Publications

2018

• mRNA decapping: finding the right structures
  
  • Charenton Clément
  • Graille Marc
  Philosophical Transactions of the Royal Society B: Biological Sciences, Royal Society, The, 2018, 373 (1762), pp.20180164. In eukaryotes, the elimination of the m7GpppN mRNA cap, a process known as decapping, is a critical, largely irreversible and highly regulated step of mRNA decay that withdraws the targeted mRNAs from the pool of translatable templates. The decapping reaction is catalysed by a multi-protein complex formed by the Dcp2 catalytic subunit and its Dcp1 cofactor, a holoenzyme that is poorly active on its own and needs several accessory proteins (Lsm1-7 complex, Pat1, Edc1-2, Edc3 and/or EDC4) to be fully efficient. Here, we discuss the several crystal structures of Dcp2 domains bound to various partners (proteins or small molecules) determined in the last couple of years that have considerably improved our current understanding of how Dcp2, assisted by its various activators, is recruited to its mRNA targets and adopts its active conformation upon substrate recognition. We also describe how, over the years, elegant integrative structural biology approaches combined to biochemistry and genetics led to the identification of the correct structure of the active Dcp1-Dcp2 holoenzyme among the many available conformations trapped by X-ray crystallography.This article is part of the theme issue '5' and 3' modifications controlling RNA degradation'. (10.1098/rstb.2018.0164)
  DOI : 10.1098/rstb.2018.0164
• Cyclization Reaction Catalyzed by Cyclodipeptide Synthases Relies on a Conserved Tyrosine Residue
  
  • Schmitt Emmanuelle
  • Bourgeois Gabrielle
  • Gondry Muriel
  • Aleksandrov Alexey
  Scientific Reports, Nature Publishing Group, 2018, 8 (1), pp.7031. Cyclodipeptide synthases (CDPSs) form various cyclodipeptides from two aminoacyl tRNAs via a stepwise mechanism with the formation of a dipeptidyl enzyme intermediate. As a final step of the catalytic reaction, the dipeptidyl group undergoes intramolecular cyclization to generate the target cyclodipeptide product. In this work, we investigated the cyclization reaction in the cyclodipeptide synthase AlbC using QM/MM methods and free energy simulations. The results indicate that the catalytic Y202 residue is in its neutral protonated form, and thus, is not likely to serve as a general base during the reaction. We further demonstrate that the reaction relies on the conserved residue Y202 serving as a proton relay, and the direct proton transfer from the amino group to S37 of AlbC is unlikely. Calculations reveal that the hydroxyl group of tyrosine is more suitable for the proton transfer than hydroxyl groups of other amino acids, such as serine and threonine. Results also show that the residues E182, N40, Y178 and H203 maintain the correct conformation of the dipeptide needed for the cyclization reaction. The mechanism discovered in this work relies on the amino groups conserved among the entire CDPS family and, thus is expected to be universal among CDPSs. Cyclodipeptide synthases (CDPSs) are a family of enzymes that use aminoacyl-tRNAs (aa-tRNAs) to synthetize cyclodipeptides, which are precursors of many secondary metabolites with diverse biological functions 1,2. The first member of this family was identified in 2002 during characterization of the albonoursin biosynthetic pathway in Streptomyces noursei and called AlbC 3. Three CDPSs are structurally characterized and all three share a common architecture, consisting of a monomer containing a Rossmann fold domain 4-7. The CDPSs display strong structural similarity to the catalytic domains of class Ic aminoacyl tRNA synthetases (aaRSs), suggesting that CDPSs evolved from the class Ic of aaRSs 1. However, there are several significant differences between CDPSs and class Ic aaRSs. The ATP-binding motifs present in aaRSs are not present in CDPSs, since there is no need to activate amino acids in CDPSs, and CDPSs are active as monomers in contrast to the TyrRS and TrpRS that function as homodimers. The catalytic mechanism has been extensively studied experimentally for the structurally characterized CDPSs, and a structure mimicking a reaction intermediate was obtained for AlbC 7. It was demonstrated that AlbC uses Phe-tRNA Phe and Leu-tRNA Leu (or a second Phe-tRNA Phe) as substrates for a ping-pong mechanism involving the formation of two successive acyl-enzyme intermediates 1. The catalytic reaction starts with the binding of the first aa-tRNA and the transfer of its aminoacyl moiety to a conserved serine residue leading to the formation of an aminoacyl enzyme intermediate. For the second step, the tRNA Phe part of the first substrate dissociates from AlbC and a second aa-tRNA binds to the enzyme. The phenylalanyl-AlbC reacts with the second aa-tRNA to form a dipeptidyl-AlbC intermediate. In the final step, the target cyclodipeptide is obtained through intramolecular cyclization. Residues important for the reaction in AlbC were identified through site-directed mutagenesis and chemical biology studies 5,7. These residues, apart from S37, the conserved residue that accepts the aminoacyl group, are Y202, Y178, E182, N40, and H203. The residues Y178 and E182 are involved in the stabilization of the aminoacyl moiety of the first substrate (named Phe1) throughout the catalytic cycle as suggested by the crystal structure of the diphenylalanyl-enzyme intermediate mimic 7. E182 was also suggested to act as a general catalytic base during the formation of the dipeptidyl-enzyme by deprotonating the ammonium group of the aminoacyl-enzyme, (10.1038/s41598-018-25479-5)
  DOI : 10.1038/s41598-018-25479-5
• Multifaceted Study on a Cytochalasin Scaffold: Lessons on Reactivity, Multidentate Catalysis, and Anticancer Properties
  
  • Zaghouani Mehdi
  • Gayraud Oscar
  • Jactel Vincent
  • Prevost Sébastien
  • Dezaire Ambre
  • Sabbah Michèle
  • Escargueil Alexandre E.
  • Lai Thanh-Lan
  • Le clainche Christophe
  • Rocques Nathalie
  • Romero Stéphane
  • Gautreau Alexis
  • Blanchard Florent
  • Frison Gilles
  • Nay Bastien
  Chemistry - A European Journal, Wiley-VCH Verlag, 2018, 24 (62), pp.16686-16691. We report an intramolecular Diels-Alder reaction efficiently accelerated by Schreiner's thiourea, to build a functionalized cytochalasin scaffold (periconiasin series) amenable to biological purpose. DFT calculation highlighted a unique multidentate cooperative hydrogen bonding in this catalysis. The deprotection end-game afforded a collection of diverse structures and showed the peculiar reactivity of the Diels-Alder cycloadducts upon functionalization. Biological studies revealed strong cytotoxicity of a few compounds on breast cancer cell lines, while preserving actin polymerization. (10.1002/chem.201804023)
  DOI : 10.1002/chem.201804023
• Archaea-guided identification of an elusive human rRNA m6A methyltransferase enzyme
  
  • van Tran Nhan
  • Muller Leslie
  • van Fassen Ernst
  • Lestini Roxane
  • Létoquart Juliette
  • de Crecy-Lagard Valerie
  • Lafontaine Denis L. J.
  • Cianferani Sarah
  • Graille Marc
  , 2018.
• tRNA accommodation during archaeal translation initiation
  
  • Coureux Pierre-Damien
  • Lazennec-Schurdevin Christine
  • Monestier Auriane
  • Mechulam Yves
  • Schmitt Emmanuelle
  , 2018, pp.O42.
• Contribution of the Yeast Saccharomyces cerevisiae Model to Understand the Mechanisms of Selenium Toxicity
  
  • Lazard Myriam
  • Dauplais Marc
  • Plateau Pierre
  , 2018, 16, pp.71-87. Selenium (Se) is an essential trace element for mammals. It is involved in redox functions as the amino acid selenocysteine, translationally inserted in the active site of a few proteins. However, at high doses it is toxic and the mechanisms underlying this toxicity are poorly understood. Because of the high level of conservation of its genes and pathways with those of higher organisms and the powerful genetic techniques that it offers, Saccharomyces cerevisiae is an attractive model organism to study the molecular basis of Se toxicity. High-throughput technologies developed in this yeast include genome-wide screening of bar-coded systematic deletion sets, as well as whole-transcriptome, -proteome, and -metabolome analysis. This chapter focuses on the contribution of S. cerevisiae to the understanding of the mechanisms of selenocompound toxicity, combining results from classical biochemistry with genome-wide analyses and more detailed gene-by-gene approaches. Experimental data demonstrate that toxicity is compound specific. Inorganic Se induces DNA damage whereas selenoamino acids cause proteotoxicity. (10.1007/978-3-319-95390-8_4)
  DOI : 10.1007/978-3-319-95390-8_4
• Computer simulations to engineer PDZ-peptide recognition
  
  • Villa Francesco
  , 2018. Protein-protein interactions (PPIs) regulate complex signaling networks in eukaryotic cells. Many binding events between several protein domains transfer information through communication pathways. Disrupting or altering the equilibrium between PPIs plays an important role inseveral diseases and the inibition of targeted PPIs is a recognized strategy for computational drug design. In the present thesis we focused on PDZ domains, which are among the most widespread signaling domains. PDZs recognize the 4-10 C-terminal amino acids of their target proteins as well as the corresponding peptides in isolation. We studied PDZ:peptide binding for the Tiam1 protein, which is a Rac GTP exchange factor involved in neuronal protrusion and axon guidance. Tiam1 activity modulates signaling for cell proliferation and migration, whose dysregulation increases growth of metastatic cancers. Its natural binder peptide is Syndecan1 (Sdc1), composed of 8 amino acids. Its last 5 Cter residues drive interactions in the binding pocket. Experimental affinities for several mutants of Sdc1 and in the protein domain constitute a complete dataset to study many ionic interactions with molecular simulations. These calculations are still challenging, despite the dramatic improvement of biomolecular modelling in the 1990's and 2000's. Upon binding, residues are transferred from a solvent-exposed environment to a solvent-poor one. This is expected to change the electron distribution within residues and nearby solvent molecules. Comparing ligands that differ by one or more ionic side-chain mutations, more sophisticated force fields where electronic polarizability is treated explicitly may be required. We developed and tested both Computational Protein Design (CPD) models and more precise free energy calculation methods based on polarizable molecular dynamics. We developed a general, high-througtput CPD protocol to optimize protein:peptide binding. The model has been implemented in on our in-house CPD package Proteus ( Simonson et al, 2014) and has been tested computing relative binding affinities for many variants of the Tiam1:Sdc1 complex. Monte Carlo sampling of equilibrium distributions of protein sequences is performed using an adaptive bias potential which flattens the energy landscape in sequence space and allows to estimate binding affinities for thousands of protein variants in limited CPU time (~1hour). We also improved our CPD implicit solvent model, implementing a more realistic description of the solute-solvent dielectric boundary. The new method, called Fluctuating Dielectric Boundary (FDB) showed a systematic improvement in the prediction of acid:base constants of several proteins. Promising results were also obtained for the complete sequence redesign of three PDZ domains. In the second part of this work we studied Tiam1:peptide affinities with more sophisticated models, based on free energy simulations with the Drude Polarizable Force field (DrudeFF). We first computed relative binding free energies for charge mutations in the Tiam1:Sdc1 complex, obtaining a clear improvement respect to equivalent calculations performed using two additive force fields. We applied the well-enstablished Dual Topology Approach: to our knowledge, this was the first example of such a calculation for a protein:peptide complex with uses the DrudeFF. Then we went on, developing the Drude polarizable models for methyl phosphate (MP) and phospho tyrosine (pTyr). We were interested in the change in binding affinity associated with phosphorylation of a Tyrosine residue of Sdc1, but Drude pTyr parameters were not yet developed. We tested our new phosphate parameters studying standard binding free energies between MP and magnesium (Mg2+) in water solution. Results showed a good agreement with experiment, improving previous calculations performed using additive force field
• Archaea-guided identification of an elusive human rRNA m6A methyltransferase enzyme
  
  • Graille Marc
  , 2018.
• Variable Neighborhood Search with Cost Function Networks to Solve Large Computational Protein Design Problems
  
  • Allouche David
  • Charpentier Antoine
  • Schiex Thomas
  • Simonson Thomas
  , 2018.
• Recruitment of the mRNA decay machineries to the 5’ end of mRNAs
  
  • Charenton Clément
  • Gaudon Plesse Claudine
  • Taverniti Valério
  • Back Régis
  • Séraphin Bertrand
  • Graille Marc
  , 2018.
• tRNA accommodation during archaeal translation initiation
  
  • Coureux Pierre-Damien
  • Lazennec-Schurdevin Christine
  • Monestier Auriane
  • Schmitt Emmanuelle
  • Mechulam Yves
  , 2018.
• Regulation of the invasion suppressor Arpin by Tankyrases
  
  • Chemeris Angelina
  , 2018. The evolutionarily conserved Arp2/3 complex plays a central role in nucleating the branched actin filament arrays that drive cell migration, endocytosis, and other processes. Recently, an inactivator of the Arp2/3 complex at the lamellipodium tip, a small protein, Arpin, was discovered and characterized. On its C-terminus, Arpin possesses an acidic (A) motif, which is homologous to the A-motif of various Nucleation Promoting Factors (NPFs). It was predicted that Arpin can bind at two binding sites to the Arp2/3 complex, similar to VCA domains of NPFs. Here, we used single particle electron microscopy to obtain a 3D reconstruction of the Arp2/3 complex bound to Arpin at a 25Å resolution. We showed that the binding of Arpin causes the standard open conformational of the Arp2/3 complex. We confirmed that there are two binding sites on the Arp2/3 complex for Arpin: one on the back of the Arp3 subunit, and the second is located between Arp2 and ARPC1 subunits. The distance between the Arp2/3 complex and Arpin (5 nm) supports the view that Arpin interacts with its partner via its unstructured C-terminal acidic tail.Next, using the pull-down assay, we identified the new Arpin binding partners, Tankyrases1/2. Interestingly, Tankyrases and the Arp2/3 complex possess overlapping amino acid sequences at Arpin binding sites. Hence, we demonstrated a competition between the ARC4 domain of Tankyrase1 and the Arp2/3 complex in a dose-dependent manner.To understand the principles of Tankyrases-Arpin interaction, we created a mutant Arpin (ArpinG218D) that lacks its ability to interact with Tankyrases, but not with the Arp2/3 complex in vitro. Interestingly, ArpinG218D was not able to inhibit the Arp2/3 complex in vivo, suggesting that Tankyrase may be necessary for Arpin-Arp2/3 complex interaction. Arpin is the turning factor of migrating cells, so we performed a migration analysis of MCF10-A cells expressing either wild type Arpin (ArpinWT) or mutant ArpinG218D in parallel with the depletion of endogenous Arpin. Cells expressing ArpinG218D had higher directional persistence, similar to the cells where the endogenous Arpin was knocked down. Thus, we suggested that mutant ArpinG218D cannot inactivate the Arp2/3 complex since it is not present at the lamellipodial tip. We compared the amount of protein for both ArpinWT and ArpinG218D in the membrane fraction of the migrating cells. A significant difference (44%) in the amount of ArpinWT and Arpin G218D was consistent with our hypothesis.Tankyrases are therapeutic targets in a variety of cancers, but currently there is no structural model available for these large and flexible proteins. In this work, we obtained for the first time two 3D reconstructions of full-length Tankyrase1 and Tankyrase1 bound to Arpin using single particle electron microscopy. The achieved resolution (27Å) was enough to detect a dramatic conformational change in Tankyrase SAM and PARP domains upon binding of Arpin molecules. In our reconstruction, three Arpins were bound to the ARC1, ARC4 and ARC5 domains of Tankyrase1. ARC5 was shown to be the most flexible part of the ARC cluster.Based on the obtained data, we suggested a model of regulation of the activity of Arpin by Tankyrases. According to our model, Tankyrases bind Arpin in the cytoplasm, change their conformational state and bring Arpin closer to the membrane in the lamellipodia. Deciphering the extracellular signals, Rac GTPase activates Arpin, which sequentially inactivates the Arp2/3 complex, while Tankyrases are released.
• Adaptive landscape flattening in amino acid sequence space for the computational design of protein:peptide binding
  
  • Villa Francesco
  • Panel Nicolas
  • Chen Xingyu
  • Simonson Thomas
  The Journal of Chemical Physics, American Institute of Physics, 2018, 149 (7), pp.072302. For the high throughput design of protein:peptide binding, one must explore a vast space of amino acid sequences in search of low binding free energies. This complex problem is usually addressed with either simple heuristic scoring or expensive sequence enumeration schemes. Far more efficient than enumeration is a recent Monte Carlo approach that adaptively flattens the energy landscape in sequence space of the unbound peptide and provides formally exact binding free energy differences. The method allows the binding free energy to be used directly as the design criterion. We propose several improvements that allow still more efficient sampling and can address larger design problems. They include the use of Replica Exchange Monte Carlo and landscape flattening for both the unbound and bound peptides. We used the method to design peptides that bind to the PDZ domain of the Tiam1 signaling protein and could serve as inhibitors of its activity. Four peptide positions were allowed to mutate freely. Almost 75 000 peptide variants were processed in two simulations of 109 steps each that used 1 CPU hour on a desktop machine. 96% of the theoretical sequence space was sampled. The relative binding free energies agreed qualitatively with values from experiment. The sampled sequences agreed qualitatively with an experimental library of Tiam1-binding peptides. The main assumption limiting accuracy is the fixed backbone approximation, which could be alleviated in future work by using increased computational resources and multi-backbone designs. (10.1063/1.5022249)
  DOI : 10.1063/1.5022249
• Towards NMR characterization of apelin encapsulated in liposomes.
  
  • Doyen Camille
  • Larquet Eric
  • Coureux Pierre-Damien
  • Frances Oriane
  • Herman Frédéric
  • Sablé Serge
  • Burnouf Jean-Pierre
  • Sizun Christina
  • Lescop Ewen
  , 2018.
• Classical Drude Polarizable Force Field Model for Methyl Phosphate and Its Interactions with Mg 2+
  
  • Villa Francesco
  • Mackerell Alexander
  • Roux Benoît
  • Simonson Thomas
  Journal of Physical Chemistry A, American Chemical Society, 2018, 122 (29), pp.6147-6155. Phosphate groups are essential components of nucleic acids and proteins, whose interactions with solvent, metal ions, and ionic side chains help control folding and binding. Methyl phosphate (MP) represents a simple analog of phosphate moieties that are post-translation modifications in proteins and present at the termini of nucleic acids, among other environments. In the present study, we optimized parameters for use in polarizable molecular dynamics simulations of MP in its mono- and dianionic forms, MP- ≡ CH3HPO4- and MP2- ≡ CH3PO42-, along with P i2- ≡ HPO42-, in the context of the classical Drude oscillator model. Parameter optimization was done in a manner consistent with the remainder of the Drude molecular mechanics force field, choosing atomic charges and polarizabilities to reproduce molecular properties from quantum mechanics as well as experimental hydration free energies. Optimized parameters were similar to existing dimethyl phosphate parameters, with a few significant differences. The developed parameters were then used to compute magnesium binding affinities in aqueous solution, using alchemical molecular dynamics free energy simulations. Good agreement with experiment was obtained, and outer sphere binding was shown to be predominant for MP- and MP2-. (10.1021/acs.jpca.8b04418)
  DOI : 10.1021/acs.jpca.8b04418
• Evaluation of AutoDock and AutoDock Vina on the CASF-2013 Benchmark
  
  • Gaillard Thomas
  Journal of Chemical Information and Modeling, American Chemical Society, 2018, 58 (8), pp.1697-1706. (10.1021/acs.jcim.8b00312)
  DOI : 10.1021/acs.jcim.8b00312
• Structural basis for partition of the cyclodipeptide synthases into two subfamilies
  
  • Bourgeois Gabrielle
  • Seguin Jérôme
  • Babin Morgan
  • Belin Pascal
  • Moutiez Mireille
  • Mechulam Yves
  • Gondry Muriel
  • Schmitt Emmanuelle
  Journal of Structural Biology, Elsevier, 2018, 203 (1), pp.17-26. Cyclodipeptide synthases (CDPSs) use two aminoacyl-tRNAs to catalyze the formation of two peptide bonds leading to cyclodipeptides that can be further used for the synthesis of diketopiperazines. It was shown that CDPSs fall into two subfamilies, NYH and XYP, characterized by the presence of specific sequence signatures. However, current understanding of CDPSs only comes from studies of enzymes from the NYH subfamily. The present study reveals the crystal structures of three CDPSs from the XYP subfamily. Comparison of the XYP and NYH enzymes shows that the two subfamilies mainly differ in the first half of their Rossmann fold. This gives a structural basis for the partition of CDPSs into two subfamilies. Despite these differences, the catalytic residues adopt similar positioning regardless of the subfamily suggesting that the XYP and NYH motifs correspond to two structural solutions to facilitate the reactivity of the catalytic serine residue. (10.1016/j.jsb.2018.03.001)
  DOI : 10.1016/j.jsb.2018.03.001
• Accurate PDZ:peptide binding specificity with additive and polarizable free energy simulations
  
  • Simonson Thomas
  , 2018. PDZ domains contain 80-100 amino acids and bind short C-terminal sequences of target proteins. Their specificity is essential for cellular signaling pathways. We studied the binding of the Tiam1 PDZ domain to peptides derived from the C-termini of its Syndecan-1 and Caspr4 targets. We used free energy perturbation (FEP) to characterize the binding energetics of one wild-type and 17 mutant complexes by simulating 21 alchemical transformations between pairs of complexes. Thirteen complexes had known experimental affinities. FEP is a powerful tool to understand protein/ligand binding. It depends, however, on the accuracy of molecular dynamics force fields and conformational sampling. Both aspects require continued testing, especially for ionic mutations. For six mutations that did not modify the net charge, we obtained excellent agreement with experiment using the additive, AMBER ff99SB force field, with a root mean square deviation (RMSD) of 0.37 kcal/mol. For six ionic mutations that modified the net charge, agreement was also good, with one large error (3 kcal/mol) and an RMSD of 0.9 kcal/mol for the other five. The large error arose from the overstabilization of a protein/peptide salt bridge by the additive force field. Four of the ionic mutations were also simulated with the polarizable Drude force field, which represents the first test of this force field for protein/ligand binding free energy changes. The large error was eliminated and the RMS error for the four mutations was reduced from 1.8 to 1.2 kcal/mol. The overall accuracy of FEP indicates it can be used to understand PDZ/peptide binding. Importantly, our results show that for ionic mutations in buried regions, electronic polarization plays a significant role.
• Computational Protein Design with an MMGBSA Energy Function
  
  • Gaillard Thomas
  • Panel Nicolas
  • Mignon David
  • Simonson Thomas
  , 2018, pp.P02. Protein design aims at conceiving new proteins or modifying existing ones to obtain a given function. Computational approaches are a valuable help for protein design, to rationalize the predictions and guide the experimental tests. Computational protein design (CPD) has sparked important methodological efforts and obtained spectacular successes such as the creation of a protein with a new fold or enzyme active site engineering. The main difficulty of CPD lies in the astronomical number of possible sequences and conformations, of the order of (20x10)^100 for a protein with 100 amino acids. Another key element for CPD success is the energy function used to evaluate and select the sequences and conformations. Our approach of CPD is based on an atomic model of the protein structure and a molecular mechanics energy function. An important aspect is the solvent treatment, represented as a dielectric continuum with a Generalized-Born term, supplemented by a term proportional to the solvent accessible surface area. The key elements of our implementation are: 1) the protein backbone is maintained fixed, 2) the side-chain conformational space is reduced to a discrete library of rotamers, 3) the energy function is decomposed into interaction pairs. The first step consists in calculating a matrix of interactions between each pair of rotamers. In the next step, the sequence-conformation space is explored with an optimization algorithm. Energy evaluations in this second step are fast thanks to the pre-calculation of the energy matrix. The implementation of our CPD procedure is presented, as well as applications to the prediction of side-chain conformations and to the design of full protein sequences.
• Le checkpoint de l’actine branchée corticale contrôle la progression du cycle cellulaire
  
  • Molinié Nicolas
  , 2018. Résumé : Le cytosquelette d’actine génère et mécanotransduit des forces. Dans cette étude, nous montrons que l’actine branchée corticale, qui dépend de RAC1, WAVE et des complexes Arp2/3 contenant ARPC1B, est spécifiquement détectée par le senseur Coronin1B, qui signale, via WISp39 et l’inhibiteur de cycline/CDK p21, à la cellule, de progresser dans le cycle cellulaire. En conséquence, la formation d’un lamellipode et la migration persistante des cellules qui en découle, est corrélée à la durée de la phase G1. L’actine branchée corticale détermine l’entrée en phase S des cellules, en intégrant les stimuli solubles des facteurs de croissance et la mécanotransduction des adhérences à la matrice extracellulaire et aux cellules voisines. Le complexe Arp2/3 est globalement sur-exprimé dans le cancer du sein. Parmi ses sous-unités, la sur-expression de l’isoforme ARPC1B est le plus fort facteur prognostique pour les patientes. En outre, l’inhibition du complexe Arp2/3 bloque la prolifération de lignées de carcinomes mammaires et de mélanomes transformées par l’oncogène RAC1, contre laquelle il n’existe pas de thérapie ciblée. La découverte du checkpoint de l’actine branchée corticale apporte ainsi de nouvelles options pronostiques, diagnostiques et thérapeutiques dans les cancers.
• Accurate PDZ:peptide binding free energies with additive and polarizable free energy simulations
  
  • Simonson Thomas
  • Villa Francesco
  • Panel Nicolas
  , 2018.
• Accurate PDZ/Peptide Binding Specificity with Additive and Polarizable Free Energy Simulations
  
  • Panel Nicolas
  • Villa Francesco
  • Fuentes Ernesto
  • Simonson Thomas
  Biophysical Journal, Biophysical Society, 2018, 114 (5), pp.1091-1102. PDZ domains contain 80-100 amino acids and bind short C-terminal sequences of target proteins. Their specificity is essential for cellular signaling pathways. We studied the binding of the Tiam1 PDZ domain to peptides derived from the C-termini of its Syndecan-1 and Caspr4 targets. We used free energy perturbation (FEP) to characterize the binding energetics of one wild-type and 17 mutant complexes by simulating 21 alchemical transformations between pairs of complexes. Thirteen complexes had known experimental affinities. FEP is a powerful tool to understand protein/ligand binding. It depends, however, on the accuracy of molecular dynamics force fields and conformational sampling. Both aspects require continued testing, especially for ionic mutations. For six mutations that did not modify the net charge, we obtained excellent agreement with experiment using the additive, AMBER ff99SB force field, with a root mean square deviation (RMSD) of 0.37 kcal/mol. For six ionic mutations that modified the net charge, agreement was also good, with one large error (3 kcal/mol) and an RMSD of 0.9 kcal/mol for the other five. The large error arose from the overstabilization of a protein/peptide salt bridge by the additive force field. Four of the ionic mutations were also simulated with the polarizable Drude force field, which represents the first test of this force field for protein/ligand binding free energy changes. The large error was eliminated and the RMS error for the four mutations was reduced from 1.8 to 1.2 kcal/mol. The overall accuracy of FEP indicates it can be used to understand PDZ/peptide binding. Importantly, our results show that for ionic mutations in buried regions, electronic polarization plays a significant role. (10.1016/j.bpj.2018.01.008)
  DOI : 10.1016/j.bpj.2018.01.008
• Directional Collective Migration in Wound Healing Assays
  
  • Molinie Nicolas
  • Gautreau Alexis
  , 2018, 1749, pp.11-19. Cell migration is suppressed by confluence in a process called contact inhibition. Relieving contact inhibition upon scratching is one of the simplest ways to induce cell migration in a variety of cell types. Wound healing is probably most relevant to epithelial monolayers, because epithelial cells generally assume a barrier function, which must be restored as fast as possible by the healing process. This versatile assay, however, can also be applied to fibroblasts and to tumor cell types. Furthermore, assessing the cell response to scratch wounding requires no special equipment or reagents. It is one of the few cell migration assays, which can even be performed without videomicroscopy, since the closure of the wound can be estimated at fixed time points. Several hours after wounding, directional collective migration is easily assessed and quantified. However, cell proliferation, which is also induced by the relief of contact inhibition, is one of the confounding factors of wound healing assays that must be taken into account. A recent alternative to the scratch-induced wound is to use special inserts to seed cells into closely spaced chambers. When the insert is removed, contact inhibition is relieved, similar to the scratch-induced wound. In this chapter, we provide the protocol of the two methods and compare their advantages and disadvantages. We also provide a protocol to estimate cell proliferation upon wound healing based on the incorporation of the nucleotide analog EdU. (10.1007/978-1-4939-7701-7_2)
  DOI : 10.1007/978-1-4939-7701-7_2
• Random Migration Assays of Mammalian Cells and Quantitative Analyses of Single Cell Trajectories
  
  • Dang Irene
  • Gautreau Alexis
  , 2018, 1749, pp.1-9. Cell migration is essential to many biological processes such as embryonic development, immune surveillance and wound healing. Random cell migration refers to the intrinsic ability of cells to migrate, often called cell motility. This basal condition contrasts with directed cell migration, where cells migrate toward a chemical or physical cue. Unlike Brownian particles, however, randomly migrating cells exhibit a directional persistence, i.e., they are more likely to sustain the movement in the direction they previously took than to change, even if this direction is randomly chosen in an isotropic environment. Here we describe how to set up time-lapse recording of mammalian cells freely moving on a two-dimensional surface coated with extracellular matrix proteins, how to acquire single cell trajectories from movies and how to extract key parameters that characterize cell motility, such as cell speed, directionality, mean square displacement, and directional persistence. (10.1007/978-1-4939-7701-7_1)
  DOI : 10.1007/978-1-4939-7701-7_1
• Role of aIF1 in Pyrococcus abyssi translation initiation
  
  • Monestier Auriane
  • Lazennec-Schurdevin Christine
  • Coureux Pierre-Damien
  • Mechulam Yves
  • Schmitt Emmanuelle
  Nucleic Acids Research, Oxford University Press, 2018, 46 (20), pp.11061-11074. In archaeal translation initiation, a preinitiation complex (PIC) made up of aIF1, aIF1A, the ternary complex (TC, e/aIF2-GTP-Met-tRNA i Met) and mRNA bound to the small ribosomal subunit is responsible for start codon selection. Many archaeal mRNAs contain a Shine-Dalgarno (SD) sequence allowing the PIC to be prepositioned in the vicinity of the start codon. Nevertheless, cryo-EM studies have suggested local scanning to definitely establish base pairing of the start codon with the tRNA anticodon. Here, using flu-orescence anisotropy, we show that aIF1 and mRNA have synergistic binding to the Pyrococcus abyssi 30S. Stability of 30S:mRNA:aIF1 strongly depends on the SD sequence. Further, toeprinting experiments show that aIF1-containing PICs display a dynamic conformation with the tRNA not firmly accommodated in the P site. AIF1-induced destabilization of the PIC is favorable for proofreading erroneous initiation complexes. After aIF1 departure, the stability of the PIC increases reflecting initiator tRNA fully base-paired to the start codon. Altogether, our data support the idea that some of the main events governing start codon selection in eukaryotes and archaea occur within a common structural and functional core. However, idiosyncratic features in loop 1 sequence involved in 30S:mRNA binding suggest adjustments of e/aIF1 functioning in the two domains. (10.1093/nar/gky850)
  DOI : 10.1093/nar/gky850