Publications

2016

• Défis algorithmiques pour les simulations biomoléculaires et la conception de protéines
  
  • Druart Karen
  , 2016. Le dessin computationnel de protéine, ou CPD, est une technique qui permet de modifier les protéines pour leur conférer de nouvelles propriétés, en exploitant leurs structures 3D et une modélisation moléculaire. Pour rendre la méthode de plus en plus prédictive, les modèles employés doivent constamment progresser. Dans cette thèse, nous avons abordé le problème de la représentation explicite de la flexibilité du squelette protéique. Nous avons développé une méthode de dessin "multi-états", qui se base sur une bibliothèque discrète de conformations du squelette, établie à l'avance. Dans un contexte de simulation Monte Carlo, le paysage énergétique d'une protéine étant rugueux, les changements de squelettes ne peuvent etre acceptés que moyennant certaines précautions. Aussi, pour explorer ces conformations, en même temps que des mutations et des mouvements de chaînes latérales, nous avons introduit un nouveau type de déplacement dans une méthode Monte Carlo existante. Il s'agit d'un déplacement "hybride", où un changement de squelette est suivi d'une courte relaxation Monte Carlo des chaînes latérales seules, après laquelle un test d'acceptation est effectué. Pour respecter une distribution de Boltzmann des états, la probabilité doit avoir une forme précise, qui contient une intégrale de chemin, difficile à calculer en pratique. Deux approximations sont explorées en détail: une basée sur un seul chemin de relaxation, ou chemin "générateur" (Single Path Approximation, ou SPA), et une plus complexe basée sur un ensemble de chemins, obtenus en permutant les étapes élémentaires du chemin générateur (Permuted Path Approximation, ou PPA). Ces deux approximations sont étudiées et comparées sur deux protéines. En particulier, nous calculons les énergies relatives des conformations du squelette en utilisant trois méthodes différentes, qui passent réversiblement d'une conformation à l'autre en empruntent des chemins très différents. Le bon accord entre les méthodes, obtenu avec de nombreuses paramétrisations différentes, montre que l'énergie libre se comporte bien comme une fonction d'état, suggérant que les états sont bien échantillonnés selon la distribution de Boltzmann. La méthode d'échantillonnage est ensuite appliquée à une boucle dans le site actif de la tyrosyl-ARNt synthétase, permettant d'identifier des séquences qui favorisent une conformation, soit ouverte, soit fermée de la boucle, permettant en principe de contrôler ou redessiner sa conformation. Nous décrivons enfin un travail préliminaire visant à augmenter encore la flexibilité du squelette, en explorant un espace de conformations continu et non plus discret. Ce changement d'espace oblige à restructurer complètement le calcul des énergies et le déroulement des simulations, augmente considérable le coût des calculs, et nécessite une parallélisation beaucoup plus agressive du logiciel de simulation.
• Structure of the active form of Dcp1–Dcp2 decapping enzyme bound to m7GDP and its Edc3 activator
  
  • Charenton Clément
  • Taverniti Valerio
  • Gaudon-Plesse Claudine
  • Back Régis
  • Séraphin Bertrand
  • Graille Marc
  Nature Structural and Molecular Biology, Nature Publishing Group, 2016, 23 (11), pp.982-986. (10.1038/nsmb.3300)
  DOI : 10.1038/nsmb.3300
• A Hybrid Monte Carlo Scheme for Multibackbone Protein Design
  
  • Druart Karen
  • Bigot Julien
  • Audit Edouard
  • Simonson Thomas
  Journal of Chemical Theory and Computation, American Chemical Society, 2016. Multistate protein design explores side chain mutations, with the backbone allowed to sample a small, predetermined library of conformations. To achieve Boltzmann sampling of sequences and conformations, we use a hybrid Monte Carlo (MC) scheme: a trial hop between backbone models is followed by a short MC segment where side chain rotamers adjust to the new backbone, before applying a Metropolis-like acceptance test. The theoretical form and a practical approximation for the acceptance test are derived. We then compute backbone conformational free energies for two SH2 and SH3 proteins using different routes and protocols, and verify that for simple test problems, the free energy behaves like a state function, a hallmark of Boltzmann sampling. (10.1021/acs.jctc.6b00421)
  DOI : 10.1021/acs.jctc.6b00421
• Direct interaction between exocyst and Wave complexes promotes cell protrusions and motility.
  
  • Biondini Marco
  • Sadou-Dubourgnoux Amel
  • Paul-Gilloteaux Perrine
  • Zago Giulia
  • Arslanhan Melis D
  • Waharte François
  • Formstecher Etienne
  • Hertzog Maud
  • Yu Jinchao
  • Guérois Raphaël
  • Gautreau Alexis
  • Scita Giorgio
  • Camonis Jacques
  • Parrini Maria Carla
  Journal of Cell Science, Company of Biologists, 2016, 129 (20), pp.3756-3769. Coordination between membrane trafficking and actin polymerization is fundamental in cell migration, but a dynamic view of the underlying molecular mechanisms is still missing. The Rac1 GTPase controls actin polymerization at protrusions by interacting with its effector, the Wave regulatory complex (WRC). The exocyst complex, which functions in polarized exocytosis, has been involved in the regulation of cell motility. Here, we show a physical and functional connection between exocyst and WRC. Purified components of exocyst and WRC directly associate in vitro, and interactions interfaces are identified. The exocyst-WRC interaction is confirmed in cells by co-immunoprecipitation and is shown to occur independently of the Arp2/3 complex. Disruption of the exocyst-WRC interaction leads to impaired migration. By using time-lapse microscopy coupled to image correlation analysis, we visualized the trafficking of the WRC towards the front of the cell in nascent protrusions. The exocyst is necessary for WRC recruitment at the leading edge and for resulting cell edge movements. This direct link between the exocyst and WRC provides a new mechanistic insight into the spatio-temporal regulation of cell migration. (10.1242/jcs.187336)
  DOI : 10.1242/jcs.187336
• Mécanisme de formation du complexe de démarrage de la traduction chez les Archées
  
  • Monestier Auriane
  , 2016. Une cellule est soumise à différents stimuli internes et externes. Pour remplir ses fonctions, elle doit donc s’adapter rapidement. Cela implique une régulation fine de l’expression génique. Celle-ci s’effectue au niveau transcriptionnel, mais également au niveau traductionnel. La traduction comprend trois phases : le démarrage, l’allongement et la terminaison. C’est au cours du démarrage de la traduction que s’effectue la sélection du codon de démarrage et donc le choix du cadre de lecture de l’ARNm. D’un point de vue cinétique, le démarrage de la traduction est l’étape limitante. Ainsi, il apparait comme une cible privilégiée pour le contrôle traductionnel.Chez les archées, le démarrage de la traduction met en jeu un complexe macromoléculaire formé de la petite sous-unité du ribosome, d’un ARNm, d’un ARN de transfert initiateur méthionylé (Met-ARNtiMet) et de trois facteurs de démarrage de la traduction (aIF1, aIF1A et aIF2). De manière intéressante, ces trois facteurs de démarrage ont chacun un orthologue eucaryote.Les ARNti archées et eucaryotes possèdent une paire de bases très conservée A1-U72, au sommet de la tige acceptrice. Cette paire de base a été montrée importante pour la discrimination des ARNt initiateurs et élongateurs. De plus, des travaux suggèrent l’importance de la géométrie de la paire A1-U72 pour l’identité initiatrice de ces ARNts. Cependant, au début de ma thèse, aucune donnée structurale n’était disponible pour expliquer comment les caractéristiques de la paire A1-U72 participaient à la sélection de l’ARNt initiateur. Dans un premier temps, mon travail de thèse a consisté en la construction d’une souche bactérienne d’E.coli utilisant comme seul source d’ARNti un variant d’ARNt initiateur bactérien (ARNtfMet) possédant une paire de base A1-U72 (ARNtfMetA1-U72). L’utilisation de cette souche nous a permis d’obtenir de grandes quantités d’ARNtfMetA1-U72 purifié. De plus, la structure cristallographique de cet ARNtA1-U72 a pu être déterminée à 2.8 Å de résolution. Un arrangement inhabituel des bases A1 et U72 a été observé.Tous les acteurs du démarrage de la traduction de l’archée P. abyssi étant disponibles au laboratoire, une étude du complexe de démarrage de la traduction archée par cryo-microscopie électronique a pu être réalisée. L’étude a permis d’identifier deux conformations de l’ARNti dans le complexe de démarrage, IC0-Premote (5.3 Å de résolution) et IC1-Pin (7.5 Å de résolution). Ces deux conformations permettent de proposer un modèle pour l’accommodation de l’ARNt initiateur lors de l’appariement au codon de démarrage.Finalement, je me suis également intéressée au rôle du facteur aIF1. La disponibilité de structures 3D et de modèles d’assemblage, ainsi que les alignements des séquences aIF1 d’archées ont permis de proposer des régions ou acides aminés pouvant être impliqués dans la liaison au ribosome et/ou dans la sélection des ARNt initiateurs lors de la formation du complexe de démarrage. Afin de pouvoir étudier l’implication de ces régions ou acides aminés, j’ai mis au point une méthode d’étude de la liaison d’aIF1 à la petite sous-unité du ribosome par anisotropie de fluorescence. Cette étude met en évidence deux résidus basiques d’aIF1 impliqués dans la liaison au ribosome. D’autre part, les rôles d’aIF1 dans la sélection du codon de démarrage de la traduction et dans la stabilisation du complexe de démarrage sur l’ARNm sont étudiés par la méthode d’empreinte du ribosome ou toeprint.
• Comparing three stochastic search algorithms for computational protein design: Monte Carlo, replica exchange Monte Carlo, and a multistart, steepest-descent heuristic
  
  • Mignon David
  • Simonson Thomas
  Journal of Computational Chemistry, Wiley, 2016, 37 (19), pp.1781 - 1793. Computational protein design depends on an energy function and an algorithm to search the sequence/conformation space. We compare three stochastic search algorithms: a heuristic, Monte Carlo (MC), and a Replica Exchange Monte Carlo method (REMC). The heuristic performs a steepest-descent minimization starting from thousands of random starting points. The methods are applied to nine test proteins from three structural families, with a fixed backbone structure, a molecular mechanics energy function, and with 1, 5, 10, 20, 30, or all amino acids allowed to mutate. Results are compared to an exact, Cost Function Network method that identifies the global minimum energy conformation (GMEC) in favorable cases. The designed sequences accurately reproduce experimental sequences in the hydrophobic core. The heuristic and REMC agree closely and reproduce the GMEC when it is known, with a few exceptions. Plain MC performs well for most cases, occasionally departing from the GMEC by 3-4 kcal/mol. With REMC, the diversity of the sequences sampled agrees with exact enumeration where the latter is possible: up to 2 kcal/mol above the GMEC. Beyond, room temperature replicas sample sequences up to 10 kcal/mol above the GMEC, providing thermal averages and a solution to the inverse protein folding problem. (10.1002/jcc.24393)
  DOI : 10.1002/jcc.24393
• PSSweb: protein structural statistics web server
  
  • Gaillard Thomas
  • Stote Roland H
  • Dejaegere Annick
  Nucleic Acids Research, Oxford University Press, 2016, 44 (W1), pp.W401 - W405. With the increasing number of protein structures available, there is a need for tools capable of automating the comparison of ensembles of structures, a common requirement in structural biology and bioinformatics. PSSweb is a web server for protein structural statistics. It takes as input an ensemble of PDB files of protein structures, performs a multiple sequence alignment and computes structural statistics for each position of the alignment. Different optional functionalities are proposed: structure superposition, Cartesian coordinate statistics, dihedral angle calculation and statistics, and a cluster analysis based on dihedral angles. An interactive report is generated, containing a summary of the results, tables, figures and 3D visualization of superposed structures. (10.1093/nar/gkw332)
  DOI : 10.1093/nar/gkw332
• Concepts and protocols for electrostatic free energies
  
  • Simonson Thomas
  • Roux Benoît
  Molecular Simulation, Taylor & Francis, 2016, 42 (13), pp.1090 - 1101. Electrostatic free energies play an essential role in numerous biomolecular processes occurring in solution. Difficulties arise when the long-range Coulomb interaction is computed for idealised infinite simulation models with periodic boundary conditions. To maintain a neutral simulation box and a finite per-box energy, a neutralising charge density or 'gellium' is commonly used, leading to amean box potential that is constrained to be rigorously equal to zero at all times. Thus, in considering quantities such as ion solvation free energy, the potential drop to move from solvent into the usual, gas phase reference state is missing. In fact, for an infinite molecular system, the electrostatic potential itself is not uniquely defined, but takes the form of an infinite series that is only conditionally convergent. This leads to several possible computational conventions that give different values for the potential and field, all mathematically valid. For experimentally measurable quantities, however, unique results are obtained when sufficiently large simulation boxes are utilised. These concepts are detailed, as well as a fundamental, linear response theoretical framework that provides qualitative understanding of the physical processes involved, especially dielectric relaxation of the environment in response to a new solute charge. Illustrative applications to ligand binding and biomolecular electron transfer are described. (10.1080/08927022.2015.1121544)
  DOI : 10.1080/08927022.2015.1121544
• Studies of the B-Z Transition of DNA: The Temperature Dependence of the Free-Energy Difference, the Composition of the Counterion Sheath in Mixed Salt, and the Preparation of a Sample of the 5 '-d[T-(m(5)C-G)(12)-T] Duplex in Pure B-DNA or Z-DNA form
  
  • Guéron Maurice
  • Plateau Pierre
  • Filoche Marcel
  Biopolymers, Wiley, 2016, 105 (7), pp.369-384. It is often envisioned that cations might coordinate at specific sites of nucleic acids and play an important structural role, for instance in the transition between B-DNA and Z-DNA. However, nucleic acid models explicitly devoid of specific sites may also exhibit features previously considered as evidence for specific binding. Such is the case of the "composite cylinder" (or CC) model which spreads out localized features of DNA structure and charge by cylindrical averaging, while sustaining the main difference between the B and Z structures, namely the better immersion of the B-DNA phosphodiester charges in the solution. Here, we analyze the non-electrostatic component of the free-energy difference between B-DNA and Z-DNA. We also compute the composition of the counterion sheath in a wide range of mixed-salt solutions and of temperatures: in contrast with the large difference of composition between the B-DNA and Z-DNA forms, the temperature dependence of sheath composition, previously unknown, is very weak. In order to validate the model, the mixed-salt predictions should be compared to experiment. We design a procedure for future measurements of the sheath composition based on Anomalous Small-Angle X-ray Scattering and complemented by P-31 NMR. With due consideration for the kinetics of the B-Z transition and for the capacity of generating at will the B or Z form in a single sample, the 5'-d[T-(m(5)C-G)(12)-T] 26-mer emerges as a most suitable oligonucleotide for this study. Finally, the application of the finite element method to the resolution of the Poisson-Boltzmann equation is described in detail. (C) 2016 Wiley Periodicals, Inc. (10.1002/bip.22824)
  DOI : 10.1002/bip.22824
• Protein side chain conformation predictions with an MMGBSA energy function
  
  • Gaillard Thomas
  • Panel Nicolas
  • Simonson Thomas
  Proteins - Structure, Function and Bioinformatics, Wiley, 2016, 84 (6), pp.803-819. (10.1002/prot.25030)
  DOI : 10.1002/prot.25030
• Aminoacetylation Reaction Catalyzed by Leucyl-tRNA Synthetase Operates via a Self-Assisted Mechanism Using a Conserved Residue and the Aminoacyl Substrate
  
  • Aleksandrov Alexey
  • Palencia Andrés
  • Cusack Stephen
  • Field Martin J
  Journal of Physical Chemistry B, American Chemical Society, 2016, 120 (19), pp.4388-4398. Leucyl-tRNA synthetase catalyzes attachment of leucine amino acid to its cognate tRNA. During the second, aminoacetylation, step of the reaction, the leucyl moiety is transferred from leucyl-adenylate to the terminal A76 adenosine of tRNA. In this work, we have investigated the aminoacetylation step catalyzed by leucyl-tRNA synthase, using ab initio quantum chemical/molecular mechanical hybrid potentials in conjunction with reaction-path-location algorithms and molecular dynamics free energy simulations. We have modeled reaction mechanisms arising from both crystallographic studies and computational work. We invoke various groups as potential proton acceptors namely, the phosphate and leucyl amino groups of leucyl-adenylate, the A76 base of tRNA, and the Asp80 and Glu532 residues of the protein and consider both metal-assisted and metal-free reactions. Free energy calculations indicate that both the phosphate group of leucyl adenylate and Glu532 are not strong bases. This agrees with the results of the quantum chemical/molecular mechanical reaction path calculations which give high free energy barriers for the studied pathways involving these groups. A self-assisted mechanism with the leucyl amino group and Asp80 as proton acceptors is the most likely. Furthermore, in this mechanism the presence of a metal ion coordinated by the phosphate group and Glu532 strongly activates the reaction. (10.1021/acs.jpcb.6b02387)
  DOI : 10.1021/acs.jpcb.6b02387
• Additive CHARMM force field for naturally occurring modified ribonucleotides
  
  • Xu You
  • Vanommeslaeghe Kenno
  • Aleksandrov Alexey
  • Mackerell Alexander D.
  • Nilsson Lennart
  Journal of Computational Chemistry, Wiley, 2016, 37 (10), pp.896-912. More than 100 naturally occurring modified nucleotides have been found in RNA molecules, in particular in tRNAs. We have determined molecular mechanics force field parameters compatible with the CHARMM36 all-atom additive force field for all these modifications using the CHARMM force field parametrization strategy. Emphasis was placed on fine tuning of the partial atomic charges and torsion angle parameters. Quantum mechanics calculations on model compounds provided the initial set of target data, and extensive molecular dynamics simulations of nucleotides and oligonucleotides in aqueous solutions were used for further refinement against experimental data. The presented parameters will allow for computational studies of a wide range of RNAs containing modified nucleotides, including the ribosome and transfer RNAs. (C) 2016 The Authors. Journal of Computational Chemistry Published by Wiley Periodicals, Inc. (10.1002/jcc.24307)
  DOI : 10.1002/jcc.24307
• Solvation of Palladium Clusters in an Ionic Liquid: A QM/MM Molecular Dynamics Study
  
  • Zvereva Elena E.
  • Katsyuba Sergey A.
  • Dyson Paul J.
  • Aleksandrov Alexey V.
  Journal of Physical Chemistry C, American Chemical Society, 2016, 120 (8), pp.4596-4604. (10.1021/acs.jpcc.5b11229)
  DOI : 10.1021/acs.jpcc.5b11229
• Protein Sequence Optimization with a Polarizable Force Field: Insights from PDZ Domains
  
  • Litman Jacob M.
  • Joo Sun Young
  • Hou Titus
  • Lucore Stephen D.
  • Panel Nicolas
  • Simonson Thomas
  • Fuentes Ernesto J.
  • Schnieders Michael J.
  , 2016, 110 (3, supplement 1), pp.345a–346a. (10.1016/j.bpj.2015.11.1859)
  DOI : 10.1016/j.bpj.2015.11.1859
• Redesigning the stereospecificity of tyrosyl-tRNA synthetase
  
  • Simonson Thomas
  • Ye-Lehmann Shixin
  • Palmai Zoltan
  • Amara Najette
  • Wydau-Dematteis Sandra
  • Bigan Erwan
  • Druart Karen
  • Moch Clara
  • Plateau Pierre
  Proteins: Structure, Function, and Genetics, Wiley, 2016, 84 (2), pp.240-253. D-Amino acids are largely excluded from protein synthesis, yet they are of great interest in biotechnology. Unnatural amino acids have been introduced into proteins using engineered aminoacyl-tRNA synthetases (aaRSs), and this strategy might be applicable to D-amino acids. Several aaRSs can aminoacylate their tRNA with a D-amino acid; of these, tyrosyl-tRNA synthetase (TyrRS) has the weakest stereospecificity. We use computational protein design to suggest active site mutations in Escherichia coli TyrRS that could increase its D-Tyr binding further, relative to L-Tyr. The mutations selected all modify one or more sidechain charges in the Tyr binding pocket. We test their effect by probing the aminoacyl-adenylation reaction through pyrophosphate exchange experiments. We also perform extensive alchemical free energy simulations to obtain L-Tyr/D-Tyr binding free energy differences. Agreement with experiment is good, validating the structural models and detailed thermodynamic predictions the simulations provide. The TyrRS stereospecificity proves hard to engineer through charge-altering mutations in the first and second coordination shells of the Tyr ammonium group. Of six mutants tested, two are active towards D-Tyr; one of these has an inverted stereospecificity, with a large preference for D-Tyr. However, its activity is low. Evidently, the TyrRS stereospecificity is robust towards charge rearrangements near the ligand. Future design may have to consider more distant and/or electrically neutral target mutations, and possibly design for binding of the transition state, whose structure however can only be modeled. (10.1002/prot.24972)
  DOI : 10.1002/prot.24972
• Protein:Ligand binding free energies: A stringent test for computational protein design
  
  • Druart Karen
  • Palmai Zoltan
  • Omarjee Eyaz
  • Simonson Thomas
  Journal of Computational Chemistry, Wiley, 2016, 37 (4), pp.404–415. A computational protein design method is extended to allow Monte Carlo simulations where two ligands are titrated into a protein binding pocket, yielding binding free energy differences. These provide a stringent test of the physical model, including the energy surface and sidechain rotamer definition. As a test, we consider tyrosyl-tRNA synthetase (TyrRS), which has been extensively redesigned experimentally. We consider its specificity for its substrate l-tyrosine (l-Tyr), compared to the analogs d-Tyr, p-acetyl-, and p-azido-phenylalanine (ac-Phe, az-Phe). We simulate l- and d-Tyr binding to TyrRS and six mutants, and compare the structures and binding free energies to a more rigorous “MD/GBSA” procedure: molecular dynamics with explicit solvent for structures and a Generalized Born + Surface Area model for binding free energies. Next, we consider l-Tyr, ac- and az-Phe binding to six other TyrRS variants. The titration results are sensitive to the precise rotamer definition, which involves a short energy minimization for each sidechain pair to help relax bad contacts induced by the discrete rotamer set. However, when designed mutant structures are rescored with a standard GBSA energy model, results agree well with the more rigorous MD/GBSA. As a third test, we redesign three amino acid positions in the substrate coordination sphere, with either l-Tyr or d-Tyr as the ligand. For two, we obtain good agreement with experiment, recovering the wildtype residue when l-Tyr is the ligand and a d-Tyr specific mutant when d-Tyr is the ligand. For the third, we recover His with either ligand, instead of wildtype Gln. © 2015 Wiley Periodicals, Inc. (10.1002/jcc.24230)
  DOI : 10.1002/jcc.24230
• Cryo-EM study of start codon selection during archaeal translation initiation
  
  • Coureux Pierre-Damien
  • Lazennec-Schurdevin Christine
  • Monestier Aurianne
  • Larquet Eric
  • Cladiere Lionel
  • Klaholz Bruno
  • Schmitt Emmanuelle
  • Mechulam Yves
  Nature Communications, Nature Publishing Group, 2016, 7. Eukaryotic and archaeal translation initiation complexes have a common structural core comprising e/aIF1, e/aIF1A, the ternary complex (TC, e/aIF2-GTP-Met-tRNAi(Met)) and mRNA bound to the small ribosomal subunit. e/aIF2 plays a crucial role in this process but how this factor controls start codon selection remains unclear. Here, we present cryo-EM structures of the full archaeal 30S initiation complex showing two conformational states of the TC. In the first state, the TC is bound to the ribosome in a relaxed conformation with the tRNA oriented out of the P site. In the second state, the tRNA is accommodated within the peptidyl (P) site and the TC becomes constrained. This constraint is compensated by codon/anticodon base pairing, whereas in the absence of a start codon, aIF2 contributes to swing out the tRNA. This spring force concept highlights a mechanism of codon/anticodon probing by the initiator tRNA directly assisted by aIF2. (10.1038/ncomms13366)
  DOI : 10.1038/ncomms13366