Biology, National Institute of Nicole Dölker1, Gorna M. 2, Gervasio F.L.1, Superti-Furga G.2 Arthritis, Musculoskeletal and SkinDiseases, National Institutes of Health, Bethesda, USA; 3Laboratory of Molec- 1Centro Nacional de Investigaciones Oncologicas (CNIO), Computational Biophysics Groups, Madrid; ular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 2CeMM-Research Center for Molecular Medicine, Vienna Bethesda, USA; Current address: Max Planck Institute of Biochemistry, Martinsried, Germany

Protein kinases constitute one of the site of the catalytic domain, it would be DNA transposons are ubiquitous in the non-specific DNA binding protein with largest protein families and are involved highly desirable to develop inhibitors that genomes of all forms of life and play an slow ATPase activity. MuB is needed to (1) in most cellular pathways. Correspond- bind to allosteric sites. A more detailed important evolutionary role in the genera- favor the assembly of the MuA-Mu ends ingly, kinase malfunction is related to an understanding of allosteric effects of other tion of gene diversity and in the shaping of complex, (2) to allosterically stimulate the important number of human diseases. protein domains on the activation of Abl is, genomic landscapes. Although typically activity of MuA, (3) to select and deliver the Protein kinases share a common canonical therefore, of paramount importance for transposons exhibit no strong selectivity target DNA and (4) to confer target immu- catalytic domain, consisting of a number the rational design of new drugs. for the target DNA site, some transposons nity. Mu. Further detailed understanding of highly conserved motifs, which have to select DNA non-randomly, avoiding self- of the mechanism of action of MuB has undergo distinct conformational changes We focus on the protein-protein interac- destruction; a phenomenon named “tar- been hampered by the bind DNA in its cen- to perform their catalytic activity. Most tions of the catalytic domain with the SH2 get immunity”, because the presence of a tral channel without seemingly altering kinases also require other domains or domain, an allosteric effector involved copy of the transposon makes nearby DNA DNA B-DNA conformation. This results in proteins for full activation. both in auto-inhibition and activation. “immune” for additional transposon inser- a unique mismatched symmetry between We have used elastic network models, nor- tions. Phage Mu is one of the most complex protein and DNA helixes not observed pre- The Abelson tyrosine kinase (Abl) is of mal mode analysis and molecular dynam- and efficient transposable elements. Two viously in other nucleoprotein filaments, special interest because of its importance ics simulations to gain insight into the phage-encoded proteins, MuA and MuB, which forces MuB monomers to interact as an anti-cancer drug target. The fusion interplay between the SH2 domain and are essential for efficient transposition. with DNA differently along the filament. protein Bcr-Abl leads to over-activation the relevant motifs at the catalytic site. We MuA is the transposase responsible for Overall, our findings dissect the structure of Abl, causing chronic myeloid leukemia propose a mechanism, by which the SH2 synapses of the two Mu ends and all the and functions of MuB to reveal the intricate (CML). Currently, Abl inhibitors are the domain modifies important functional DNA cutting and joining steps. However, control that an AAA+ ATPase can impose on only pharmaceutical treatment for CML, modes of the catalytic domain. MuA is highly inefficient in the absence a complex reaction system. but a significant proportion of patients MuB, which is a small ATP-dependent develop resistance against currently This proposal was verified experimentally known inhibitors. As most resistance by introducing specific mutants the rel- mutants are located around the active evant motifs identified during the com- putational study.

94 POSTER SESSION Allosteric Regulation of Cell Signalling 95 Modulation of αvβ3 integrin allostery by IFIT proteins as new effectors in antiviral 8 9 binding of isoDGR containing cyclopeptides defence: investigating RNA binding and inter- molecular interactions in the IFIT complex

Maria W. Górna, Andreas Pichlmair*, Tamara Theil, Elena Rudashevskaya, Keiryn L. Ben- Michela Ghitti1, Andrea Spitaleri1, Barbara Valentinis2, Catia Traversari2, Gian-Paolo Rizzardi2 nett, Jacques Colinge and Giulio Superti-Furga CeMM- Research Center for Molecular and Giovanna Musco1 1Fondazione Telethon, c/o Ospedale San Raffaele;2 Molmed Spa, Milan, Italy Medicine, Vienna, Austria; *Max Planck Institute of Biochemistry, Martinsried, Germany

Nowadays most of pharmaceutical efforts unwanted signals. Neglecting this aspect Virus-derived RNA bearing a 5' triphos- repeats (IFIT) as top candidates. We have in drug design are focused on the discovery, could lead to the development of drugs that phate group (ppp-RNA) and present in the shown that two of the IFIT family members development and optimization of new induce agonist-like activities and adverse cytoplasm is detected by the cell as non-self (IFIT1 and IFIT5) can specifically recog- ligands that efficiently bind to receptors paradoxical effects. In this context, 1 we RNA, which results in strong induction of an nize ppp-RNA, and that IFIT proteins play a involved in human disease. Even though exploited a combination of computational antiviral response. So far, the best described role in antiviral defence. IFITs also interact traditional computational techniques are and biochemical studies to determine the ppp-RNA sensor is RIG-I, which initiates with each other to form a macromolecular able to find small molecules that maximize biologically active conformation of a small a signaling cascade that leads to produc- complex. Here, we investigate in further binding free energy, they are not able to library of cyclopeptides, to discriminate tion of the type I interferon. However, we detail which RNA features are required for predict how small differences in ligand in silico binders from non-binders, to hypothesized that there should also exist efficient binding by IFITs, and dissect the binding could reflect into structural and understand at molecular level integrin interferoninduced effector proteins that act proteinprotein interactions between the functional response of the receptor. For allosteric changes induced by ligand bind- on ppp-RNA. We previously performed an IFIT family members. Our data suggest that this reason, receptors that undergo a sub- ing and to identify real antagonist among affinity-purification/mass-spectrometry IFITs form a large multivalent molecular stantial conformational transition upon the selected binders. Our study reveals the screen for proteins preferentially binding platform for RNA that determines the fate ligand binding should be precluded from importance of ligand-receptor complex to ppp-RNA and identified the interferon- of certain viral RNAs. structure based drug design. dynamics in drug design studies aiming at induced proteins with tetratricopeptide developing new real integrin antagonists, Here we present a case study on Integrin confirming that ligand induced confor- avb3. One major problem with integrin mational change of the receptor and its ligands is their potential to activate con- structure-function relationship should formational changes, which can initiate play a crucial step in common drug design strategies.

96 POSTER SESSION Allosteric Regulation of Cell Signalling 97 Focal Adhesion Kinase Distinguishing between induced fit 10 11 as a cellular mechano-sensor and conformational selection with the Torsional Network Model

Jing Zhou and Frauke Gräter Heidelberg Institute for Theoretical Studies (HITS) gGmbH, Germany Javier Klett and Ugo Bastolla CSIC-Centro de Biología Molecular Severo Ochoa, Madrid, Spain

Focal adhesion kinase (FAK) is a crucial site induces the maximum activity of FAK. Since the first studies on protein dynamics, 1 Bucher D, Grant BJ, McCammon component of focal adhesion sites, which We tested if mechanical forces as they are it has been of interest how this event occurs JA.Biochemistry. 2011 Dec exerts its activity by transducing signals present at focal adhesion sites can induce upon ligand binding. Nowadays, apart form 6;50(48):10530-9. Epub 2011 Nov 10. between the cytosol and the extracellular an allosteric switch to an active state of the classic “lock and key” paradigm, two 2 Changeux JP, Edelstein S. F1000 Biol matrix. The translocation of FAK to focal FAK with an exposed phosphorylation site, new paradigms are under debate: confor- Rep. 2011;3:19. Epub 2011 Sep 1. adhesion sites and its functional activa- using Molecular Dynamics simulations. mational selection and induced fit models. 3 Mendez R, Bastolla U.Phys Rev Lett. 2010 tion by tyrosine phosphorylation leads to [1][2] Jun 4;104(22):228103. Epub 2010 Jun 3. the formation of large enormous multi- We indeed find mechanical forces propa- molecular complexes. Theses complexes gated onto FAK when tethered between In our work we aim to propose a way to can trigger different signaling pathways, the membrane and the cytoskeleton can distinguish whether protein motion is including the MAPK pathway. remove the auto-inhibitory FERM domain accounted by any of these two paradigms from the kinase domain of FAK. This func- using our in-house elastic network model The three-dimensional structure of FAK tional activation of FAK can then trigger a TNM.[3] consists of a tyrosine kinase domain and sequence of downstream signaling events. two large non-catalytic regions. The The mechanotransduction mechanism N-terminal FERM domain is involved in of FAK can explain how FAK acts as a force auto-inhibition of the kinase by blocking sensor, translating mechanical forces at a phosphorylation site (Tyr576/577)[1]. the focal adhesion site into a biochemical The exposure of this phosphorylation signal.

98 POSTER SESSION Allosteric Regulation of Cell Signalling 99 Dynamic fingerprint Molecular recognition mechanisms 12 13 of Imatinib sensitive kinases of microtubule plus end tracking proteins

Rubén M Buey1*, Renu Mohan2, Indrani Sen3, Kris Leslie4, Oliver Kortt5, John H Missimer3, Ines Kretzschmar5, Rudolf Volkmer5, Anna Akhmanova2 and Michel O steinmetz3 1Universidad de Salamanca, Salamanca, Spain; 2Utrecht University, Utrecht, NL; 3Paul Scherrer Institute, Villigen, Silvia Lovera, Giorgio Saladino, Nicole Doelker and Francesco L. Gervasio CNIO, Madrid, Spain CH; 4Erasmus Medical Center, Roterdam, NL; 5Institute for Medical Inmunology, Berlin, DE

Protein kinases constitute one of the larg- the case of Lck, quite effectively inhib- Microtubule (MT) plus-end tracking pro- ends. Using a combination of structural est and most functionally diverse protein ited by Imatinib (IC50=9 uM) 3, despite teins (+TIPs) comprise a structurally and and biochemical techniques, we propose families, involved in most cellular path- the high identity with Src kinase (70%). functionally diverse family of proteins that a structural model for EB proteins in solu- ways. They are of crucial importance for Recently, in our work concerning the two preferentially accumulate at MT growing tion. Furthermore, our biochemical data signal transduction, and their deregula- kinases c-Abl and c-Src, we have shown ends. +TIPs form dynamic networks where suggest an important role for long-range tion is related to numerous human dis- that the difference in the Imatinib activity most of the protein-protein interactions are electrostatic interactions between the CC eases, including cancer. Among kinases, can be attributed to a larger flexibility of mediated by End-Binding proteins (EBs). domain of EBs and the MT lattice, which c-Abl and c-Src are of particular interest c-Abl4. Now in this follow up work we have EBs were recently recognized to be master allows the EBs to discriminate between for cancer research. The BCR-Abl fusion extended the analysis to the other cited regulators of +TIP networks because: they the MT-lattice and MT-tips. protein, is the initial cause of chronic kinases, analyzing their flexibility profiles. are able to autonomously track MT tips and myeloid leukemia disease (CML) 1. CML is Performing classical molecular dynamic to recruit a large variety of other +TIPs to We also present insights into the molecular treated with the powerful anti-cancer drug simulations, followed by RMSF analysis, we this location. EBs are modular proteins com- recognition mechanism of EBs by +TIPs Imatinib, which acts as ATP-competitor. have identified regions with a higher flex- prised of two functional domains connected containing a ‘MT-tip localization signal’ Imatinib, however, is not so selective and ibility common to all the kinases sensitive by a long linker: a calponin-homology (CH) (MtLS), which comprises the short linear targets numerous kinases2, beside c-Abl to Imatinib (c-Abl, c-KIT and also Lck) and domain, responsible for MT binding, and sequence motif SxIP. By means of an exhaus- and c-Src, like c-KIT, PDGFR, Syk, Lck and a more rigid profile in the same regions for a coiled-coil (CC) domain, responsible for tive functional analysis, we have derived the many others, showing towards some of those not responding to the drug (c-Src partner binding. sequence determinants of a canonical MtLS them a lack of activity. For instance it and Syk). From these preliminary results and correlated the EB-binding and MT tip effectively inhibitsBCR -Abl (IC50=0.2 we could recognize flexibility as a dynamic Here, we present structural and func- tracking activities of differentS xIP-contain- uM) but not Src 3, no matter their high fingerprint of all the studied kinases sensi- tional data describing how the concerted ing +TIPs. Moreover, we have investigated sequence homology (47%), neither that tive to Imatinib. action of the CH, linker, and CC domains of how phosphorylation regulates the EB-SxIP the bound structure of Src with Imatinib EBs accounts for the autonomous MT tip interaction and, consequently, MT-tip local- is very similar to that of Abl with Imatinib. 1 Rowley, J.D. Nature 1973, 243, 290–293. tracking, regulation of MT dynamics, and ization. Our data establish a favorable basis 2 Lee SJ, Wang JYJ. Journal of Biology. 2009. In the same way, the drug inhibits c-KIT 3 Deininger, M.; Buchdunger, E.; Druker, recruitment of numerous partners to MT for computational approaches to search for (IC50=0.41 uM) but not Syk 3, although B. J. Blood 2005, 105, 2640–2653. novel +TIPs in entire genomes. they share the same sequence identity 4 Lovera S, Sutto L, Boubeva R, et al. Journal of the American Chemical Society. 2011:8-11. of 40% with both Abl and Src. Striking is

100 POSTER SESSION Allosteric Regulation of Cell Signalling 101 A New Phosphorylation/Acetylation A novel potential of mean force 14 15 Switch in the Regulation of Cortactin to study protein flexibility and its Role in Cell Spreading

Eugenia Meiler, Elvira Nieto-Pelegrín and Narcisa Martinez-Quiles Universidad Complutense de Madrid, Spain Raúl Méndez and Ugo Bastolla Centro de Biología Molecular, Madrid, Spain

Cortactin is a multidomain protein that new regulatory mechanism: a competition Understanding protein flexibility is of key between any two atoms. But this state is plays a central role in the remodeling between the acetylation and the tyrosine- importance in order to understand protein never achieved in protein structures. Here of the actin cytoskeleton in virtue of its phosphorylation of cortactin. This switch function. Any computational modeling we present a novel PMF that is totally inde- capacity to active the Arp2/3 complex and was confirmed with the endogenous pro- of protein flexibility requires an appro- pendent of any reference state. Considering NWASP protein (1). Cortactin is an onco- tein. Furthermore, we analyzed the effect priate energy function, which can either only directly interacting atom pairs (such protein overexpressed in different human of the phosphorylation in cortactin–Focal rely on first principles or on statistics of as that there are no intermediate atom), it carcinomas and currently considered a Adhesion Kinase (FAK) complex forma- known protein structures. Knowledge- computes the interaction energy between bona fide invadopodia marker. In addi- tion during cell spreading (3). Finally we based potentials (KBP) are nevertheless, atoms of type a and b, as the minus log of tion, cortactin is targeted by pathogens present new unpublished data of how the simpler and easier to compute than clas- the probability density function to find a to invade or to adhere to host cells (2). cortactin-FAK complex relates to N-WASP sical Molecular Mechanics force fields, but pair of atoms of type a, b in direct contact Although cortactin was cloned as a Src sub- protein. still useful to describe protein folded states. plus a normalization constant that is deter- strate how this post-translational modifi- There are two main ways of obtaining KBPs: mined by equaling the average interaction cation regulates the activity of the protein 1 Martinez-Quiles N, Ho HY, Kirschner MW, 1) Optimizing the likelihood of the protein energy to the propensity of atoms of type Ramesh N, Geha RS. Erk/Src phosphorylation remains unclear. Moreover the protein is of cortactin acts as a switch on-switch off native structure vs. the artificially gener- a, b to establish direct contacts. In order regulated by acetylation but how these two mechanism that controls its ability to activate ated decoys [1]; 2) Deriving Boltzmann-like to obtain an analytical function, a least post-translational modifications relate to N-WASP. Mol Cell Biol. 2004 Jun;24(12):5269-80. statistics as Potential of the Mean Force squares non-linear fit of the computed val- 2 Nieto-Pelegrin E, Martinez-Quiles N. Distinct each other was unknown. Therefore to phosphorylation requirements regulate cortactin (PMF) [2]. The former strategy can be lim- ues is performed to a quadruple exponential analyze the biochemical consequences activation by TirEPEC and its binding to N-WASP. ited because of the high dimensionality of function (Morselike). Finally we will ensure of cortactin phosphorylation on tyrosines, Cell Commun Signal. 2009 May 6;7:11. the parameter space and/or the coarse- that native protein structures are in the 3 Meiler E, Nieto-Pelegrín E, Martinez-Quiles we used a fusion-expression system that N. Cortactin tyrosine phosphorylation grained energy model adopted to cope actual energy minimum of the DICAM force forces the interaction between cortactin promotes its deacetylation and inhibits cell with this problem. In the case of the PMF, field by optimizing the parameters over a and Src kinase in cells. We describe here a spreading. PLoS One. 2012;7(3):e33662. the observed atomic contact frequencies set of well determined protein structures, must be normalized versus a reference state, so that torsional fluctuations due toDICAM defined as the one with no interactions are minimized.

102 POSTER SESSION Allosteric Regulation of Cell Signalling 103 Free energy of the interdomain Sequence dependent allostery in PDZ 16 17 assembly of SH2 and catalytic domain domains dictates their binding selectivity in c-Abl combining metadynamics and all-atom structure-based modeling.

Montemiglio, L.C., Gianni, S., Haq, R.S., Brunori, M. and Jemth, P. Ilaria Mereu, Ludovico Sutto and Francesco L. Gervasio CNIO, Madrid, Spain University of Rome, Dept. of Biochemical Sciences, Rome, Italy

We report on a computational analysis scales are well beyond the capabilities of The crowded and heterogeneous cellular distinct roles of the various PDZ domains in of the dynamical and structural effects standard all-atom molecular dynamics environment demands the interactions cellular signaling require selectivity. due to the rearrangements of the sh2 and simulations. This method provides us with between its numerous components to be kinase domains of c-Abl. Our approach the potential to gain new insights in the highly specific, to avoid potentially harmful We used double mutant cycles (4), involv- is based on a hybrid structure-based mechanisms regulating the activity of the side reactions. Given the vast number of ing site-directed mutagenesis of both PDZ all-atom model [1] combined with the whole c-Abl kinase in atomistic details and ligands competing for binding to a limited domain and peptide ligand, in conjunc- metadynamics method to enhance con- a free-energy landscape interpretation of number of domain families, it is often puz- tion with binding kinetics to unveil the fine formational sampling [2]. The combined some of the known cancer-driving and zling how specificity can be achieved (1). energetic details of the specific long-range benefits of these two approaches allow the resistant point mutations of c-Abl. In monomeric proteins selectivity may networks involved in peptide recognition. study of events whose time and length be modulated by intradomain allostery, We performed the analysis on two homolo- whereby a remote residue is energetically gous PDZ-ligand complexes and found that connected to the functional binding site the energetically coupled residues differ for via side chain or backbone interactions. these complexes. This result demonstrates Whereas several intradomain energetic that amino acid sequence rather than topol- pathways have been predicted in modular ogy dictates the allosteric pathways. Fur- protein domains, there is a paucity of exper- thermore, our data support a mechanism imental data to validate their existence. whereby the whole domain, not only the binding pocket, is optimized for a specific Here, we have identified such functional ligand. Such cross-talk between binding energetic networks in one of the most com- sites and remote residues may be used to mon protein-protein interaction modules, tune target selectivity. the PDZ domain, involved in the control of several cellular events (2,3). Although this 1 Kaneko T (2011) Trends Biochem. Sci. 36, 183–90; 2 Fanning AS (1999) J. Clin. Invest. 103, 767-72; protein family displays a simple topology 3 Jemth P (2007) Biochemistry 46, 8701-08; and a rather conserved binding pocket, the 4 Horovitz A (1990) J. Mol. Biol. 214, 613–17.

104 POSTER SESSION Allosteric Regulation of Cell Signalling 105 Dynamic properties of c-SRC Allosteric communication pathways 18 19 tyrosine Kinase by NMR and molecular in KIX domain-a Metadynamics study dynamic calculations

Maria Morando1, Nicola D’Amelio1, Moreno Lelli2 and Francesco Gervasio1 Palazzesi F. 1, Barducci A. 2 and Parrinello M. 3 1, 3 Department of Chemistry and Applied Bio- 1Biophysical Computational group, Spanish National Cancer Research Centre sciences, ETH Zurich, and Facoltà di Informatica, Istituto di Scienze Computazionali, Università (CNIO), Madrid, Spain; 2Centre de RMN à Très Hauts Champs, Lyon, France della Svizzera Italiana, Lugano Switzerland; 2ITP EPFL, Lausanne, Switzerland

Tyrosine kinases (TK) play a fundamen- ranging from an open form, to a closed Here we present an investigation on the demanding conformational transition is tal role in cellular signaling pathways. conformation, which completely blocks origins of allosteric dynamical changes that unaccessible for the typical MD simulation Their deregulation is linked to several the substrate binding site. The αC- helix in are caused by the interaction between MLL timescale. In order to avoid this limitation, diseases including Cancer and diabetes. the N-lobe can swing out of its active con- protein with KIX and C-Myb domains and we employ enhanced MD methodologies In particular two highly homologous TK, formation (so- called αC-in), interrupt- its biophysical characterization. In previ- such as Metadynamics. Indeed, to achieve namely c-ABL and c-SRC, are causative ing a crucial salt bridge in the active site ous NMR studies [1], the dynamic ensemble the sampling of the conformations ensem- of Chronic Myeloid Leukaemia (fusion and leading to a reoriented conformation of the accessible states of this system were ble, Well-Tempered Ensemble [2] combined gene Bcr-Abl) and metastasis (c-SRC proto (called αC-out). The DFG motif is located studied using relaxation dispersion tech- with Parallel Tempering simulations were oncogene). Despite the high homology at the N-terminal end of the A-loop and niques. Dispersion profiles obtained for performed. Using this advanced technique (47%), they show a different sensitiv- has been proposed to have a role in the the binary state (MLL:KIX) indicated the we have properly characterized the atomis- ity to the anticancer drug Imatinib. The catalytic activity of the kinase. Finally the presence of a conformational transition tic configuration of the excited state for the catalytic domain of TK, responsible of this P-loop, also located in the N-lobe, is known (on the milliseconds timescale) between KIX domain in the binary complex. Other interaction, comprises an N-terminal to adopt a distinct kinked conformation in two configurations: a so called “ground” important outcomes were obtained from lobe, containing mostly β-sheets, and a the Abl-Imatinib complex, while in c-Src it state (B) which is highly populated, and an the understanding of the structural nature larger, α-helical C-terminal lobe. At the remains unchanged upon Imatinib bind- “excited” state (B*) which is scarcely popu- of the communication pathway and the interface between the lobes, a number of ing. Its importance is further underlined lated. Interestingly, the chemical shifts of B* energetics associated with them. Indeed highly conserved residues form the active by the fact that many mutations which state were seen to be similar to the ternary we shed a light on the signal transmission site with the ATP binding pocket. Generally, confer resistance to Imatinib are located complex (MLL:KIX:c-Myb). This observa- along the allosteric pathway and how the kinase activation is mainly controlled by in the P-loop. tion suggested the pre-existence of state residues pass the information from one conformational changes in four conserved B*, which is likely to bind to c-Myb, in the site to the other. structural motifs at the active site: the Here we present the pico-nanosecond ensemble of binary complex conformations. activation loop (A-loop), the Asp-Phe-Gly dynamics measured by NMR relaxation However, relaxation dispersion NMR tech- 1 S. Bruschweiler, P. Schanda, K. Kloiber, B. Brutscher, G. Kontaxis, R. Konrat, (DFG) motif,the αC-helix and the P-loop. experiments on the catalytic domain of niques, could not resolve the structure of and M. Tollinger, J. Am. Chem. The A-loop is a highly flexible region con- cSRC. These preliminary results will be the higher energy conformation. Molecular Soc., 131, 3063-3068 (2009) necting the two lobes, which can adopt compared with MD calculations. dynamics (MD) is a natural choice for under- 2 M. Bonomi and M. Parrinello, Phys. Rev. Lett. 104, 190601 (2010) an ensemble of different conformations standing the molecular origins of dynamics allostery in this system. However, this time

106 POSTER SESSION Allosteric Regulation of Cell Signalling 107 Deciphering CAD, a 1.5 MegaDa Crystal structures of Schistosoma mansoni 20 21 anti-tumoral target that controls Peroxiredoxin I: insight into the mechanism of de synthesis of pyrimidines assembly of stress-regulated chaperones Fulvio Saccoccia, Francesco Angelucci, Giovanna Boumis, Maurizio Brunori, Patrizio Di Micco, Ilias Koutris, Adriana E. Miele, Veronica Morea, David Williams and Andrea Bellelli Dept. of Biochemical Santiago Ramón-Maiques, Araceli Grande-García, Nada Lallous and Alba Ruiz-Ramos Sciences “A. Rossi Fanelli”, “Sapienza” University of Rome and Istituto Pasteur-Fondazione Cenci Bolo- Structural Bases of Genome Integrity Group; Structural Biology and Biocomputing Programme; gnetti. CNR – National Research Council of Italy, Institute of Molecular Biology and Pathology, Rome, Spanish National Cancer Research Centre (CNIO), Madrid, Spain Italy; Department of Immunology/Microbiology, Rush University Medical Center, Chicago, USA

CAD is a 1.5x106 Da macromolecular in cancer, there is no direct structural Schistosomiasis poses one of the great- involved in the detoxification pathway of complex that controls the initial steps of information about CAD or about any of its est health challenges for countries in the S. mansoni: TGR; Thioredoxin (Trx); Glu- the de novo synthesis of pyrimidines, is domains, other than it associates form- tropical and sub-tropical area, where it kills tathione Peroxidase (Gpx) and Peroxire- implicated in tumor development, and ing a homohexamer that travels between 280000 people/year out of an estimate of doxin1 (Prx1). Thus specificity of new drugs is a target for anti-tumoral drugs. CAD is nucleus and cytoplasm depending on the 200 million infected. The adult worms of may in principle be achieved by targeting formed by a multifunctional polypeptide functional situation or the stage of the both sex live in the veins of the bowel and this pathway. 2-Cys peroxiredoxins (Prxs) of 253 kDa with three enzymatic functions, cell cycle. The structures of CPS, ATC and the bladder of the host, where females lay play two different roles depending on the Carbamyl phosphate synthetase II (CPS DHO homologs in bacteria or archea have eggs causing inflammation and hemor- physiological status of the cell. They are II), Aspartate transcarbamylase (ATC) been determined but there is no structural rages, and in chronic form leads to severe thioredoxin-dependent peroxidases under and Dihydroorotase (DHO), each of them information of any eukaryotic counterpart complications. Praziquantel, the drug of low oxidative stress and ATP-independent catalyzing one of the three initial reactions that could be applied to CAD. Given its choice against all Schistosome species, chaperones upon exposure to high peroxide of the pathway. Up-regulation of CAD activ- large size and modular nature, we aim to is effective only on the adult worms and concentrations. These alternative func- ity is needed in proliferating and tumoral determine the structure of CAD combining unfortunately some less sensitive strains tions have been associated with changes cells to allow the fast synthesis of pyrimi- X-ray crystallography and single particle are emerging; thus it is mandatory to dis- in the oligomerization state from low- dines required for replication. Thus, CAD electron microscopy. Here we report the cover new specific drugs. The aim of our (LMW) to high-molecularweight (HMW) is a target for the design of anti-tumoral expression and purification of full-length project is to obtain a complete characteri- species. Here we present the structures of drugs like PALA, a bi-substrate inhibitor CAD. We also present the crystal structures zation of the 3D structure of the enzymes Schistosoma mansoni PrxI in both states: of the ATC activity. The activity of CAD is of the DHO and ATC domains of human involved in the detoxification pathway of the LMW decamer and the HMW 20-mer allosterically regulated, being feed-back CAD. These results provide the first view S. mansoni. This pathway is peculiar in so formed by two stacked decamers. The latter inhibited by UTP, the final product of the of eukaryotic DHO and ATC enzymes, and far as the parasite relies on a single enzyme, is the structure of a 2-Cys Prx chaperonic pathway, and activated by the nucleotide give us, for the first time, high-resolution thioredoxin glutathione reductase (TGR), to form. Comparison of the structures sheds precursor PRPP. The allosteric regulation information for trying to decipher the maintain redox homeostasis, contrary to light on the mechanism by which chemical of CAD is also modulated by phosphoryla- architecture of CAD. humans that possess two separate enzymes. stressors, such as high H(2)O(2) as high tion of MAP kinase and PKA signaling cas- By X-ray crystallography we have solved the concentration at acidic pH, are sensed and cades during cell cycle. Despite its key role 3D structure of the following enzymes, all translated into a functional switch in this in cell metabolism and its implications protein family.

108 POSTER SESSION Allosteric Regulation of Cell Signalling 109 Insights on the molecular structure Rigidity-based Allosteric 22 23 of the plant Salt-Overly-Sensitive 1 (SOS1) Communication Na+/H+ antiporter

María José Sánchez-Barrena1, Rafael Núñez-Ramírez2, Irene Villalta3, Francisco J. Quintero3, Juan Francisco Vega2, Jose M. Pardo3, Javier Martinez-Salazar2, Armando Albert1 1Departamento de Cristalografía y Biología Estructural, Instituto de Química Física Adnan Sljoka York University, Toronto, Canada

Due to their sessile nature, plants have of SOS1. In addition, we have performed Given the 3-D structure of a protein, under- conformational change that is transmitted to endure adverse environmental condi- electron microscopy and single particle standing how it functions depends in criti- through the protein to cause a rearrange- tions. The Arabidopsis thaliana Na+/H+ reconstruction of negatively stained full cal ways on predicting which parts are rigid ment and alteration of the shape of the antiporter SOS1 is essential to maintain length and active AtSOS1. Our studies show and which are flexible. The rigidity and active site. In this study we introduce sev- low intracellular levels of the toxic Na+ that the protein is a homodimer that con- flexibility analysis of the molecular graph, eral rigidity-based allostery modes of com- under salt stress. Available data show that tains a membrane domain similar to that using a fast combinatorial rigidity algo- munication and corresponding algorithms the plant SOS2 protein kinase and its inter- found in other antiporters of the family, rithm – the pebble game algorithm (flexweb. which can detect rigidity and shape changes acting activator, the SOS3 calcium-binding and an elongated, large and structured asu.edu), can rapidly decompose a protein between two distant binding pockets in protein, function together in decoding cytosolic domain. Both the transmem- into flexible and rigid regions. allosteric proteins. Starting with a GPCR calcium signals elicited by salt stress and brane and cytosolic moieties contribute structure, we will show how ligand (agonist) regulating the phosphorylation state and to the dimerization of the antiporter. The This rigidity approach is applied to study- binding induced rigidity changes at the the activity of SOS1. Molecular genetic close contacts between the transmem- ing allostery in recently solved GPCR pro- extracellular binding pocket triggers rigid- studies have shown that the activation brane and the cytosolic domains provide teins. It is widely believed that the binding ity changes which propagate to the binding implies a domain reorganization of the a link between regulation and transport of a ligand at the allosteric site triggers a regions at the cytoplasmic domain. antiporter cytosolic moiety indicating activity of the antiporter. Structural stud- that there is a clear relationship between ies on the different components of Salt function and molecular structure of the Overly Sensitive pathway are letting us antiporter. To provide information on this understand how the molecular machinery issue, we have carried out in vivo and in for salt tolerance works and opens path vitro studies on the oligomerization state towards biotechnological applications.

110 POSTER SESSION Allosteric Regulation of Cell Signalling 111 Germ-line DICER1 mutation and associated An hybrid structure-based model 24 25 loss of heterozygosity in a pineoblastoma for large scale conformational transition

Archana Srivastava1,2, Nelly Sabbaghian1,2, Nancy Hamel1,3, Steffen Albrecht4, John R. Priest5 in protein kinases and William D Foulkes1,3 1Program in Cancer Genetics, Department of Oncology and Human Genetics, McGill University, Montreal, Quebec Canada; 2Lady Davis Institute and Segal Cancer Centre, Jewish Gen- eral Hospital, McGill University, Montreal, Quebec, Canada; 3Research Institute, McGill University Health Centre, Montreal, Quebec, Canada; 4Department of Pathology, Montreal Children’s Hospital and McGill L. Sutto1, I. Mereu1, F.L. Gervasio1 1Structural Biology and Biocomputing Programme, University, Montreal, Quebec, Canada; 5Minneapolis, Minnesota, USA Spanish National Cancer Research Center (CNIO), Madrid, Spain

The pineal gland, so named by Galen well as primitive neuroectodermal tumors, Structure based models are successful at the local backbone and sidechain interac- because of its resemblance to a pine Wilms tumor, cystic nephroma, pulmo- conjugating the essence of the energy land- tions is necessary. The proposed model nut, is a midline structure situated on nary sequestration, juvenile intestinal scape theory of protein folding with an easy merges a precise description of these inter- the posterior wall of the third ventricle, polyps, and familial multinodular goiter. and efficient implementation. Recently actions with a structurebased non-bonded deep between the cerebral hemispheres. Based on this profile, we hypothesized their realm expanded beyond single protein potential that takes into account different A pineoblastoma is a supratentorial primi- that such mutations might also occur in structure, been used profitably to widely conformers. We present the results for tive neuroectodermal tumour arising in children with pineoblastoma. study large conformational transitions. Still, the activation of the catalytic domain of a the pineal gland. It is known to occur in when dealing with conformational transi- human kinase for which we reconstructed RB-1 mutation carriers, but is rarely seen Here we describe a novel germ-line DICER1 tion between two well-defined structures the transition free energy and the descrip- in association with other hereditary syn- mutation in a 6 year old child with a highly an unbiased and realistic description of tion of the activation loop flexibility. dromes. aggressive pineoblastoma. In addition, we observed loss of heterozygosity (LOH) of DICER1 is an RNAse endoribonuclease the wild-type allele, an event not previous- responsible for the production of micro- ly reported in DICER1-associated tumours. RNAs (miRNAs). In addition, DICER1 is Our observations suggest that: a) pineob- involved in regulation of chromatin, DNA lastoma are associated with germ-line replication timing, genome stability, and DICER1 mutations, thus further extend- cellular senescence. We and others have ing the disease profile ofDICER1 kindred, recently demonstrated that germ-line and b) unexpectedly, tumour cells can DICER1 mutations are associated with a thrive in the absence of functional DICER1. cancer syndrome presenting embryonal These findings will further help identify tumours such as pleuropulmonary blasto- at risk families, a clinically important task ma (PPB), ovarian sex cord stromal tumors given the association between DICER1 (especially Sertoli-Leydig cell tumors), mutations and PPB which can be fatal to cervical embryonal rhabdomyosarcoma as affected infants.

112 POSTER SESSION Allosteric Regulation of Cell Signalling 113 Allosteric Regulation of Cell SignallinG CNIO FRONTIERS MEETINGS 2013 Chromosome Confirmed speakers Instability and Angelika Amon, Daniela Cimini, Don W. Aneuploidy in Cancer: Cleveland, Duane A. Compton, Philip Hieter, Randall King, Rong Li, David Pellman, Charles from Mechanisms Swanton, Stephen Taylor, Jan van Deursen, to Therapeutics Ashok Venkitaraman and Todd Waldman. 27/05/2013-29/05/2013

Organisers summary Robert Benezra, Memorial Sloan-Kettering The conference will focus on recent progress Cancer Center, New York, USA. in understanding how aneuploidy contributes Ana Losada, CNIO, Madrid, Spain. to initiation and progression of human cancers. Marcos Malumbres, CNIO, Madrid, Spain. Some of the subjects discussed will be: René Medema, The Netherlands Cancer • Mechanisms of aneuploidy generation. Institute, Amsterdam, The Netherlands. • the effect of aneuploidy on cell pro- liferation and survival and how cancer cells tolerate it and profit from it. • cin genes in human cancer. • Modeling aneuploidy in mouse • targeting aneuploidy as a therapeutic opportunity.

Allosteric Regulation of Cell Signalling 117 Allosteric Regulation of Cell SignallinG Previous CNIO Frontiers Meetings and CNIO Cancer Conferences 2011 2010

Recapturing Pluripotency: Cancer pharmacogenetics: links between celLular Personalizing medicine reprogramming and cancer 22/11/2010-24/11/2010 07/11/2011-09/11/2011 Organisers: Javier Benítez, William E. Evans, Organisers: Maria A. Blasco, Konrad Miguel Martín and Magnus Ingelman-Sundberg Hochedlinger, Manuel Serrano, Inder Verma Molecular cancer therapeutics Canceromatics II: Multilevel 08/03/2010-10/03/2010 Interpretation of Cancer Genome Organisers: Gail Eckhardt, Roy S. 28/03/2011-30/03/2011 Herbst and Manuel Hidalgo Organisers: Søren Brunak, Stephen Chanock, Núria Malats, Chris Sander, Alfonso Valencia

Breast Cancer 07/02/2011-09/02/2011 Organisers: Joaquín Arribas, José Baselga, Miguel Ángel Piris, Lajos Pusztai and Jorge Reis-Filho

Allosteric Regulation of Cell Signalling 121 2009 2008 2007 2006

The energy of cancer Signalling upstream of mTOR Links between cancer, replication Telomeres and telomerase-CNIO / 02/11/2009-04/11/2009 03/11/2008-05/11/2008 stress and genomic integrity Joséf Steiner Cancer Conference Organisers: Toren Finkel, David M. Sabatini, Organisers: Dario R. Alessi, Tomi P. Mäkelä 05/11/2007-07/11/2007 13/11/2006-15/11/2006 Manuel Serrano and David A. Sinclair and Montserrat Sánchez-Céspedes Organisers: Oskar Fernández-Capetillo, Jiri Organisers: Maria A. Blasco and Jerry Shay Lukas, Juan Méndez and André Nussenzweig Cancer-om-atics: Structure and mechanisms Medicinal chemistry in oncology Multilevelinterpretation of essential complexes Myc and the transcriptional control 02/10/2006-04/10/2006 of cancer genome data for cell survival of proliferation and oncogenesis Organisers: Fernando Albericio, James R. 06/07/2009-08/07/2009 23/06/2008-25/06/2008 11/06/2007-13/06/2007 Bischoff,C arlos García-Echeverria and Andrew Organisers: Søren Brunak, Núria Malats, Organisers: Niko Grigorieff,E va Organisers: Robert N. Eisenman, Mortlock Chris Sander and Alfonso Valencia Nogales and Jose María Valpuesta Martin Eilers and Javier León Inflammation and cancer Stem cells and cancer Development and cancer Molecular mechanisms 22/05/2006-24/05/2006 23/02/2009-25/02/2009 04/02/2008-06/02/2008 in lymphoid neoplasm Organisers: Curtis Harris, Raymond DuBois, Organisers: Elaine Fuchs, Maria A. Blasco, Organisers: Konrad Basler, Ginés Morata, 19/02/2007-21/02/2007 Jorge Moscat and Manuel Serrano Eduard Batlle and Mirna Pérez-Moreno Eduardo Moreno and Miguel Torres Organisers: Elias Campo, Riccardo Dalla- Favera, Elaine S. Jaffe and MiguelA ngel Piris PTEN and the AKT route 08/05/2006-10/05/2006 Organisers: Ana Carrera, Pier Paolo Pandolfi andP eter Vogt

122 Previous CNIO Frontiers Meetings and CNIO Cancer Conferences Allosteric Regulation of Cell Signalling 123 2005 2004 2003 2002

Cancer and aging Cadherins, catenins and cancer Apoptosis and cancer Mechanisms of invasion 07/11/2005-09/11/2005 29/11/2004-01/12/2004 01/12/2003-03/12/2003 and metastasis Organisers: Maria A. Blasco, Kathy Collins, Organisers: Amparo Cano, Hans Clevers, Organisers: Gabriel Nuñez, Marisol 18/11/2002-20/11/2002 Jan Hoeijmakers and Manuel Serrano José Palacios and Franz Van Roy Soengas and Scott Lowe Organisers: Joan Massagué and Richard Hynes

MAP kinases and cancer Structural biology of Small GTPases in human The cell cycle and cancer 30/05/2005-01/06/2005 cancer targets carcinogenesis 30/09/2002-02/10/2002 Organisers: Philip Cohen, Roger Davis, 27/09/2004-29/09/2004 16/06/2003-18/06/2003 Organisers: Marcos Malumbres, Worcester, Chris Marshall and Ángel Nebreda Organisers: Ernest Laue, Guillermo Organisers: Juan Carlos Lacal, Charles Sherr and Jiri Bartek Montoya and Alfred Wittinghofer Channing Der and Shuh Narumiya Animal tumour models Cancer epigenetics: DNA and functional genomics Targeted search for methylation and chromatin 07/03/2005-09/03/2005 anticancer drugs 29/05/2002-31/05/2002 Organisers: Allan Balmain, Mariano 17/03/2003-19/03/2003 Organisers: Manel Esteller Barbacid, Anton Berns and Tyler Jacks Organisers: Amancio Carnero and Stephen B. Baylin and David H. Beach

124 Previous CNIO Frontiers Meetings and CNIO Cancer Conferences Allosteric Regulation of Cell Signalling 125 FRONTIERS IN TUMOUR HETEROGENEITY AND PLASTICITY

27/10/2013-30/10/2013

Organisers Keynote speakers Mirna Pérez-Moreno, CNIO, Madrid. Kornelia Polyak and José Baselga. Scott Lowe, MSKCC, New York, USA. Erwin Wagner, CNIO, Madrid, Spain.

Confirmed speakers Cedric Blanpain, Snorri Thorgeirsson, Co.organisers from Christoph Klein, Elaine Mardis, Nature Publishing Group Charles Swanton, John Condeelis, Barbara Marte, Nature. Ruslan Medzhitov, Karen Vousden, Nicola McCarthy, Nature Mike Hemann, Victoria Seewaldt, Reviews Cancer. Sean Morrison, Angela Nieto, Lillian Alexia-Ileana Zaromytidou, Nature Siu, Jeff Settleman, Gail Eckhardt Cell Biology. Nicholas Barker and Hiroyuki Mano.

126 Allosteric Regulation of Cell Signalling Allosteric Regulation of Cell Signalling 127 NOTES Allosteric Regulation of Cell Signalling NOTES Allosteric Regulation of Cell Signalling NOTES Allosteric Regulation of Cell Signalling NOTES Allosteric Regulation of Cell Signalling The Spanish Ministry of Economy and Competitiveness contributed to the funding of this meeting through "Acción complementaria BIO2011-15251-E"

Centro Nacional de Investigaciones Oncológicas (CNIO) Spanish National Cancer Research Centre Melchor Fernández Almagro, 3 28029 Madrid, Spain www.cnio.es

Coordination and edition Peter Klatt, Mercedes Moro and Virginia de la Cruz, CNIO, Madrid, Spain Design by underbau Photographic archive CNIO

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NOTES