Publications

2022

• Molecular mechanism of Arp2/3 complex inhibition by Arpin
  
  • Fregoso Fred
  • van Eeuwen Trevor
  • Simanov Gleb
  • Rebowski Grzegorz
  • Boczkowska Malgorzata
  • Zimmet Austin
  • Gautreau Alexis
  • Dominguez Roberto
  Nature Communications, Nature Publishing Group, 2022, 13 (1), pp.628. Abstract Positive feedback loops involving signaling and actin assembly factors mediate the formation and remodeling of branched actin networks in processes ranging from cell and organelle motility to mechanosensation. The Arp2/3 complex inhibitor Arpin controls the directional persistence of cell migration by interrupting a feedback loop involving Rac-WAVE-Arp2/3 complex, but Arpin’s mechanism of inhibition is unknown. Here, we describe the cryo-EM structure of Arpin bound to Arp2/3 complex at 3.24-Å resolution. Unexpectedly, Arpin binds Arp2/3 complex similarly to WASP-family nucleation-promoting factors (NPFs) that activate the complex. However, whereas NPFs bind to two sites on Arp2/3 complex, on Arp2-ArpC1 and Arp3, Arpin only binds to the site on Arp3. Like NPFs, Arpin has a C-helix that binds at the barbed end of Arp3. Mutagenesis studies in vitro and in cells reveal how sequence differences within the C-helix define the molecular basis for inhibition by Arpin vs. activation by NPFs. (10.1038/s41467-022-28112-2)
  DOI : 10.1038/s41467-022-28112-2
• Analyse structurale et fonctionnelle du complexe Upf1 : Nmd4 impliqué dans une voie de dégradation des ARNm, la voie NMD
  
  • Barbarin Irène
  , 2022. L’ARN messager (ARNm) produit à partir de l’ADN sert de matrice au ribosome pour la production de protéines dans les cellules. Le rôle majeur de l’ARNm dans le transfert de l’information explique la présence de nombreux mécanismes de contrôle qualité le concernant.Parmi ces contrôles qualités se trouve la voie « non-sense mediated mRNA decay » (NMD). Celle-ci permet la dégradation des ARNm porteurs d’un codon stop précoce (PTC). L’élimination de ces ARNm aberrants permet d’éviter l’accumulation de protéines tronquées dans la cellule. Bien que mis en évidence en 1979, le mécanisme de la voie NMD, à ce jour, ne fait pas consensus. De nombreuses études ont été menées chez différents organismes (humain, levure, ver) et certains arguments se contredisent rendant difficile l’établissement d’un modèle unique.La protéine Upf1 est reconnue comme étant l’acteur central de la voie NMD. Cette enzyme a de nombreux partenaires. Son implication au sein de deux complexes distincts et successifs chez la levure a été mise en évidence en 2018. Le premier complexe composé des protéines historiques de la voie NMD : les protéines Upf1, Upf2 et Upf3, serait responsable de la discrimination entre un codon stop précoce et un codon stop normal. Le second organisé autour d’Upf1 permettrait la dégradation de l’ARNm cible. Au sein de ce second complexe dit effecteur, se trouve les protéines du complexe de decapping connues pour altérer l’intégrité de l’ARNm mature ainsi que les protéines Nmd4 et Ebs1. Le rôle précis de ces dernières n’est pas connu mais elles possèdent toutes deux des homologues humains fortement étudiés. Par analyse de séquence, Ebs1 est proche de la protéine humaine hSMG7 qui est responsable du recrutement du complexe de déadénlyation CCR4-NOT. L’homologue humain de Nmd4 serait hSMG6, responsable de coupure endonucléolytique dégradant l’ARN ciblé.Cette thèse se concentre sur l’étude de la protéine de levure Nmd4. L’étude de la protéine seule a permis de valider son repliement prévu en domaine de type PIN. Ce type de repliement est caractéristique des endonucléases. Cependant la non-conservation d’un résidu conservé dans le site catalytique met en doute la conservation de cette activité présente chez l’Homme. Dans un second temps, l’interaction entre Nmd4 et le domaine central de la protéine Upf1 a été validée, le complexe correspondant ayant été reconstitué. La structure de ce complexe a été résolue par cristallographie aux rayons X et a permis de mettre en évidence le rôle majeur de l’extrémité C-terminale de Nmd4 dans cette interaction. Plusieurs techniques ont permis de valider ce rôle majeur, notamment la diminution de cette interaction par mutagenèse dirigée confirmant l’implication d’un motif précis.De manière intéressante la protéine humaine SMG6 (homologue de Nmd4) interagit avec Upf1 de deux manières différentes : selon des interactions phospho-dépendantes mais également selon des interactions phospho-indépendantes. Une analyse bio-informatique couplée par une étude in cellulo a permis de mettre en évidence que le motif d’interaction responsable de l’interaction chez la levure, permet également une interaction phospho-indépendante entre hSMG6 et hUPF1. Cette conservation entre les deux organismes permet de commencer à unifier les modèles proposés.
• The SARS-CoV-2 protein NSP2 impairs the microRNA-induced silencing capacity of human cells
  
  • Zou Limei
  • Moch Clara
  • Graille Marc
  • Chapat Clément
  , 2022. Abstract The coronavirus SARS-CoV-2 is the cause of the ongoing pandemic of COVID-19. Given the absence of effective treatments against SARS-CoV-2, there is an urgent need for a molecular understanding of how the virus influences the machineries of the host cell. The SARS-CoV-2 generates 16 Non-Structural Proteins (NSPs) through proteolytic cleavage of a large precursor protein. In the present study, we focused our attention on the SARS-CoV-2 protein NSP2, whose role in the viral pathogenicity is poorly understood. Recent proteomic studies shed light on the capacity of NSP2 to bind the 4EHP-GIGYF2 complex, a key factor involved in microRNA-mediated silencing of gene expression in human cells. In order to gain a better understanding of the function of NSP2, we attempted to identify the molecular basis of its interaction with 4EHP-GIGYF2. Our data demonstrate that NSP2 physically associates with the endogenous 4EHP-GIGYF2 complex in the cytoplasm. Using co-immunoprecipitation and in vitro interaction assays, we identified both 4EHP and a central segment in GIGYF2 as binding sites for NSP2. We also provide functional evidence that NSP2 impairs the function of GIGYF2 in mediating mRNA silencing using reporter-based assays, thus leading to a reduced activity of microRNAs. Altogether, these data reveal the profound impact of NSP2 on the post-transcriptional silencing of gene expression in human cells, pointing out 4EHP-GIGYF2 targeting as a possible strategy of SARS-CoV-2 to take over the silencing machinery and to suppress host defenses. (10.1101/2022.01.25.477753)
  DOI : 10.1101/2022.01.25.477753
• Translational regulation by RACK1 in astrocytes represses KIR4.1 expression and regulates neuronal activity
  
  • Oudart Marc
  • Avila-Gutierrez Katia
  • Moch Clara
  • Dossi Elena
  • Milior Giampaolo
  • Boulay Anne-Cécile
  • Gaudey Mathis
  • Moulard Julien
  • Lombard Bérangère
  • Loew Damarys
  • Bemelmans Alexis-Pierre
  • Rouach Nathalie
  • Chapat Clément
  • Cohen-Salmon Martine
  , 2022. Summary The regulation of translation in astrocytes, the main glial cells in the brain, remains poorly characterized. We developed a high-throughput proteomic screen for polysome-associated proteins in astrocytes and focused on the ribosomal protein receptor of activated protein C kinase 1 (RACK1), a critical factor in translational regulation. In astrocyte somata and perisynaptic astrocytic processes (PAPs), RACK1 preferentially bound to a number of mRNAs, including Kcnj10 , encoding the inward rectifying potassium (K + ) channel KIR4.1, a critical astrocytic regulator of neurotransmission. By developing an astrocyte-specific, conditional RACK1 knock-out mouse model, we showed that RACK1 repressed the production of KIR4.1 in hippocampal astrocytes and PAPs. Reporter-based assays revealed that RACK1 controlled Kcnj10 translation through the transcript’s 5’ untranslated region. Upregulation of KIR4.1 in the absence of RACK1 modified the astrocyte territory volume and neuronal activity attenuatin burst frequency and duration in the hippocampus. Hence, astrocytic RACK1 represses KIR4.1 translation and influences neuronal activity. (10.1101/2022.07.16.500292)
  DOI : 10.1101/2022.07.16.500292
• Medical contrast agents as promising tools for biomacromolecular SAXS experiments
  
  • Gabel Frank
  • Engilberge Sylvain
  • Schmitt Emmanuelle
  • Thureau Aurélien
  • Mechulam Yves
  • Pérez Javier
  • Girard Eric
  Acta crystallographica Section D : Structural biology [1993-...], International Union of Crystallography, 2022, 78 (9), pp.1120-1130. Small-angle X-ray scattering (SAXS) has become an indispensable tool in structural biology, complementing atomic-resolution techniques. It is sensitive to the electron-density difference between solubilized biomacromolecules and the buffer, and provides information on molecular masses, particle dimensions and interactions, low-resolution conformations and pair distance-distribution functions. When SAXS data are recorded at multiple contrasts, i.e. at different solvent electron densities, it is possible to probe, in addition to their overall shape, the internal electron-density profile of biomacromolecular assemblies. Unfortunately, contrast-variation SAXS has been limited by the range of solvent electron densities attainable using conventional co-solutes (for example sugars, glycerol and salt) and by the fact that some biological systems are destabilized in their presence. Here, SAXS contrast data from an oligomeric protein and a protein–RNA complex are presented in the presence of iohexol and Gd-HPDO3A, two electron-rich molecules that are used in biomedical imaging and that belong to the families of iodinated and lanthanide-based complexes, respectively. Moderate concentrations of both molecules allowed solvent electron densities matching those of proteins to be attained. While iohexol yielded higher solvent electron densities (per mole), it interacted specifically with the oligomeric protein and precipitated the protein–RNA complex. Gd-HPDO3A, while less efficient (per mole), did not disrupt the structural integrity of either system, and atomic models could be compared with the SAXS data. Due to their elevated solubility and electron density, their chemical inertness, as well as the possibility of altering their physico-chemical properties, lanthanide-based complexes represent a class of molecules with promising potential for contrast-variation SAXS experiments on diverse biomacromolecular systems. (10.1107/S2059798322007392)
  DOI : 10.1107/S2059798322007392
• Small photo-activatable molecules to control biological processes with light
  
  • Joyeux Benjamin
  • Gamet Antoine
  • Nay Bastien
  • Romero Stéphane
  • Marteau Maurine
  • Gautreau Alexis
  • Le Clainche Christophe
  • Changenet-Barret, Pascale
  • Perrin Auriane
  • Hache François
  • Réfrégiers Matthieu
  • Wien Frank
  , 2022.
• The SARS-CoV-2 protein NSP2 impairs the silencing capacity of the human 4EHP-GIGYF2 complex
  
  • Zou Limei
  • Moch Clara
  • Graille Marc
  • Chapat Clément
  iScience, Elsevier, 2022, 25 (7), pp.104646. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. (10.1016/j.isci.2022.104646)
  DOI : 10.1016/j.isci.2022.104646
• PPP2R1A regulates migration persistence through the WAVE Shell Complex
  
  • Wang Yanan
  , 2022. During cell migration, the RAC1-WAVE-ARP2/3 signaling pathway induces the network of branched actin, that serves as a motor for lamellipodia protrusion. This pathway is finely regulated by numerous feed-back and feed-forward signals that control the protrusion lifetime and migration persistence. We screened in MCF10A human breast epithelial cells for proteins that associate with the WAVE complex during persistent migration, but whose association with WAVE is modulated when the downstream production of branched actin is inhibited. The differential proteomics screen identified PPP2R1A (a regulatory subunit of the PP2A trimeric phosphatase) as the strongest hit and a novel WAVE-associated factor required for migration persistence in normal and cancer human cells, in various conditions. The differential proteomics screen identified PPP2R1A (a regulatory subunit of the PP2A trimeric phosphatase) as the strongest hit and a novel WAVE-associated factor required for migration persistence in normal and cancer human cells, in various conditions. Our observation that PPP2R1A interacts with four WAVE complex subunits, but not with WAVE/WASF, led to a purification and characterization of a “WAVE shell complex (WSC)”, a novel variant of WAVE containing the migration regulatory protein NHSL1 that turned out to be necessary for the existence of WSC. Interestingly, PPP2R1A is mutated on hotspots in different cancer types, and these mutations abolish its interaction with NHSL1 and WSC, suggesting a critical role of for this pathway not only in normal cells, but also in cancer progression.
• Role of aIF5B in archaeal translation initiation
  
  • Kazan Ramy
  • Bourgeois Gabrielle
  • Lazennec-Schurdevin Christine
  • Larquet Eric
  • Mechulam Yves
  • Coureux Pierre-Damien
  • Schmitt Emmanuelle
  Nucleic Acids Research, Oxford University Press, 2022. In eukaryotes and in archaea late steps of translation initiation involve the two initiation factors e/aIF5B and e/aIF1A. In eukaryotes, the role of eIF5B in ribosomal subunit joining is established and structural data showing eIF5B bound to the full ribosome were obtained. To achieve its function, eIF5B collaborates with eIF1A. However, structural data illustrating how these two factors interact on the small ribosomal subunit have long been awaited. The role of the archaeal counterparts, aIF5B and aIF1A, remains to be extensively addressed. Here, we study the late steps of Pyrococcus abyssi translation initiation. Using in vitro reconstituted initiation complexes and light scattering, we show that aIF5B bound to GTP accelerates subunit joining without the need for GTP hydrolysis. We report the crystallographic structures of aIF5B bound to GDP and GTP and analyze domain movements associated to these two nucleotide states. Finally, we present the cryo-EM structure of an initiation complex containing 30S bound to mRNA, Met-tRNAiMet, aIF5B and aIF1A at 2.7 Å resolution. Structural data shows how archaeal 5B and 1A factors cooperate to induce a conformation of the initiator tRNA favorable to subunit joining. Archaeal and eukaryotic features of late steps of translation initiation are discussed. (10.1093/nar/gkac490)
  DOI : 10.1093/nar/gkac490
• Role of aIF5B in archaeal translation initiation
  
  • Kazan Ramy
  , 2022. Translation initiation universally occurs with accurate selection of the start codon that defines the reading frame on the mRNA. The mechanism involves a macromolecular complex composed of the small ribosomal subunit, the mRNA, a specialized methionylated initiator tRNA and initiation factors (IFs). Once the start codon is selected at the P site on the small ribosomal subunit and the large subunit is associated, the IFs are released and an elongation competent ribosome is formed. Although the general principles are the same in the three domains of life, the molecular mechanisms are different in bacteria, eukaryotes and archaea as illustrated by the different number and types of the initiation factors.In eukaryotes and in archaea, late steps of translation initiation involve the two initiation factors a/eIF1A and a/eIF5B. Importantly, a/eIF5B and a/eIF1A are orthologues of the bacterial proteins IF1 and IF2 respectively. Therefore, late steps of translation initiation have a universal character.We determined the cryo-EM structure of an archaeal translation initiation complex containing the small ribosomal subunit, a model mRNA, the methionylated initiator tRNA and the two initiation factors aIF5B and aIF1A. The two initiation factors are very well defined in the cryo-EM map. aIF5B is bound to the methionine group of the initiator tRNA by its domain IV while domains I, II, and III contacts the body part of the small ribosomal subunit in the uS12, h5 region. For the first time, interaction between archaeal aIF1A and aIF5B is observed. The structure allows us to model the late steps of translation initiation and to understand how aIF5B facilitates the joining of the large ribosomal subunit. Our results are compared to the eukaryotic and bacterial cases.
• Role of aIF5B in archaeal translation initiation
  
  • Kazan Ramy
  • Bourgeois Gabrielle
  • Lazennec-Schurdevin Christine
  • Larquet Eric
  • Mechulam Yves
  • Coureux Pierre-Damien
  • Schmitt Emmanuelle
  , 2022. In eukaryotes and in archaea late steps of translation initiation involve the two initiation factors e/aIF5B and e/aIF1A. In eukaryotes, the role of eIF5B in ribosomal subunit joining is established and structural data showing eIF5B bound to the full ribosome were obtained. To achieve its function, eIF5B collaborates with eIF1A. However, structural data illustrating how these two factors interact on the small ribosomal subunit have long been awaited. The role of the archaeal counterparts, aIF5B and aIF1A, remains to be extensively addressed. Here, we study the late steps of Pyrococcus abyssi translation initiation. Using in vitro reconstituted initiation complexes and light scattering, we show that aIF5B bound to GTP accelerates subunit joining without the need for GTP hydrolysis. We report the crystallographic structures of aIF5B bound to GDP and GTP and analyze domain movements associated to these two nucleotide states. Finally, we present the cryo-EM structure of an initiation complex containing 30S bound to mRNA, Met-tRNA i Met , aIF5B and aIF1A at 2.7 Å resolution. Structural data shows how archaeal 5B and 1A factors cooperate to induce a conformation of the initiator tRNA favorable to subunit joining. Archaeal and eukaryotic features of late steps of translation initiation are discussed. (10.1101/2022.05.01.490067)
  DOI : 10.1101/2022.05.01.490067
• Nucleation, stabilization, and disassembly of branched actin networks
  
  • Gautreau Alexis
  • Fregoso Fred
  • Simanov Gleb
  • Dominguez Roberto
  Trends in Cell Biology, 2022, 32 (5), pp.421-432. Arp2/3 complex is an actin filament nucleation and branching machinery conserved in all eukaryotes from yeast to human. Arp2/3 complex branched networks generate pushing forces that drive cellular processes ranging from membrane remodeling to cell and organelle motility. Several molecules regulate these processes by directly inhibiting or activating Arp2/3 complex and by stabilizing or disassembling branched networks. Here, we review recent advances in our understanding of Arp2/3 complex regulation, including high-resolution cryoelectron microscopy (cryo-EM) structures that illuminate the mechanisms of Arp2/3 complex activation and branch formation, and novel cellular pathways of branch formation, stabilization, and debranching. We also identify major gaps in our understanding of Arp2/3 complex inhibition and branch stabilization and disassembly. (10.1016/j.tcb.2021.10.006)
  DOI : 10.1016/j.tcb.2021.10.006
• Cg1246, a new player in mycolic acid biosynthesis in Corynebacterium glutamicum
  
  • de Sousa-d'Auria Célia
  • Constantinesco Florence
  • Bayan Nicolas
  • Constant Patricia
  • Tropis Maryelle
  • Daffé Mamadou
  • Graille Marc
  • Houssin Christine
  Microbiology, Microbiology Society, 2022, 168. Mycolic acids are key components of the complex cell envelope of Corynebacteriales. These fatty acids, conjugated to trehalose or to arabinogalactan form the backbone of the mycomembrane. While mycolic acids are essential to the survival of some species, such as Mycobacterium tuberculosis, their absence is not lethal for Corynebacterium glutamicum, which has been extensively used as a model to depict their biosynthesis. Mycolic acids are first synthesized on the cytoplasmic side of the inner membrane and transferred onto trehalose to give trehalose monomycolate (TMM). TMM is subsequently transported to the periplasm by dedicated transporters and used by mycoloyltransferase enzymes to synthesize all the other mycolate-containing compounds. Using a random transposition mutagenesis, we recently identified a new uncharacterized protein (Cg1246) involved in mycolic acid metabolism. Cg1246 belongs to the DUF402 protein family that contains some previously characterized nucleoside phosphatases. In this study, we performed a functional and structural characterization of Cg1246. We showed that absence of the protein led to a significant reduction in the pool of TMM in C. glutamicum, resulting in a decrease in all other mycolate-containing compounds. We found that, in vitro, Cg1246 has phosphatase activity on organic pyrophosphate substrates but is most likely not a nucleoside phosphatase. Using a computational approach, we identified important residues for phosphatase activity and constructed the corresponding variants in C. glutamicum. Surprisingly complementation with these non-functional proteins fully restored the defect in TMM of the Δcg1246 mutant strain, suggesting that in vivo, the phosphatase activity is not involved in mycolic acid biosynthesis. (10.1099/mic.0.001171)
  DOI : 10.1099/mic.0.001171
• The X-ray crystallography phase problem solved thanks to AlphaFold and RoseTTAFold models: a case-study report
  
  • Barbarin-Bocahu Irène
  • Graille Marc
  Acta crystallographica Section D : Structural biology [1993-...], International Union of Crystallography, 2022, 78 (4). The breakthrough recently made in protein structure prediction by deep-learning programs such as AlphaFold and RoseTTAFold will certainly revolutionize biology over the coming decades. The scientific community is only starting to appreciate the various applications, benefits and limitations of these protein models. Yet, after the first thrills due to this revolution, it is important to evaluate the impact of the proposed models and their overall quality to avoid the misinterpretation or overinterpretation of these models by biologists. One of the first applications of these models is in solving the `phase problem' encountered in X-ray crystallography in calculating electron-density maps from diffraction data. Indeed, the most frequently used technique to derive electron-density maps is molecular replacement. As this technique relies on knowledge of the structure of a protein that shares strong structural similarity with the studied protein, the availability of high-accuracy models is then definitely critical for successful structure solution. After the collection of a 2.45 Å resolution data set, we struggled for two years in trying to solve the crystal structure of a protein involved in the nonsense-mediated mRNA decay pathway, an mRNA quality-control pathway dedicated to the elimination of eukaryotic mRNAs harboring premature stop codons. We used different methods (isomorphous replacement, anomalous diffraction and molecular replacement) to determine this structure, but all failed until we straightforwardly succeeded thanks to both AlphaFold and RoseTTAFold models. Here, we describe how these new models helped us to solve this structure and conclude that in our case the AlphaFold model largely outcompetes the other models. We also discuss the importance of search-model generation for successful molecular replacement. (10.1107/S2059798322002157)
  DOI : 10.1107/S2059798322002157
• How much can physics do for protein design?
  
  • Michael Eleni
  • Simonson Thomas
  Current Opinion in Structural Biology, Elsevier, 2022, 72, pp.46-54. Physics and physical chemistry are an important thread in computational protein design, complementary to knowledge-based tools. They provide molecular mechanics scoring functions that need little or no ad hoc parameter readjustment, methods to thoroughly sample equilibrium ensembles, and different levels of approximation for conformational flexibility. They led recently to the successful redesign of a small protein using a physics-based folded state energy. Adaptive Monte Carlo or molecular dynamics schemes were discovered where protein variants are populated as per their ligand-binding free energy or catalytic efficiency. Molecular dynamics have been used for backbone flexibility. Implicit solvent models have been refined, polarizable force fields applied, and many physical insights obtained. (10.1016/j.sbi.2021.07.011)
  DOI : 10.1016/j.sbi.2021.07.011
• Structural and molecular determinants for the interaction of ExbB from Serratia marcescens and HasB, a TonB paralog
  
  • Biou Valérie
  • Adaixo Ricardo Jorge Diogo
  • Chami Mohamed
  • Coureux Pierre-Damien
  • Laurent Benoist
  • Enguéné Véronique Yvette Ntsogo
  • de Amorim Gisele Cardoso
  • Izadi-Pruneyre Nadia
  • Malosse Christian
  • Chamot-Rooke Julia
  • Stahlberg Henning
  • Delepelaire Philippe
  Communications Biology, Nature Publishing Group, 2022, 5 (1), pp.355. Abstract ExbB and ExbD are cytoplasmic membrane proteins that associate with TonB to convey the energy of the proton-motive force to outer membrane receptors in Gram-negative bacteria for iron uptake. The opportunistic pathogen Serratia marcescens ( Sm ) possesses both TonB and a heme-specific TonB paralog, HasB. ExbB Sm has a long periplasmic extension absent in other bacteria such as E. coli (Ec) . Long ExbB’s are found in several genera of Alphaproteobacteria, most often in correlation with a hasB gene. We investigated specificity determinants of ExbB Sm and HasB. We determined the cryo-EM structures of ExbB Sm and of the ExbB-ExbD Sm complex from S. marcescens . ExbB Sm alone is a stable pentamer, and its complex includes two ExbD monomers. We showed that ExbB Sm extension interacts with HasB and is involved in heme acquisition and we identified key residues in the membrane domain of ExbB Sm and ExbB Ec , essential for function and likely involved in the interaction with TonB/HasB. Our results shed light on the class of inner membrane energy machinery formed by ExbB, ExbD and HasB. (10.1038/s42003-022-03306-y)
  DOI : 10.1038/s42003-022-03306-y
• A Computational Model for the PLP-Dependent Enzyme Methionine γ-Lyase
  
  • Chen Xingyu
  • Briozzo Pierre
  • Machover David
  • Simonson Thomas
  Frontiers in Molecular Biosciences, Frontiers Media, 2022, 9, pp.886358. Pyridoxal-5′-phosphate (PLP) is a cofactor in the reactions of over 160 enzymes, several of which are implicated in diseases. Methionine γ-lyase (MGL) is of interest as a therapeutic protein for cancer treatment. It binds PLP covalently through a Schiff base linkage and digests methionine, whose depletion is damaging for cancer cells but not normal cells. To improve MGL activity, it is important to understand and engineer its PLP binding. We develop a simulation model for MGL, starting with force field parameters for PLP in four main states: two phosphate protonation states and two tautomeric states, keto or enol for the Schiff base moiety. We used the force field to simulate MGL complexes with each form, and showed that those with a fullydeprotonated PLP phosphate, especially keto, led to the best agreement with MGL structures in the PDB. We then confirmed this result through alchemical free energy simulations that compared the keto and enol forms, confirming a moderate keto preference, and the fully-deprotonated and singly-protonated phosphate forms. Extensive simulations were needed to adequately sample conformational space, and care was needed to extrapolate the protonation free energy to the thermodynamic limit of a macroscopic, dilute protein solution. The computed phosphate pK a was 5.7, confirming that the deprotonated, −2 form is predominant. The PLP force field and the simulation methods can be applied to all PLP enzymes and used, as here, to reveal fine details of structure and dynamics in the active site. (10.3389/fmolb.2022.886358)
  DOI : 10.3389/fmolb.2022.886358
• Computational Peptide Science
  
  • Simonson Thomas
  , 2022, 2405. This volume details current and new computational methodologies to study peptides. Chapters guide readers through antimicrobial peptides, foldability, amyloid sheet formation, membrane-active peptides, organized peptide assemblies, protein-peptide interfaces, prediction of peptide-MHC complexes, advanced free energy simulations for peptide binding, and methods for high throughput peptide or miniprotein design. Written in the format of the highly successful Methods in Molecular Biology series, each chapter includes an introduction to the topic, lists necessary materials, software, and reagents, includes tips on troubleshooting and known pitfalls, and step-by-step, readily reproducible protocols. Authoritative and cutting-edge, Computational Peptides Science: Methods and Protocols aims to provide concepts, methods, and guidelines to help both novices and experienced workers benefit from today's new opportunities and challenges. (10.1007/978-1-0716-1855-4)
  DOI : 10.1007/978-1-0716-1855-4
• Grid batch-dependent tuning of glow discharge parameters
  
  • Kazan Ramy
  • Bourgeois Gabrielle
  • Carisetti Dominique
  • Florea Ileana
  • Larquet Eric
  • Maurice Jean-Luc
  • Mechulam Yves
  • Ozanam François
  • Schmitt Emmanuelle
  • Coureux Pierre-Damien
  Frontiers in Molecular Biosciences, Frontiers Media, 2022, 9, pp.910218. Sample preparation on cryo-EM grids can give various results, from very thin ice and homogeneous particle distribution (ideal case) to unwanted behavior such as particles around the "holes" or complexes that do not entirely correspond to the one in solution (real life). We recently run into such a case and finally found out that variations in the 3D reconstructions were systematically correlated with the grid batches that were used. We report the use of several techniques to investigate the grids' characteristics, namely TEM, SEM, Auger spectroscopy and Infrared Interferometry. This allowed us to diagnose the origin of grid preparation problems and to adjust glow discharge parameters. The methods used for each approach are described and the results obtained on a common specific case are reported. (10.3389/fmolb.2022.910218)
  DOI : 10.3389/fmolb.2022.910218