Publications

2017

• Computational protein design : un outil pour l'ingénierie des protéines et la biologie synthétique
  
  • Mignon David
  , 2017. Le « Computational protein design » ou CPD est la recherche des séquences d’acides aminés compatibles avec une structure protéique ciblée. L’objectif est de concevoir une fonction nouvelle et/ou d’ajouter un nouveau comportement. Le CPD est en développement dans de notre laboratoire depuis plusieurs années, avec le logiciel Proteus qui a plusieurs succès à son actif.Notre approche utilise un modèle énergétique basé sur la physique et s’appuie sur la différence d’énergie entre l’état plié et l’état déplié de la protéine. Au cours de cette thèse, nous avons enrichi Proteus sur plusieurs points, avec notamment l’ajout d’une méthode d’exploration Monte Carlo avec échange de répliques ou REMC. Nous avons comparé trois méthodes stochastiques pour l’exploration de l’espace de la séquence : le REMC, le Monte Carlo simple et une heuristique conçue pour le CPD, le «Multistart Steepest Descent » ou MSD. Ces comparaisons portent sur neuf protéines de trois familles de structures : SH2, SH3 et PDZ. En utilisant les techniques d’exploration ci-dessus, nous avons été en mesure d’identifier la conformation du minimum global d’énergie ou GMEC pour presque tous les tests dans lesquels jusqu’à 10 positions de la chaîne polypeptidique étaient libres de muter (les autres conservant leurs types natifs). Pour les tests avec 20 positions libres de muter, le GMEC a été identifié dans 2/3 des cas. Globalement, le REMC et le MSD donnent de très bonnes séquences en termes d’énergie, souvent identiques ou très proches du GMEC. Le MSD a obtenu les meilleurs résultats sur les tests à 30 positions mutables. Le REMC avec huit répliques et des paramètres optimisés a donné le plus souvent le meilleur résultat lorsque toutes les positions peuvent muter. De plus, comparé à une énumération exacte des séquences de faible énergie, le REMC fournit un échantillon de séquences de grande diversité.Dans la seconde partie de ce travail, nous avons testé notre modèle pour la conception de domaines PDZ. Pour l’état plié,nous avons utilisé deux variantes d’un modèle de solvant GB. La première utilise une frontière diélectrique protéine/solvant effective moyenne ; la seconde, plus rigoureuse, utilise une frontière exacte qui fluctue le long de la trajectoire MC. Pour caractériser l’état déplié, nous utilisons un ensemble de potentiels chimiques d’acide aminé ou énergies de références. Ces énergies de références sont déterminées par maximisation d’une fonction de vraisemblance afin de reproduire les fréquences d’acides aminés des domaines PDZ naturels. Les séquences conçues par Proteus ont été comparées aux séquences naturelles. Nos séquences sont globalement similaires aux séquences Pfam, au sens des scoresBLOSUM40, avec des scores particulièrement élevés pour les résidus au cœur de la protéine. La variante de GB la plus rigoureuse donne toujours des séquences similaires à des homologues naturels modérément éloignés et l’outil de reconnaissance de plis Super family appliqué à ces séquences donne une reconnaissance parfaite. Nos séquences ont également été comparées à celles du logiciel Rosetta. La qualité, selon les mêmes critères que précédemment, est très comparable, mais les séquences Rosetta présentent moins de mutations que les séquences Proteus.
• Computational design of fully overlapping coding schemes for protein pairs and triplets
  
  • Opuu Vaitea
  • Silvert Martin
  • Simonson Thomas
  Scientific Reports, Nature Publishing Group, 2017, 7 (1). Gene pairs that overlap in their coding regions are rare except in viruses. They may occur transiently in gene creation and are of biotechnological interest. We have examined the possibility to encode an arbitrary pair of protein domains as a dual gene, with the shorter coding sequence completely embedded in the longer one. For 500 × 500 domain pairs (X, Y), we computationally designed homologous pairs (X′, Y′) coded this way, using an algorithm that provably maximizes the sequence similarity between (X′, Y′) and (X, Y). Three schemes were considered, with X′ and Y′ coded on the same or complementary strands. For 16% of the pairs, an overlapping coding exists where the level of homology of X′, Y′ to the natural proteins represents an E-value of 10 −10 or better. Thus, for an arbitrary domain pair, it is surprisingly easy to design homologous sequences that can be encoded as a fully-overlapping gene pair. The algorithm is general and was used to design 200 triple genes, with three proteins encoded by the same DNA segment. The ease of design suggests overlapping genes may have occurred frequently in evolution and could be readily used to compress or constrain artificial genomes. (10.1038/s41598-017-16221-8)
  DOI : 10.1038/s41598-017-16221-8
• Introduction to computational protein design and accurate energy models
  
  • Simonson Thomas
  , 2017.
• Crystallographers rescued by HDX-MS: integrative structural biology to study enzymes modifying factors involved in translation
  
  • Graille Marc
  , 2017.
• Structural and functional insights into eukaryotic methyltransferase complexes modifying various RNAs
  
  • Graille Marc
  • Létoquart Juliette
  • Bourgeois Gabrielle
  • Tran N.V.
  • Roychowdhury Amlan
  , 2017.
• Étude computationnelle du domaine PDZ de Tiam1
  
  • Panel Nicolas
  , 2017. Les interactions protéine-protéine sont souvent contrôlées par de petits domaines protéiques qui régulent les chemins de signalisation au sein des cellules eucaryotes. Les domaines PDZ sont parmi les domaines les plus répandus et les plus étudiés. Ils reconnaissent spécifiquement les 4 à 10 acides aminés C-terminaux de leurs partenaires. Tiam1 est un facteur d'échange de GTP de la protéine Rac1 qui contrôle la migration et la prolifération cellulaire et dont le domaine PDZ lie les protéines Syndecan-1 (Sdc1), Caspr4 et Neurexine. Des petits peptides ou des molécules peptidomimétiques peuvent potentiellement inhiber ou moduler son activité et être utilisés à des fins thérapeutiques. Nous avons appliqué des approches de dessin computationnel de protéine (CPD) et de calcul d'énergie libre par simulations dynamique moléculaire (DM) pour comprendre et modifier sa spécificité. Le CPD utilise un modèle structural et une fonction d'énergie pour explorer l'espace des séquences et des structures et identifier des variants protéiques ou peptidiques stables et fonctionnels. Nous avons utilisé le programme de CPD Proteus, développé au laboratoire, pour redessiner entièrement le domaine PDZ de Tiam1. Les séquences générées sont similaires à celles des domaines PDZ naturels, avec des scores de similarité et de reconnaissance de pli comparables au programme Rosetta, un outil de CPD très utilisé. Des séquences contenant environ 60 positions mutées sur 90, ont été testées par simulations de DM et des mesures biophysiques. Quatre des cinq séquences testées expérimentalement (par nos collaborateurs) montrent un dépliement réversible autour de 50°C. Proteus a également déterminer correctement la spécificité de la liaison de quelques variants protéiques et peptidiques. Pour étudier plus finement la spécificité, nous avons paramétré un modèle d'énergie libre semi-empirique de Poisson-Boltzmann ayant la forme d'une énergie linéaire d'interaction, ou PB/LIE, appliqué à des conformations issues de simulations de DM en solvant explicite de complexes PDZ:peptide. Avec trois paramètres ajustables, le modèle reproduit correctement les affinités expérimentales de 41 variants, avec une erreur moyenne absolue de 0,4~kcal/mol, et donne des prédictions pour 10 nouveaux variants. Le modèle PB/LIE a ensuite comparé à la méthode non-empirique de calcul d'énergie libre par simulations alchimiques, qui n'a pas de paramètre ajustable et qui prédit correctement l'affinité de 12 complexes Tiam1:peptide. Ces outils et les résultats obtenus devraient nous permettre d'identifier des peptides inhibiteurs et auront d'importantes retombées pour l'ingénierie des interactions PDZ:peptide.
• A unique surface on Pat1 C-terminal domain directly interacts with Dcp2 decapping enzyme and Xrn1 5′–3′ mRNA exonuclease in yeast
  
  • Charenton Clément
  • Gaudon-Plesse Claudine
  • Fourati Zaineb
  • Taverniti Valério
  • Back Régis
  • Kolesnikova Olga
  • Séraphin Bertrand
  • Graille Marc
  Proceedings of the National Academy of Sciences of the United States of America, National Academy of Sciences, 2017, 114 (45), pp.E9493-E9501. The Pat1 protein is a central player of eukaryotic mRNA decay that has also been implicated in translational control. It is commonly considered a central platform responsible for the recruitment of several RNA decay factors. We demonstrate here that a yeast-specific C-terminal region from Pat1 interacts with several short motifs, named helical leucine-rich motifs (HLMs), spread in the long C-terminal region of yeast Dcp2 decapping enzyme. Structures of Pat1-HLM complexes reveal the basis for HLM recognition by Pat1. We also identify a HLM present in yeast Xrn1, the main 5'-3' exonuclease involved in mRNA decay. We show further that the ability of yeast Pat1 to bind HLMs is required for efficient growth and normal mRNA decay. Overall, our analyses indicate that yeast Pat1 uses a single binding surface to successively recruit several mRNA decay factors and show that interaction between those factors is highly polymorphic between species. (10.1073/pnas.1711680114)
  DOI : 10.1073/pnas.1711680114
• Simple models for nonpolar solvation: Parameterization and testing
  
  • Michael Eleni
  • Polydorides Savvas
  • Simonson Thomas
  • Archontis Georgios
  Journal of Computational Chemistry, Wiley, 2017, 38 (29), pp.2509-2519. Implicit solvent models are important for many biomolecular simulations. The polarity of aqueous solvent is essential and qualitatively captured by continuum electrostatics methods like Generalized Born (GB). However, GB does not account for the solvent-induced interactions between exposed hydrophobic sidechains or solute-solvent dispersion interactions. These "nonpolar" effects are often modeled through surface area (SA) energy terms, which lack realism, create mathematical singularities, and have a many-body character. We have explored an alternate, Lazaridis-Karplus (LK) gaussian energy density for nonpolar effects and a dispersion (DI) energy term proposed earlier, associated with GB electrostatics. We parameterized several combinations of GB, SA, LK, and DI energy terms, to reproduce 62 small molecule solvation free energies, 387 protein stability changes due to point mutations, and the structures of 8 protein loops. With optimized parameters, the models all gave similar results, with GBLK and GBDILK giving no performance loss compared to GBSA, and mean errors of 1.7 kcal/mol for the stability changes and 2 Å deviations for the loop conformations. The optimized GBLK model gave poor results in MD of the Trpcage mini-protein, but parameters optimized specifically for MD performed well for Trpcage and three other small proteins. Overall, the LK and DI nonpolar terms are valid alternatives to SA treatments for a range of applications. (10.1002/jcc.24910)
  DOI : 10.1002/jcc.24910
• On the Relation between Chemical Oscillations and Self-Replication
  
  • Bigan Erwan
  • Plateau Pierre
  Artificial Life, Massachusetts Institute of Technology Press (MIT Press), 2017, 23 (4), pp.453-480. One proposed scenario for the emergence of biochemical oscillations is that they may have provided the basic mechanism behind cellular self-replication by growth and division. However, alternative scenarios not requiring any chemical oscillation have also been proposed. Each of the various protocell models proposed to support one or another scenario comes with its own set of specific assumptions, which makes it difficult to ascertain whether chemical oscillations are required or not for cellular self-replication. This article compares these two cases within a single whole-cell model framework. This model relies upon a membrane embedding a chemical reaction network (CRN) synthesizing all the cellular constituents, including the membrane, by feeding from an external nutrient. Assuming the osmolarity is kept constant, the system dynamics are governed by a set of nonlinear differential equations coupling the chemical concentrations and the surface-area-to-volume ratio. The resulting asymptotic trajectories are used to determine the cellular shape by minimizing the membrane bending energy (within an approximate predefined family of shapes). While the stationary case can be handled quite generally, the oscillatory one is investigated using a simple oscillating CRN example, which is used to identify features that are expected to hold for any network. It is found that cellular self-replication can be reached with or without chemical oscillations, and that a requirement common to both stationary and oscillatory cases is that a minimum spontaneous curvature of the membrane is required for the cell to divide once its area and volume are both doubled. The oscillatory case can result in a greater variety of cellular shape trajectories but raises additional constraints for cellular division and self-replication: (i) the ratio of doubling time to oscillation period should be an integer, and (ii) if the oscillation amplitude is sufficiently high, then the spontaneous curvature must be below a maximum value to avoid early division before the end of the cycle. Because of these additional stringent constraints, it is likely that early protocells did not rely upon chemical oscillations. Biochemical oscillations typical of modern evolved cells may have emerged later through evolution for other reasons (e.g., metabolic advantage) and must have required additional feedback mechanisms for such a self-replicating system to be robust against even slight environmental variations (e.g., temperature fluctuations). (10.1162/ARTL_a_00241)
  DOI : 10.1162/ARTL_a_00241
• Comparing pairwise-additive and many-body generalized Born models for acid/base calculations and protein design
  
  • Villa Francesco
  • Mignon David
  • Polydorides Savvas
  • Simonson Thomas
  Journal of Computational Chemistry, Wiley, 2017, 38 (28), pp.2396-2410. Generalized Born (GB) solvent models are common in acid/base calculations and protein design. With GB, the interaction between a pair of solute atoms depends on the shape of the protein/solvent boundary and, therefore, the positions of all solute atoms, so that GB is a many-body potential. For compute-intensive applications, the model is often simplified further, by introducing a mean, native-like protein/solvent boundary, which removes the many-body property. We investigate a method for both acid/base calculations and protein design that uses Monte Carlo simulations in which side chains can explore rotamers, bind/release protons, or mutate. The fluctuating protein/solvent dielectric boundary is treated in a way that is numerically exact (within the GB framework), in contrast to a mean boundary. Its originality is that it captures the many-body character while retaining the residue-pairwise complexity given by a fixed boundary. The method is implemented in the Proteus protein design software. It yields a slight but systematic improvement for acid/base constants in nine proteins and a significant improvement for the computational design of three PDZ domains. It eliminates a source of model uncertainty, which will facilitate the analysis of other model limitations. (10.1002/jcc.24898)
  DOI : 10.1002/jcc.24898
• Take off your cap: coordinated recruitment of enzymes for eukaryotic mRNA decay
  
  • Graille Marc
  , 2017.
• Full Protein Sequence Redesign with an MMGBSA Energy Function
  
  • Gaillard Thomas
  • Simonson Thomas
  Journal of Chemical Theory and Computation, American Chemical Society, 2017, 13 (10), pp.4932-4943. Computational protein design aims to create proteins with novel properties. A key element is the energy or scoring function used to select the sequences and conformations. We study the performance of an "MMGBSA" energy function, which combines molecular mechanics terms, a generalized Born and surface area (GBSA) solvent model, with approximations that make the model pairwise additive. Our approach is implemented in the Proteus software. The use of a physics-based energy function ensures a certain model transferability and explanatory power. As a first test, we redesign the sequence of nine proteins, one position at a time, with the rest of the protein having its native sequence and crystallographic conformation. As a second test, all positions are designed together. The contributions of individual energy terms are evaluated, and various parametrizations are compared. We find that the GB term significantly improves the results compared to simple Coulomb electrostatics but is affected by pairwise decomposition errors when all positions are designed together. The SA term, with distinct energy coefficients for nonpolar and polar atoms, makes a decisive contribution to obtain realistic protein sequences and can partially compensate for the absence of a GB term. With the best GBSA protocol, we obtain nativelike protein cores and Superfamily recognition of almost all of our sequences. (10.1021/acs.jctc.7b00202)
  DOI : 10.1021/acs.jctc.7b00202
• A Simple PB/LIE Free Energy Function Accurately Predicts the Peptide Binding Specificity of the Tiam1 PDZ Domain
  
  • Panel Nicolas
  • Sun Young Joo
  • Fuentes Ernesto
  • Simonson Thomas
  Frontiers in Molecular Biosciences, Frontiers Media, 2017, 4. PDZ domains generally bind short amino acid sequences at the C-terminus of target proteins, and short peptides can be used as inhibitors or model ligands. Here, we used experimental binding assays and molecular dynamics simulations to characterize 51 complexes involving the Tiam1 PDZ domain and to test the performance of a semi-empirical free energy function. The free energy function combined a Poisson-Boltzmann (PB) continuum electrostatic term, a van der Waals interaction energy, and a surface area term. Each term was empirically weighted, giving a Linear Interaction Energy or "PB/LIE" free energy. The model yielded a mean unsigned deviation of 0.43 kcal/mol and a Pearson correlation of 0.64 between experimental and computed free energies, which was superior to a Null model that assumes all complexes have the same affinity. Analyses of the models support several experimental observations that indicate the orientation of the α 2 helix is a critical determinant for peptide specificity. The models were also used to predict binding free energies for nine new variants, corresponding to point mutants of the Syndecan1 and Caspr4 peptides. The predictions did not reveal improved binding; however, they suggest that an unnatural amino acid could be used to increase protease resistance and peptide lifetimes in vivo. The overall performance of the model should allow its use in the design of new PDZ ligands in the future. (10.3389/fmolb.2017.00065)
  DOI : 10.3389/fmolb.2017.00065
• Recognition of l-β-homomethionine by methionyl-trna synthetase
  
  • Nigro Giuliano
  • Schmitt Emmanuelle
  • Marlière Philippe
  • Mechulam Yves
  , 2017.
• The role of a trimeric coiled coil protein in WASH complex assembly
  
  • Visweshwaran Sai Prasanna
  , 2017. The Arp2/3 complex generates branched actin networks, which produces a pushing force that helps the cell to remodel its membranes. The WASH complex activates the Arp2/3 complex at the surface of endosomes and thereby, facilitates the membrane scission of the transport intermediates containing internalized receptors such as α5β1 integrins. Hence, by promoting integrin recycling, the WASH complex plays a crucial role in tumor cell invasion during cancer progression. However, how cells assemble the WASH complex at first is unknown. Here we report the identification of the first assembly factor of the WASH complex. We identified HSBP1 in a proteomics screen for proteins binding to precursor forms of subunits, but not to the fully assembled WASH complex. Through biochemical reconstitution and molecular modeling, we found that HSBP1 associates with the precursor CCDC53 trimer, dissociates it and forms a heterotrimer that will eventually contribute a single CCDC53 molecule to the assembling WASH complex. The role of HSBP1 in WASH complex assembly is well conserved since WASH is similarly destabilized upon HSBP1 knock-down in mammalian cells or upon HSBP1 knock-out in Dictyostelium amoeba. In line with the defective assembly of the WASH complex, the HSBP1 knock-out closely phenocopies WASH knock-out in amoeba. In human mammary carcinoma cells, HSBP1 depletion results in impaired integrin recycling to the plasma membrane leading to the defective development of focal adhesions and reduced invasion abilities. Moreover, HSBP1 was found to localize at the centrosome and was required for the polarization associated with the migration. On the other end, in mammary breast tumors, we found that HSBP1 was often overexpressed and that its overexpression was associated with increased levels of the WASH complex and with poor prognosis for breast cancer patients. Hence, HSBP1 is a conserved assembly factor that controls the levels of the WASH complex.
• Study of trm112, a unique methyltransferase activator at the interface between ribosome synthesis and function
  
  • Tran Van Nhan
  , 2017. Methylation is a widely distributed modification found in a variety of substrates involved in different steps of eukaryotic protein translation. Methylation reactions are catalyzed by enzymes called methyltransferases (MTases) generally using S-adenosyl-L- methionine (SAM or AdoMet) as the methyl donor. The effects of methylation on translation are perfectly illustrated by the Trm112 protein, which is an activating platform, essential for the function of four SAM-dependent MTases (Trm9, Trm11, Bud23 and Mtq2) modifying factors participated in protein synthesis. The Trm9-Trm112 and Trm11-Trm112 complexes methylate some tRNAs to form mcm5U34 and m2G10 respectively. The Bud23-Trm112 complex modifies 18S rRNA to form m7G1715 while the Mtq2-Trm112 complex methylates class I translation termination factor eRF1 at glutamine side chain of GGQ motif. Until now, the study of Trm112 network in eukaryotes has been quite clear structurally and functionally, however, little is known for corresponding proteins in Archaea.My PhD project aims to characterize the Trm112 network in archaea using Haloferax volcanii as a model organism and to decipher the mechanisms of substrate modification by Trm112-MTase complexes. This will help understanding the roles of these enzymes in protein synthesis from an evolutionary point of view.Towards this goal, I have generated several H. volcanii strains (Δtrm112, Δtrm112 Trm112-Flag, …). Co-immunoprecipitation of Trm112-Flag coupled to mass spectrometry allowed me identifying a significant number of methyltransferases (MTases), including putative orthologues of eukaryotic Trm112 partners, as potential interactors. I have next validated these new partners by biochemical approaches (co-purification, enzymatic assays, …) and determined the crystal structure for one Trm112-MTase complex. I have then convincing evidences that H. volcanii Trm12 has more MTase partners than the eukaryotic one. My work opens new routes towards the characterization of the role of Trm112 in archaea but has also led to the identification of a new MTase partner of the eukaryotic Trm112.
• Structural and functional studies of four Trm112-Methyltransferase holoenzymes modifying RNA and proteins involved in translation
  
  • Graille Marc
  , 2017.
• Recent advances in the mechanism of selenoamino acids toxicity in eukaryotic cells
  
  • Lazard Myriam
  • Dauplais Marc
  • Blanquet Sylvain
  • Plateau Pierre
  Biomolecular concepts, De Gruyter, 2017, 8 (2), pp.93-104. Selenium is an essential trace element due to its incorporation into selenoproteins with important biological functions. However, at high doses it is toxic. Selenium toxicity is generally attributed to the induction of oxidative stress. However, it has become apparent that the mode of action of seleno-compounds varies, depending on its chemical form and speciation. Recent studies in various eukaryotic systems, in particular the model organism Saccharomyces cerevisiae, provide new insights on the cytotoxic mechanisms of selenomethionine and selenocysteine. This review first summarizes current knowledge on reactive oxygen species (ROS)-induced genotoxicity of inorganic selenium species. Then, we discuss recent advances on our understanding of the molecular mechanisms of selenocysteine and selenomethionine cytotoxicity. We present evidences indicating that both oxidative stress and ROS-independent mechanisms contribute to selenoamino acids cytotoxicity. These latter mechanisms include disruption of protein homeostasis by selenocysteine misincorporation in proteins and/or reaction of selenols with protein thiols. (10.1515/bmc-2017-0007)
  DOI : 10.1515/bmc-2017-0007
• PSSweb: protein structural statistics web server
  
  • Gaillard Thomas
  • Stote R.H.
  • Dejaegere A.
  , 2017. 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. The server is available at http://pssweb.org.
• The structure of an E. coli tRNA f Met A 1-U 72 variant shows an unusual conformation of the A 1-U 72 base pair
  
  • Monestier Auriane
  • Aleksandrov Alexey
  • Coureux Pierre-Damien
  • Panvert Michel
  • Mechulam Yves
  • Schmitt Emmanuelle
  RNA, Cold Spring Harbor Laboratory Press, 2017, 23 (5), pp.673-682. Translation initiation in eukaryotes and archaea involves a methionylated initiator tRNA delivered to the ribosome in a ternary complex with e/aIF2 and GTP. Eukaryotic and archaeal initiator tRNAs contain a highly conserved A 1-U 72 base pair at the top of the acceptor stem. The importance of this base pair to discriminate initiator tRNAs from elongator tRNAs has been established previously using genetics and biochemistry. However, no structural data illustrating how the A 1-U 72 base pair participates in the accurate selection of the initiator tRNAs by the translation initiation systems are available. Here, we describe the crystal structure of a mutant E. coli initiator tRNA f Met A 1-U 72 , aminoacylated with methionine, in which the C 1 : A 72 mismatch at the end of the tRNA acceptor stem has been changed to an A 1-U 72 base pair. Sequence alignments show that the mutant E. coli tRNA is a good mimic of archaeal initiator tRNAs. The crystal structure, determined at 2.8 Å resolution, shows that the A 1-U 72 pair adopts an unusual arrangement. A 1 is in a syn conformation and forms a single H-bond interaction with U 72. This interaction requires protonation of the N1 atom of A 1. Moreover, the 5 ′ phosphoryl group folds back into the major groove of the acceptor stem and interacts with the N7 atom of G 2. A possible role of this unusual geometry of the A 1-U 72 pair in the recognition of the initiator tRNA by its partners during eukaryotic and archaeal translation initiation is discussed. (10.1261/rna.057877.116)
  DOI : 10.1261/rna.057877.116
• Computational Design of the Tiam1 PDZ Domain and Its Ligand Binding
  
  • Mignon David
  • Panel Nicolas
  • Chen Xingyu
  • Fuentes Ernesto
  • Simonson Thomas
  Journal of Chemical Theory and Computation, American Chemical Society, 2017, 13 (5), pp.2271-2289. PDZ domains direct protein-protein interactions and serve as models for protein design. Here, we optimized a protein design energy function for the Tiam1 and Cask PDZ domains that combines a molecular mechanics energy, Generalized Born solvent, and an empirical unfolded state model. Designed sequences were recognized as PDZ domains by the Superfamily fold recognition tool and had similarity scores comparable to natural PDZ sequences. The optimized model was used to redesign the two PDZ domains, by gradually varying the chemical potential of hydrophobic amino acids; the tendency of each position to lose or gain a hydrophobic character represents a novel hydrophobicity index. We also redesigned four positions in the Tiam1 PDZ domain involved in peptide binding specificity. The calculated affinity differences between designed variants reproduced experimental data and suggest substitutions with altered specificities. (10.1021/acs.jctc.6b01255)
  DOI : 10.1021/acs.jctc.6b01255
• The Arp2/3 inhibitory protein Arpin is dispensable for chemotaxis
  
  • Dang Irene
  • Linkner Joern
  • Yan Jun
  • Irimia Daniel
  • Faix Jan
  • Gautreau Alexis
  Biology of the Cell, Wiley, 2017, 109 (4), pp.162-166. Arpin is an Arp2/3 inhibitory protein, which decreases the protrusion lifetime and hence directional persistence in the migration of diverse cells. Arpin is activated by the small GTPase Rac, which controls cell protrusion, thus closing a negative feedback loop that renders the protrusion intrinsically unstable. Because of these properties, it was proposed that Arpin might play a role in directed migration, where directional persistence has to be fine-tuned. We report here, however, that Arpin-depleted tumour cells and Arpin knock-out Dictyostelium amoeba display no obvious defect in chemotaxis. These results do not rule out a potential role of Arpin in other systems, but argue against a general role of Arpin in chemotaxis. (10.1111/boc.201600064)
  DOI : 10.1111/boc.201600064
• Exposure to selenomethionine causes selenocysteine misincorporation and protein aggregation in Saccharomyces cerevisiae.
  
  • Plateau Pierre
  • Saveanu Cosmin
  • Lestini Roxane
  • Dauplais Marc
  • Decourty Laurence
  • Jacquier Alain
  • Blanquet Sylvain
  • Lazard Myriam
  Scientific Reports, Nature Publishing Group, 2017, 7, pp.44761. Selenomethionine, a dietary supplement with beneficial health effects, becomes toxic if taken in excess. To gain insight into the mechanisms of action of selenomethionine, we screened a collection of ≈5900 Saccharomyces cerevisiae mutants for sensitivity or resistance to growth-limiting amounts of the compound. Genes involved in protein degradation and synthesis were enriched in the obtained datasets, suggesting that selenomethionine causes a proteotoxic stress. We demonstrate that selenomethionine induces an accumulation of protein aggregates by a mechanism that requires de novo protein synthesis. Reduction of translation rates was accompanied by a decrease of protein aggregation and of selenomethionine toxicity. Protein aggregation was supressed in a ∆cys3 mutant unable to synthetize selenocysteine, suggesting that aggregation results from the metabolization of selenomethionine to selenocysteine followed by translational incorporation in the place of cysteine. In support of this mechanism, we were able to detect random substitutions of cysteinyl residues by selenocysteine in a reporter protein. Our results reveal a novel mechanism of toxicity that may have implications in higher eukaryotes. (10.1038/srep44761)
  DOI : 10.1038/srep44761
• DNA binding specificities of Escherichia coli Cas1–Cas2 integrase drive its recruitment at the CRISPR locus
  
  • Moch Clara
  • Fromant Michel
  • Blanquet Sylvain
  • Plateau Pierre
  Nucleic Acids Research, Oxford University Press, 2017, 45 (5), pp.2714-2723. Prokaryotic adaptive immunity relies on the capture of fragments of invader DNA (protospacers) followed by their recombination at a dedicated acceptor DNA locus. This integrative mechanism, called adaptation, needs both Cas1 and Cas2 proteins. Here, we studied in vitro the binding of an Escherichia coli Cas1-Cas2 complex to various protospacer and acceptor DNA molecules. We show that, to form a long-lived ternary complex containing Cas1-Cas2, the acceptor DNA must carry a CRISPR locus, and the protospacer must not contain 3΄-single-stranded overhangs longer than 5 bases. In addition, the acceptor DNA must be supercoiled. Formation of the ternary complex is synergistic, in such that the binding of Cas1-Cas2 to acceptor DNA is reinforced in the presence of a protospacer. Mutagenesis analysis at the CRISPR locus indicates that the presence in the acceptor plasmid of the palindromic motif found in CRISPR repeats drives stable ternary complex formation. Most of the mutations in this motif are deleterious even if they do not prevent cruciform structure formation. The leader sequence of the CRISPR locus is fully dispensable. These DNA binding specificities of the Cas1-Cas2 integrase are likely to play a major role in the recruitment of this enzyme at the CRISPR locus. (10.1093/nar/gkw1309)
  DOI : 10.1093/nar/gkw1309
• Equivalence of M- and P-Summation in Calculations of Ionic Solvation Free Energies
  
  • Simonson Thomas
  • Hummer Gerhard
  • Roux Benoît
  Journal of Physical Chemistry A, American Chemical Society, 2017, 121 (7), pp.1525-1530. Condensed-phase simulations commonly use periodic boundary conditions (PBCs) to represent the thermodynamic limit. For the vapor to liquid transfer of an ion, the gas/liquid boundary and its associated potential change are then missing. Furthermore, the electric potential and field at a given point are given by conditionally convergent infinite series, for which different summation schemes give different results. Nevertheless, standard simulation protocols can be used to compute experimental quantities unambiguously. In particular, using an auxiliary test particle and a multistep solvation path, we show that particle-based, Ewald, and common molecule-based summation schemes for the potential and field are all essentially equivalent. However, all methods require prior knowledge of the gas/liquid boundary potential to compute ionic solvation free energies using PBC protocols for both force-field and quantum-mechanical models. (10.1021/acs.jpca.6b12691)
  DOI : 10.1021/acs.jpca.6b12691