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1. Broad sarbecovirus neutralization by combined memory B cell antibodies to ancestral SARS-CoV-2

Author

Cyril Planchais, Ignacio Fernández, Benjamin Chalopin, Timothée Bruel, Pierre Rosenbaum, Maxime Beretta, Jordan D. Dimitrov, Laurine Conquet, Flora Donati, Matthieu Prot, Françoise Porrot, Delphine Planas, Isabelle Staropoli, Florence Guivel-Benhassine, Eduard Baquero, Sylvie van der Werf, Ahmed Haouz, Etienne Simon-Lorière, Xavier Montagutelli, Bernard Maillère, Félix A. Rey, Pablo Guardado-Calvo, Hervé Nozach, Olivier Schwartz, and Hugo Mouquet

Subjects
immunology, virology, Science
Abstract

Summary: Antibodies play a pivotal role in protecting from SARS-CoV-2 infection, but their efficacy is challenged by the continuous emergence of viral variants. In this study, we describe two broadly neutralizing antibodies cloned from the memory B cells of a single convalescent individual after infection with ancestral SARS-CoV-2. Cv2.3194, a resilient class 1 anti-RBD antibody, remains active against Omicron sub-variants up to BA.2.86. Cv2.3132, a near pan-Sarbecovirus neutralizer, targets the heptad repeat 2 membrane proximal region. When combined, Cv2.3194 and Cv2.3132 form a complementary SARS-CoV-2 neutralizing antibody cocktail exhibiting a local dose-dependent synergy. Thus, remarkably robust neutralizing memory B cell antibodies elicited in response to ancestral SARS-CoV-2 infection can withstand viral evolution and immune escape. The cooperative effect of such antibody combination may confer a certain level of protection against the latest SARS-CoV-2 variants.

Published
2024
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2. Prodomain-driven enzyme dimerization: a pH-dependent autoinhibition mechanism that controls Plasmodium Sub1 activity before merozoite egress

Author

Mariano Martinez, Anthony Bouillon, Sébastien Brûlé, Bertrand Raynal, Ahmed Haouz, Pedro M. Alzari, and Jean-Christophe Barale

Subjects
malaria, Plasmodium, subtilin-like protease, pro-domain, enzyme regulation, 3D structure, Microbiology, QR1-502
Abstract

ABSTRACT Malaria symptoms are associated with the asexual multiplication of Plasmodium falciparum within human red blood cells (RBCs) and fever peaks coincide with the egress of daughter merozoites following the rupture of the parasitophorous vacuole (PV) and the RBC membranes. Over the last two decades, it has emerged that the release of competent merozoites is tightly regulated by a complex cascade of events, including the unusual multi-step activation mechanism of the pivotal subtilisin-like protease 1 (Sub1) that takes place in three different cellular compartments and remains poorly understood. Following an initial auto-maturation in the endoplasmic reticulum (ER) between its pro- and catalytic domains, the Sub1 prodomain (PD) undergoes further cleavages by the parasite aspartic protease plasmepsin X (PmX) within acidic secretory organelles that ultimately lead to full Sub1 activation upon discharge into the PV. Here, we report the crystal structure of full-length P. falciparum Sub1 (PfS1FL) and demonstrate, through structural, biochemical, and biophysical studies, that the atypical Plasmodium-specific Sub1 PD directly promotes the assembly of inactive enzyme homodimers at acidic pH, whereas Sub1 is primarily monomeric at neutral pH. Our results shed new light into the finely tuned Sub1 spatiotemporal activation during secretion, explaining how PmX processing and full activation of Sub1 can occur in different cellular compartments, and uncover a robust mechanism of pH-dependent subtilisin autoinhibition that plays a key role in P. falciparum merozoites egress from infected host cells.IMPORTANCEMalaria fever spikes are due to the rupture of infected erythrocytes, allowing the egress of Plasmodium sp. merozoites and further parasite propagation. This fleeting tightly regulated event involves a cascade of enzymes, culminating with the complex activation of the subtilisin-like protease 1, Sub1. Differently than other subtilisins, Sub1 activation strictly depends upon the processing by a parasite aspartic protease within acidic merozoite secretory organelles. However, Sub1 biological activity is required in the pH neutral parasitophorous vacuole, to prime effectors involved in the rupture of the vacuole and erythrocytic membranes. Here, we show that the unusual, parasite-specific Sub1 prodomain is directly responsible for its acidic-dependent dimerization and autoinhibition, required for protein secretion, before its full activation at neutral pH in a monomeric form. pH-dependent Sub1 dimerization defines a novel, essential regulatory element involved in the finely tuned spatiotemporal activation of the egress of competent Plasmodium merozoites.

Published
2024
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3. DNA-binding mechanism and evolution of replication protein A

Author

Clément Madru, Markel Martínez-Carranza, Sébastien Laurent, Alessandra C. Alberti, Maelenn Chevreuil, Bertrand Raynal, Ahmed Haouz, Rémy A. Le Meur, Marc Delarue, Ghislaine Henneke, Didier Flament, Mart Krupovic, Pierre Legrand, and Ludovic Sauguet

Subjects
Science
Abstract

Abstract Replication Protein A (RPA) is a heterotrimeric single stranded DNA-binding protein with essential roles in DNA replication, recombination and repair. Little is known about the structure of RPA in Archaea, the third domain of life. By using an integrative structural, biochemical and biophysical approach, we extensively characterize RPA from Pyrococcus abyssi in the presence and absence of DNA. The obtained X-ray and cryo-EM structures reveal that the trimerization core and interactions promoting RPA clustering on ssDNA are shared between archaea and eukaryotes. However, we also identified a helical domain named AROD (Acidic Rpa1 OB-binding Domain), and showed that, in Archaea, RPA forms an unanticipated tetrameric supercomplex in the absence of DNA. The four RPA molecules clustered within the tetramer could efficiently coat and protect stretches of ssDNA created by the advancing replisome. Finally, our results provide insights into the evolution of this primordial replication factor in eukaryotes.

Published
2023
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4. The crystal structure of a simian Foamy Virus receptor binding domain provides clues about entry into host cells

Author

Ignacio Fernández, Lasse Toftdal Dynesen, Youna Coquin, Riccardo Pederzoli, Delphine Brun, Ahmed Haouz, Antoine Gessain, Félix A. Rey, Florence Buseyne, and Marija Backovic

Subjects
Science
Abstract

Foamy viruses are ancient retroviruses that are prevalent in non-human primates. Fernández et al. report an X-ray structure of a protein domain from a simian Foamy virus that mediates the attachment to cells, providing insights into viral entry.

Published
2023
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5. Dynamics and structural changes of calmodulin upon interaction with the antagonist calmidazolium

Author

Corentin Léger, Irène Pitard, Mirko Sadi, Nicolas Carvalho, Sébastien Brier, Ariel Mechaly, Dorothée Raoux-Barbot, Maryline Davi, Sylviane Hoos, Patrick Weber, Patrice Vachette, Dominique Durand, Ahmed Haouz, J. Iñaki Guijarro, Daniel Ladant, and Alexandre Chenal

Subjects
Calmodulin, CaM, Calmidazolium, CDZ, Calmodulin antagonist, Structure, Biology (General), QH301-705.5
Abstract

Abstract Background Calmodulin (CaM) is an evolutionarily conserved eukaryotic multifunctional protein that functions as the major sensor of intracellular calcium signaling. Its calcium-modulated function regulates the activity of numerous effector proteins involved in a variety of physiological processes in diverse organs, from proliferation and apoptosis, to memory and immune responses. Due to the pleiotropic roles of CaM in normal and pathological cell functions, CaM antagonists are needed for fundamental studies as well as for potential therapeutic applications. Calmidazolium (CDZ) is a potent small molecule antagonist of CaM and one the most widely used inhibitors of CaM in cell biology. Yet, CDZ, as all other CaM antagonists described thus far, also affects additional cellular targets and its lack of selectivity hinders its application for dissecting calcium/CaM signaling. A better understanding of CaM:CDZ interaction is key to design analogs with improved selectivity. Here, we report a molecular characterization of CaM:CDZ complexes using an integrative structural biology approach combining SEC-SAXS, X-ray crystallography, HDX-MS, and NMR. Results We provide evidence that binding of a single molecule of CDZ induces an open-to-closed conformational reorientation of the two domains of CaM and results in a strong stabilization of its structural elements associated with a reduction of protein dynamics over a large time range. These CDZ-triggered CaM changes mimic those induced by CaM-binding peptides derived from physiological protein targets, despite their distinct chemical natures. CaM residues in close contact with CDZ and involved in the stabilization of the CaM:CDZ complex have been identified. Conclusion Our results provide molecular insights into CDZ-induced dynamics and structural changes of CaM leading to its inhibition and open the way to the rational design of more selective CaM antagonists. Graphical abstract Calmidazolium is a potent and widely used inhibitor of calmodulin, a major mediator of calcium-signaling in eukaryotic cells. Structural characterization of calmidazolium-binding to calmodulin reveals that it triggers open-to-closed conformational changes similar to those induced by calmodulin-binding peptides derived from enzyme targets. These results provide molecular insights into CDZ-induced dynamics and structural changes of CaM leading to its inhibition and open the way to the rational design of more selective CaM antagonists.

Published
2022
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6. Interactions of the protein tyrosine phosphatase PTPN3 with viral and cellular partners through its PDZ domain: insights into structural determinants and phosphatase activity

Author

Mariano Genera, Baptiste Colcombet-Cazenave, Anastasia Croitoru, Bertrand Raynal, Ariel Mechaly, Joël Caillet, Ahmed Haouz, Nicolas Wolff, and Célia Caillet-Saguy

Subjects
protein tyrosine phosphatase PTPN3, X-ray crystallography, PDZ domains, PDZ-binding motif, enzyme kinetics, bioinformatics, Biology (General), QH301-705.5
Abstract

The human protein tyrosine phosphatase non-receptor type 3 (PTPN3) is a phosphatase containing a PDZ (PSD-95/Dlg/ZO-1) domain that has been found to play both tumor-suppressive and tumor-promoting roles in various cancers, despite limited knowledge of its cellular partners and signaling functions. Notably, the high-risk genital human papillomavirus (HPV) types 16 and 18 and the hepatitis B virus (HBV) target the PDZ domain of PTPN3 through PDZ-binding motifs (PBMs) in their E6 and HBc proteins respectively. This study focuses on the interactions between the PTPN3 PDZ domain (PTPN3-PDZ) and PBMs of viral and cellular protein partners. We solved the X-ray structures of complexes between PTPN3-PDZ and PBMs of E6 of HPV18 and the tumor necrosis factor-alpha converting enzyme (TACE). We provide new insights into key structural determinants of PBM recognition by PTPN3 by screening the selectivity of PTPN3-PDZ recognition of PBMs, and by comparing the PDZome binding profiles of PTPN3-recognized PBMs and the interactome of PTPN3-PDZ. The PDZ domain of PTPN3 was known to auto-inhibit the protein’s phosphatase activity. We discovered that the linker connecting the PDZ and phosphatase domains is involved in this inhibition, and that the binding of PBMs does not impact this catalytic regulation. Overall, the study sheds light on the interactions and structural determinants of PTPN3 with its cellular and viral partners, as well as on the inhibitory role of its PDZ domain on its phosphatase activity.

Published
2023
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7. Structure, function, and evolution of the Orthobunyavirus membrane fusion glycoprotein

Author

Jan Hellert, Andrea Aebischer, Ahmed Haouz, Pablo Guardado-Calvo, Sven Reiche, Martin Beer, and Félix A. Rey

Subjects
CP: Microbiology, Biology (General), QH301-705.5
Abstract

Summary: La Crosse virus, responsible for pediatric encephalitis in the United States, and Schmallenberg virus, a highly teratogenic veterinary virus in Europe, belong to the large Orthobunyavirus genus of zoonotic arthropod-borne pathogens distributed worldwide. Viruses in this under-studied genus cause CNS infections or fever with debilitating arthralgia/myalgia syndromes, with no effective treatment. The main surface antigen, glycoprotein Gc (∼1,000 residues), has a variable N-terminal half (GcS) targeted by the patients’ antibody response and a conserved C-terminal moiety (GcF) responsible for membrane fusion during cell entry. Here, we report the X-ray structure of post-fusion La Crosse and Schmallenberg virus GcF, revealing the molecular determinants for hairpin formation and trimerization required to drive membrane fusion. We further experimentally confirm the role of residues in the fusion loops and in a vestigial endoplasmic reticulum (ER) translocation sequence at the GcS-GcF junction. The resulting knowledge provides essential molecular underpinnings for future development of potential therapeutic treatments and vaccines.

Published
2023
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8. Molecular basis of the interaction of the human tyrosine phosphatase PTPN3 with the hepatitis B virus core protein

Author

Mariano Genera, Barbara Quioc-Salomon, Antonin Nourisson, Baptiste Colcombet-Cazenave, Ahmed Haouz, Ariel Mechaly, Mariette Matondo, Magalie Duchateau, Alexander König, Marc P. Windisch, Christine Neuveut, Nicolas Wolff, and Célia Caillet-Saguy

Subjects
Medicine, Science
Abstract

Abstract Interactions between the hepatitis B virus core protein (HBc) and host cell proteins are poorly understood, although they may be essential for the propagation of the virus and its pathogenicity. HBc has a C-terminal PDZ (PSD-95, Dlg1, ZO-1)-binding motif (PBM) that is responsible for interactions with host PDZ domain-containing proteins. In this work, we focused on the human protein tyrosine phosphatase non-receptor type 3 (PTPN3) and its interaction with HBc. We solved the crystal structure of the PDZ domain of PTPN3 in complex with the PBM of HBc, revealing a network of interactions specific to class I PDZ domains despite the presence of a C-terminal cysteine in this atypical PBM. We further showed that PTPN3 binds the HBc protein within capsids or as a homodimer. We demonstrate that overexpression of PTPN3 significantly affects HBV infection in HepG2 NTCP cells. Finally, we performed proteomics studies on both sides by pull-down assays and screening of a human PDZ domain library. We identified a pool of human PBM-containing proteins that might interact with PTPN3 in cells and that could be in competition with the HBc PBM during infection, and we also identified potential cellular partners of HBc through PDZ-PBM interactions. This study opens up many avenues of future investigations into the pathophysiology of HBV.

Published
2021
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9. Interactions of Severe Acute Respiratory Syndrome Coronavirus 2 Protein E With Cell Junctions and Polarity PSD-95/Dlg/ZO-1-Containing Proteins

Author

Yanlei Zhu, Flavio Alvarez, Nicolas Wolff, Ariel Mechaly, Sébastien Brûlé, Benoit Neitthoffer, Sandrine Etienne-Manneville, Ahmed Haouz, Batiste Boëda, and Célia Caillet-Saguy

Subjects
SARS-CoV-2, envelope protein, host–pathogen interactions, PDZ-binding motif, PDZ-containing protein, protein–protein interaction, Microbiology, QR1-502
Abstract

The C-terminus of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) protein E contains a PBM (PDZ-binding motif) targeting PDZ (PSD-95/Dlg/ZO-1) domains, which is identical to the PBM of SARS-CoV. The latter is involved in the pathogenicity of the virus. Recently, we identified 10 human PDZ-containing proteins showing significant interactions with SARS-CoV-2 protein E PBM. We selected several of them involved in cellular junctions and cell polarity (TJP1, PARD3, MLLT4, and LNX2) and MPP5/PALS1 previously shown to interact with SARS-CoV E PBM. Targeting cellular junctions and polarity components is a common strategy by viruses to hijack cell machinery to their advantage. In this study, we showed that these host PDZ domains TJP1, PARD3, MLLT4, LNX2, and MPP5/PALS1 interact in a PBM-dependent manner in vitro and colocalize with the full-length E protein in cellulo, sequestrating the PDZ domains to the Golgi compartment. We solved three crystal structures of complexes between human LNX2, MLLT4, and MPP5 PDZs and SARS-CoV-2 E PBM highlighting its binding preferences for several cellular targets. Finally, we showed different affinities for the PDZ domains with the original SARS-CoV-2 C-terminal sequence containing the PBM and the one of the beta variant that contains a mutation close to the PBM. The acquired mutations in the E protein localized near the PBM might have important effects both on the structure and the ion-channel activity of the E protein and on the host machinery targeted by the variants during the infection.

Published
2022
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10. Essential dynamic interdependence of FtsZ and SepF for Z-ring and septum formation in Corynebacterium glutamicum

Author

Adrià Sogues, Mariano Martinez, Quentin Gaday, Mathilde Ben Assaya, Martin Graña, Alexis Voegele, Michael VanNieuwenhze, Patrick England, Ahmed Haouz, Alexandre Chenal, Sylvain Trépout, Rosario Duran, Anne Marie Wehenkel, and Pedro M. Alzari

Subjects
Science
Abstract

The mechanisms of Z-ring assembly and regulation in bacteria are poorly understood, particularly in non-model organisms. Here, Sogues et al. study the interaction between FtsZ and SepF in Corynebacterium glutamicum, showing an essential interdependence of these proteins for formation of a functional Z-ring.

Published
2020
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11. Structure of the prefusion-locking broadly neutralizing antibody RVC20 bound to the rabies virus glycoprotein

Author

Jan Hellert, Julian Buchrieser, Florence Larrous, Andrea Minola, Guilherme Dias de Melo, Leah Soriaga, Patrick England, Ahmed Haouz, Amalio Telenti, Olivier Schwartz, Davide Corti, Hervé Bourhy, and Félix A. Rey

Subjects
Science
Abstract

Rabies virus (RABV) infection of unvaccinated individuals can be treated with post exposure prophylaxis. Here, Hellert et al. analyze the structure of a broadly neutralizing human antibody in complex with its target domain in the RABV glycoprotein and demonstrate that it blocks the acid-induced conformational change required for membrane fusion.

Published
2020
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12. Human herpesvirus 8 molecular mimicry of ephrin ligands facilitates cell entry and triggers EphA2 signaling.

Author

Taylor P Light, Delphine Brun, Pablo Guardado-Calvo, Riccardo Pederzoli, Ahmed Haouz, Frank Neipel, Félix A Rey, Kalina Hristova, and Marija Backovic

Subjects
Biology (General), QH301-705.5
Abstract

Human herpesvirus 8 (HHV-8) is an oncogenic virus that enters cells by fusion of the viral and endosomal cellular membranes in a process mediated by viral surface glycoproteins. One of the cellular receptors hijacked by HHV-8 to gain access to cells is the EphA2 tyrosine kinase receptor, and the mechanistic basis of EphA2-mediated viral entry remains unclear. Using X-ray structure analysis, targeted mutagenesis, and binding studies, we here show that the HHV-8 envelope glycoprotein complex H and L (gH/gL) binds with subnanomolar affinity to EphA2 via molecular mimicry of the receptor's cellular ligands, ephrins (Eph family receptor interacting proteins), revealing a pivotal role for the conserved gH residue E52 and the amino-terminal peptide of gL. Using FSI-FRET and cell contraction assays, we further demonstrate that the gH/gL complex also functionally mimics ephrin ligand by inducing EphA2 receptor association via its dimerization interface, thus triggering receptor signaling for cytoskeleton remodeling. These results now provide novel insight into the entry mechanism of HHV-8, opening avenues for the search of therapeutic agents that could interfere with HHV-8-related diseases.

Published
2021
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13. A High‐Affinity Calmodulin‐Binding Site in the CyaA Toxin Translocation Domain is Essential for Invasion of Eukaryotic Cells

Author

Alexis Voegele, Mirko Sadi, Darragh Patrick O'Brien, Pauline Gehan, Dorothée Raoux‐Barbot, Maryline Davi, Sylviane Hoos, Sébastien Brûlé, Bertrand Raynal, Patrick Weber, Ariel Mechaly, Ahmed Haouz, Nicolas Rodriguez, Patrice Vachette, Dominique Durand, Sébastien Brier, Daniel Ladant, and Alexandre Chenal

Subjects
adenylate cyclase, Bordetella pertussis, calmodulin, CyaA toxin, membrane translocation, protein membrane interaction, Science
Abstract

Abstract The molecular mechanisms and forces involved in the translocation of bacterial toxins into host cells are still a matter of intense research. The adenylate cyclase (CyaA) toxin from Bordetella pertussis displays a unique intoxication pathway in which its catalytic domain is directly translocated across target cell membranes. The CyaA translocation region contains a segment, P454 (residues 454–484), which exhibits membrane‐active properties related to antimicrobial peptides. Herein, the results show that this peptide is able to translocate across membranes and to interact with calmodulin (CaM). Structural and biophysical analyses reveal the key residues of P454 involved in membrane destabilization and calmodulin binding. Mutational analysis demonstrates that these residues play a crucial role in CyaA translocation into target cells. In addition, calmidazolium, a calmodulin inhibitor, efficiently blocks CyaA internalization. It is proposed that after CyaA binding to target cells, the P454 segment destabilizes the plasma membrane, translocates across the lipid bilayer and binds calmodulin. Trapping of CyaA by the CaM:P454 interaction in the cytosol may assist the entry of the N‐terminal catalytic domain by converting the stochastic motion of the polypeptide chain through the membrane into an efficient vectorial chain translocation into host cells.

Published
2021
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14. Orthobunyavirus spike architecture and recognition by neutralizing antibodies

Author

Jan Hellert, Andrea Aebischer, Kerstin Wernike, Ahmed Haouz, Emiliana Brocchi, Sven Reiche, Pablo Guardado-Calvo, Martin Beer, and Félix A. Rey

Subjects
Science
Abstract

Orthobunyaviruses (OBVs) cause severe disease in humans and farm animals, but the molecular basis for infection is not fully understood. Here, the authors present crystal structures of free and antibody-bound OBV envelope glycoproteins and show that their domains enable efficient immunization in a mouse model.

Published
2019
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15. Defective lytic transglycosylase disrupts cell morphogenesis by hindering cell wall de-O-acetylation in Neisseria meningitidis

Author

Allison Hillary Williams, Richard Wheeler, Ala-Eddine Deghmane, Ignacio Santecchia, Ryan E Schaub, Samia Hicham, Maryse Moya Nilges, Christian Malosse, Julia Chamot-Rooke, Ahmed Haouz, Joseph P Dillard, William P Robins, Muhamed-Kheir Taha, and Ivo Gomperts Boneca

Subjects
Neisseria meningitidis, peptidoglycan, Lytic transglycosylase, cell division, cell separation, X-ray crystallography, Medicine, Science, Biology (General), QH301-705.5
Abstract

Lytic transglycosylases (LT) are enzymes involved in peptidoglycan (PG) remodeling. However, their contribution to cell-wall-modifying complexes and their potential as antimicrobial drug targets remains unclear. Here, we determined a high-resolution structure of the LT, an outer membrane lipoprotein from Neisseria species with a disordered active site helix (alpha helix 30). We show that deletion of the conserved alpha-helix 30 interferes with the integrity of the cell wall, disrupts cell division, cell separation, and impairs the fitness of the human pathogen Neisseria meningitidis during infection. Additionally, deletion of alpha-helix 30 results in hyperacetylated PG, suggesting this LtgA variant affects the function of the PG de-O-acetylase (Ape 1). Our study revealed that Ape 1 requires LtgA for optimal function, demonstrating that LTs can modulate the activity of their protein-binding partner. We show that targeting specific domains in LTs can be lethal, which opens the possibility that LTs are useful drug-targets.

Published
2020
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16. Integrative Study of the Structural and Dynamical Properties of a KirBac3.1 Mutant: Functional Implication of a Highly Conserved Tryptophan in the Transmembrane Domain

Author

Charline Fagnen, Ludovic Bannwarth, Iman Oubella, Dania Zuniga, Ahmed Haouz, Eric Forest, Rosa Scala, Saïd Bendahhou, Rita De Zorzi, David Perahia, and Catherine Vénien-Bryan

Subjects
crystal structure of KirBac3.1 W46R, HDX-mass spectrometry, molecular dynamics, normal modes, electrophysiology, gain of function Kir, Biology (General), QH301-705.5, Chemistry, QD1-999
Abstract

ATP-sensitive potassium (K-ATP) channels are ubiquitously expressed on the plasma membrane of cells in several organs, including the heart, pancreas, and brain, and they govern a wide range of physiological processes. In pancreatic β-cells, K-ATP channels composed of Kir6.2 and SUR1 play a key role in coupling blood glucose and insulin secretion. A tryptophan residue located at the cytosolic end of the transmembrane helix is highly conserved in eukaryote and prokaryote Kir channels. Any mutation on this amino acid causes a gain of function and neonatal diabetes mellitus. In this study, we have investigated the effect of mutation on this highly conserved residue on a KirBac channel (prokaryotic homolog of mammalian Kir6.2). We provide the crystal structure of the mutant KirBac3.1 W46R (equivalent to W68R in Kir6.2) and its conformational flexibility properties using HDX-MS. In addition, the detailed dynamical view of the mutant during the gating was investigated using the in silico method. Finally, functional assays have been performed. A comparison of important structural determinants for the gating mechanism between the wild type KirBac and the mutant W46R suggests interesting structural and dynamical clues and a mechanism of action of the mutation that leads to the gain of function.

Published
2021
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17. Covalently linked dengue virus envelope glycoprotein dimers reduce exposure of the immunodominant fusion loop epitope

Author

Alexander Rouvinski, Wanwisa Dejnirattisai, Pablo Guardado-Calvo, Marie-Christine Vaney, Arvind Sharma, Stéphane Duquerroy, Piyada Supasa, Wiyada Wongwiwat, Ahmed Haouz, Giovanna Barba-Spaeth, Juthathip Mongkolsapaya, Félix A. Rey, and Gavin R. Screaton

Subjects
Science
Abstract

The immunodominant epitope of dengue virus envelope protein (E) induces poorly neutralizing antibodies, which poses a problem for vaccine development. Here, the authors engineer covalently locked E dimers exposing an epitope that has been shown to induce potent and broadly neutralizing antibodies.

Published
2017
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18. Structural basis for haem piracy from host haemopexin by Haemophilus influenzae

Author

Silvia Zambolin, Bernard Clantin, Mohamed Chami, Sylviane Hoos, Ahmed Haouz, Vincent Villeret, and Philippe Delepelaire

Subjects
Science
Abstract

Haemophilus influenzae requires haem, and acquires it from host haemoproteins including haemopexin. Here, the authors examine the haem transport system consisting of HxuA, HxuB and HxuC via the structures of HxuA in complex with haemopexin.

Published
2016
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19. Designed mono- and di-covalent inhibitors trap modeled functional motions for Trypanosoma cruzi proline racemase in crystallography.

Author

Patricia de Aguiar Amaral, Delphine Autheman, Guilherme Dias de Melo, Nicolas Gouault, Jean-François Cupif, Sophie Goyard, Patricia Dutra, Nicolas Coatnoan, Alain Cosson, Damien Monet, Frederick Saul, Ahmed Haouz, Philippe Uriac, Arnaud Blondel, and Paola Minoprio

Subjects
Arctic medicine. Tropical medicine, RC955-962, Public aspects of medicine, RA1-1270
Abstract

Chagas disease, caused by Trypanosoma cruzi, affects millions of people in South America and no satisfactory therapy exists, especially for its life threatening chronic phase. We targeted the Proline Racemase of T. cruzi, which is present in all stages of the parasite life cycle, to discover new inhibitors against this disease. The first published crystal structures of the enzyme revealed that the catalytic site is too small to allow any relevant drug design. In previous work, to break through the chemical space afforded to virtual screening and drug design, we generated intermediate models between the open (ligand free) and closed (ligand bound) forms of the enzyme. In the present work, we co-crystallized the enzyme with the selected inhibitors and found that they were covalently bound to the catalytic cysteine residues in the active site, thus explaining why these compounds act as irreversible inhibitors. These results led us to the design of a novel, more potent specific inhibitor, NG-P27. Co-crystallization of this new inhibitor with the enzyme allowed us to confirm the predicted protein functional motions and further characterize the chemical mechanism. Hence, the catalytic Cys300 sulfur atom of the enzyme attacks the C2 carbon of the inhibitor in a coupled, regiospecific-stereospecific Michael reaction with trans-addition of a proton on the C3 carbon. Strikingly, the six different conformations of the catalytic site in the crystal structures reported in this work had key similarities to our intermediate models previously generated by inference of the protein functional motions. These crystal structures span a conformational interval covering roughly the first quarter of the opening mechanism, demonstrating the relevance of modeling approaches to break through chemical space in drug design.

Published
2018
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20. Evolutionary diversification of the HAP2 membrane insertion motifs to drive gamete fusion across eukaryotes.

Author

Juliette Fedry, Jennifer Forcina, Pierre Legrand, Gérard Péhau-Arnaudet, Ahmed Haouz, Mark Johnson, Felix A Rey, and Thomas Krey

Subjects
Biology (General), QH301-705.5
Abstract

HAPLESS2 (HAP2) is a broadly conserved, gamete-expressed transmembrane protein that was shown recently to be structurally homologous to viral class II fusion proteins, which initiate fusion with host cells via insertion of fusion loops into the host membrane. However, the functional conformation of the HAP2 fusion loops has remained unknown, as the reported X-ray structure of Chlamydomonas reinhardtii HAP2 lacked this critical region. Here, we report a structure-guided alignment that reveals diversification of the proposed HAP2 fusion loops. Representative crystal structures show that in flowering plants, HAP2 has a single prominent fusion loop projecting an amphipathic helix at its apex, while in trypanosomes, three small nonpolar loops of HAP2 are poised to interact with the target membrane. A detailed structure-function analysis of the Arabidopsis HAP2 amphipathic fusion helix defines key residues that are essential for membrane insertion and for gamete fusion. Our study suggests that HAP2 may have evolved multiple modes of membrane insertion to accommodate the diversity of membrane environments it has encountered during eukaryotic evolution.

Published
2018
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21. Bulgecin A: The Key to a Broad‐Spectrum Inhibitor That Targets Lytic Transglycosylases

Author

Allison H. Williams, Richard Wheeler, Constance Thiriau, Ahmed Haouz, Muhamed‐Kheir Taha, and Ivo G. Boneca

Subjects
peptidoglycan, Neisseria, bulgecin, lytic transglycosylase, beta‐lactam, Therapeutics. Pharmacology, RM1-950
Abstract

Lytic transglycosylases (Lts) are involved in recycling, cell division, and metabolism of the peptidoglycan. They have been understudied for their usefulness as potential antibacterial targets due to their high redundancy in Gram‐negative bacteria. Bulgecin A is an O‐sulphonated glycopeptide that targets primarily soluble lytic tranglycosylases (Slt). It has been shown that bulgecin A increases the efficacy of β‐lactams that target penicillin bindings proteins (PBPs). Here, we present the high‐resolution crystal structure of LtgA from Neisseria meningitidis strain MC58, a membrane bound homolog of Escherichia coli Slt, in complex with bulgecin A. The LtgA‐bulgecin A complex reveals the mechanism of inhibition by bulgecin A at near atomic resolution. We further demonstrate that bulgecin A is not only a potent inhibitor of LtgA, but most importantly, it restores the efficacy of β‐lactam antibiotics in strains of N. meningitidis and Neisseria gonorrhoeae that have reduced susceptibility to β‐lactams. This is particularly relevant for N. gonorrhoeae where no vaccines are available. This work illustrates how best to target dangerous pathogens using a multiple drug target approach, a new and alternative approach to fighting antibiotic resistance.

Published
2017
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22. The MksG nuclease is the executing part of the bacterial plasmid defense system MksBEFG

Author

Manuela Weiß, Giacomo Giacomelli, Mathilde Ben Assaya, Finja Grundt, Ahmed Haouz, Feng Peng, Stéphanie Petrella, Anne Marie Wehenkel, and Marc Bramkamp

Subjects
Genetics
Abstract

Cells are continuously facing the risk of taking up foreign DNA that can compromise genomic integrity. Therefore, bacteria are in a constant arms race with mobile genetic elements such as phages, transposons and plasmids. They have developed several active strategies against invading DNA molecules that can be seen as a bacterial ‘innate immune system’. Here, we investigated the molecular arrangement of the Corynebacterium glutamicum MksBEFG complex, which is homologous to the MukBEF condensin system. We show here that MksG is a nuclease that degrades plasmid DNA. The crystal structure of MksG revealed a dimeric assembly through its C-terminal domain that is homologous to the TOPRIM domain of the topoisomerase II family of enzymes and contains the corresponding ion binding site essential for DNA cleavage in topoisomerases. The MksBEF subunits exhibit an ATPase cycle in vitro and we reason that this reaction cycle, in combination with the nuclease activity provided by MksG, allows for processive degradation of invading plasmids. Super-resolution localization microscopy revealed that the Mks system is spatially regulated via the polar scaffold protein DivIVA. Introduction of plasmids results in an increase in DNA bound MksG, indicating an activation of the system in vivo.

Published
2023

23. Trypanosoma brucei RRP44: a versatile enzyme for processing structured and non-structured RNA substrates

Author

Giovanna Cesaro, Heloisa Tramontin da Soler, Eloise Pavão Guerra-Slompo, Ahmed Haouz, Pierre Legrand, Nilson Ivo Tonin Zanchin, Beatriz Gomes Guimaraes, Fiocruz Paraná - Instituto Carlos Chagas / Carlos Chagas Institute [Curitiba, Brésil] (ICC), Fundação Oswaldo Cruz / Oswaldo Cruz Foundation (FIOCRUZ), Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), Federal University of Paraná, Cristallographie (Plateforme) - Crystallography (Platform), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Synchrotron SOLEIL (SSOLEIL), Centre National de la Recherche Scientifique (CNRS), ICC-CNPq-PROEP [442323/2019-0], Fiocruz-INOVA [3501948026], CAPES-COFECUB [CAPES 862/2015 – COFECUB Me862-15], Nilson I.T. Zanchin and Beatriz G. Guimarães are CNPq research fellows [304167/2019-3, 304788/2018-0], and studentships for the graduate students Giovanna Cesaro and Heloisa T. da Soler were provided by CNPq and ICC-Fiocruz, respectively. Funding for open access charge: FIOTEC-Fundação de apoio à Fiocruz.

Subjects
Genetics, [SDV.MP.PAR]Life Sciences [q-bio]/Microbiology and Parasitology/Parasitology, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM]
Abstract

Rrp44/Dis3 is a conserved eukaryotic ribonuclease that acts on processing and degradation of nearly all types of RNA. It contains an endo- (PIN) and an exonucleolytic (RNB) domain and, its depletion in model organisms supports its essential function for cell viability. In Trypanosoma brucei, depletion of Rrp44 (TbRRP44) blocks maturation of ribosomal RNA, leading to disruption of ribosome synthesis and inhibition of cell proliferation. We have determined the crystal structure of the exoribonucleolytic module of TbRRP44 in an active conformation, revealing novel details of the catalytic mechanism of the RNB domain. For the first time, the position of the second magnesium involved in the two-metal-ion mechanism was determined for a member of the RNase II family. In vitro, TbRRP44 acts preferentially on non-structured uridine-rich RNA substrates. However, we demonstrated for the first time that both TbRRP44 and its homologue from Saccharomyces cerevisiae can also degrade structured substrates without 3’-end overhang, suggesting that Rrp44/Dis3 ribonucleases may be involved in degradation of a wider panel of RNA than has been assumed. Interestingly, deletion of TbRRP44 PIN domain impairs RNA binding to different extents, depending on the type of substrate.

Published
2023

24. A novel nuclease is the executing part of a bacterial plasmid defense system

Author

Manuela Weiß, Giacomo Giacomelli, Mathilde Ben Assaya, Finja Grundt, Ahmed Haouz, Feng Peng, Stéphanie Petrella, Anne Marie Wehenkel, Marc Bramkamp, Christian-Albrechts University of Kiel, Ludwig-Maximilians-Universität München (LMU), Institut für Allgemeine Mikrobiologie, Christian-Albrechts-Universität zu Kiel (CAU), Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Cristallogenèse et Diffraction des Rayons X (Plate-forme/PF6), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), University of Vienna [Vienna], Ludwig-Maximilians University [Munich] (LMU), and Kiel University

Subjects
[SDV]Life Sciences [q-bio]
Abstract

Cells are continuously facing the risk of taking up foreign DNA that can compromise genomic and cellular integrity. Therefore, bacteria are in a constant arms race with mobile genetic elements such as phages, transposons and plasmids. They have developed several active strategies against invading DNA molecules that can be seen as a bacterial ‘innate immune system’. Anti-phage systems are usually organized in ‘defense islands’ and can consist of restriction-modification (R-M) systems, CRISPR-Cas, and abortive infection (Abi) systems. Despite recent advances in the field, much less is known about plasmid defense systems. We have recently identified the MksBEFG system in Corynebacterium glutamicum as a novel plasmid defense system which comprises homologues of the condensin system MukFEB. Here, we investigated the molecular arrangement of the MksBEFG complex. Importantly, we identified MksG as a novel nuclease that degrades plasmid DNA and is, thus, the executing part of the system. The crystal structure of MksG revealed a dimeric assembly through its DUF2220 C-terminal domains. This domain is homologous to the TOPRIM domain of the topoisomerase II family of enzymes and contains the corresponding divalent ion binding site that is essential for DNA cleavage in topoisomerases, explaining the in vitro nuclease activity of MksG. We further show that the MksBEF subunits exhibit an ATPase cycle similar to MukBEF in vitro and we reason that this reaction cycle, in combination with the nuclease activity provided by MksG, allows for processive degradation of invading plasmids. Super-resolution localization microscopy revealed that the Mks system is spatially regulated via to the polar scaffold protein DivIVA. Introduction of plasmids increases the diffusion rate and alters the localization of MksG, indicating an activation of the system in vivo.

Published
2022

25. FtsEX-independent control of RipA-mediated cell separation in

Author

Quentin, Gaday, Daniela, Megrian, Giacomo, Carloni, Mariano, Martinez, Bohdana, Sokolova, Mathilde, Ben Assaya, Pierre, Legrand, Sebastien, Brûlé, Ahmed, Haouz, Anne Marie, Wehenkel, and Pedro M, Alzari

Subjects
Bacterial Proteins, Cell Wall, Actinomycetales, Humans, N-Acetylmuramoyl-L-alanine Amidase, Peptidoglycan, Cell Separation, Phylogeny
Abstract

The bacterial cell wall is a multi-layered mesh, whose major component is peptidoglycan (PG), a sugar polymer cross-linked by short peptide stems. During cell division, a careful balance of PG synthesis and degradation, precisely coordinated both in time and space, is necessary to prevent uncontrolled destruction of the cell wall. In

Published
2022

26. Structure and dynamic association of an assembly platform subcomplex of the bacterial type II secretion system

Author

Régine Dazzoni, Yuanyuan Li, Aracelys López-Castilla, Sébastien Brier, Ariel Mechaly, Florence Cordier, Ahmed Haouz, Michael Nilges, Olivera Francetic, Benjamin Bardiaux, Nadia Izadi-Pruneyre, Bioinformatique structurale - Structural Bioinformatics, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Biochimie des Interactions Macromoléculaires / Biochemistry of Macromolecular Interactions, Plateforme Technologique de RMN Biologique et HDX-MS - Biological NMR and HDX-MS Technological Platform, Cristallographie (Plateforme) - Crystallography (Platform), This work was funded by the French Agence Nationale de la Recherche (ANR Synergy-T2SS ANR-19-CE11-0020-01) and the Fondation pour la Recherche Médicale (Equipe FRM 2017M.DEQ20170839114). YL was funded by the Pasteur Paris University (PPU) international PhD program and the China National Biotec Group Company Limited, and by a doctoral fellowship from the China Scholarship Council. We thank Ingrid Guilvout and Maylis Lejeune for their constant help. We acknowledge Iñaki Guijarro, Rémy Le Meur, Bertrand Raynal, Sébastien Brûlé and Christophe Thomas of C2RT for their help and assistance. The 800-MHz NMR spectrometer and the optima AUC of the Institut Pasteur were partially funded by the Région Ile de France (SESAME 2014 NMRCHR grant no 4014526) and DIM one health, respectively. The authors are grateful to the staff of the Institut Pasteur Crystallography platform for robot-driven crystallization screening. We acknowledge the Synchrotron SOLEIL (St Aubin, France) staff for assistance and advice during data collection on PROXIMA-1 and PROXIMA-2A beamlines. This work used the computational and storage service (TARS cluster) provided by the IT Department at Institut Pasteur, Paris., ANR-19-CE11-0020,SYNERGY_T2SS,Structure et fonction moléculaire du pseudopilus dans la sécrétion de protéines par lla voiè de type 2(2019), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Systèmes transmembranaires bactériens - Bacterial transmembrane systems, and This work was supported by the French Agence Nationale de la Recherche (ANR Synergy-T2SS ANR-19-CE11-0020-01) and the Fondation pour la Recherche Médicale (Equipe FRM 2017M.DEQ20170839114). Y.L. was funded by the Pasteur Paris University (PPU) international PhD program.

Subjects
protein-protein interaction, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Structural Biology, Assembly platform, Type II Secretion System, [SDV]Life Sciences [q-bio], Type II Secretion System Assembly platform Type IV pili protein-protein interaction Ferredoxin-like fold, Ferredoxin-like fold, Type IV pili, Molecular Biology, Ferredoxin-like domain, NMR, X-ray crystallography
Abstract

Type II secretion systems (T2SS) allow diderm bacteria to secrete hydrolytic enzymes, adhesins or toxins important for growth and virulence. In T2SS, secretion of folded proteins from the periplasm to the cell surface requires assembly of periplasmic filaments called pseudopili. Like the related type IV pili, pseudopili are polymerized in the inner membrane through addition of subunits at the filament base, mediated by the essential assembly platform (AP). To understand the structure and molecular role of the AP, we focused on its components PulL and PulM from the Klebsiella oxytoca T2SS. By combining biophysical methods, NMR and X-ray crystallography we studied the structure and associations of their periplasmic domains. We describe the first structure of the heterodimer complex formed by the PulL and PulM ferredoxin-like domains and show how their structural complementarity and plasticity favor their association during the secretion process. Cysteine scanning and cross-linking of transmembrane segments provided additional constraints to build a structural model of the PulL–PulM complex and assembly in the cellular context. Together with the relative abundance of PulL, PulM and their partners our findings suggest a model of the AP as a dynamic hub that orchestrates pseudopilus polymerization.

Published
2022

27. Structural basis for the allosteric inhibition of UMP kinase from Gram‐positive bacteria, a promising antibacterial target

Author

Patrick Walter, Ariel Mechaly, Julien Bous, Ahmed Haouz, Patrick England, Joséphine Lai‐Kee‐Him, Aurélie Ancelin, Sylviane Hoos, Bruno Baron, Stefano Trapani, Patrick Bron, Gilles Labesse, Hélène Munier‐Lehmann, Chimie et Biocatalyse, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Cristallographie (Plateforme) - Crystallography (Platform), Centre de Biologie Structurale [Montpellier] (CBS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM), Biophysique Moléculaire (Plate-forme), Biophysique Moléculaire (plateforme) - Molecular Biophysics (platform), This work was supported by the European Union’s Framework Programme for Research and Innovation Horizon 2020 (2014-2020) under the Marie Skłodowska-Curie Grant Agreement No. 675555, Accelerated Early staGe drug discovery (AEGIS) project (to HML). The project was also supported in part by the Centre National de la Recherche Scientifique (CNRS), the Institut National de la Santé Et de la Recherche Médicale (INSERM), the Institut Pasteur and the French Infrastructure for Integrated Structural Biology (FRISBI) ANR-10-INBS-05. PW was the recipient of a PhD fellowship from the EU H2020 (2014-2020) under the Marie Skłodowska-Curie Grant Agreement No. 675555, Accelerated Early staGe drug discovery (AEGIS)., ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), and European Project: 675555,H2020,H2020-MSCA-ITN-2015,AEGIS(2016)

Subjects
crystal structure, allostery, [SDV]Life Sciences [q-bio], Cryoelectron Microscopy, Uridine Triphosphate, Cell Biology, nucleotide metabolism, Gram-Positive Bacteria, Biochemistry, Anti-Bacterial Agents, Adenosine Triphosphate, effector, Allosteric Regulation, cryo-EM single particle analysis, Humans, Amino Acid Sequence, Guanosine Triphosphate, enzyme regulation, Nucleoside-Phosphate Kinase, Uridine Monophosphate, Molecular Biology, UMP kinase
Abstract

International audience; Tuberculosis claims significantly more than one million lives each year. A feasible way to face the issue of drug resistance is the development of new antibiotics. Bacterial uridine 5’-monophosphate (UMP) kinase is a promis- ing target for novel antibiotic discovery as it is essential for bacterial sur- vival and has no counterpart in human cells. The UMP kinase from M. tuberculosis is also a model of particular interest for allosteric regula- tion with two effectors, GTP (positive) and UTP (negative). In this study, using X-ray crystallography and cryo-electron microscopy, we report for the first time a detailed description of the negative effector UTP-binding site of a typical Gram-positive behaving UMP kinase. Comparison between this snapshot of low affinity for Mg-ATP with our previous 3D-structure of the GTP-bound complex of high affinity for Mg-ATP led to a better understanding of the cooperative mechanism and the allosteric regulation of UMP kinase. Thermal shift assay and circular dichroism experiments corroborate our model of an inhibition by UTP linked to higher flexibility of the Mg-ATP-binding domain. These new structural insights provide valuable knowledge for future drug discovery strategies targeting bacterial UMP kinases.

Published
2022

28. DNA-binding mechanism and evolution of Replication Protein A

Author

Clément Madru, Markel Martinez-Carranza, Sébastien Laurent, Alessandra C. Alberti, Maelenn Chevreuil, Bertrand Raynal, Ahmed Haouz, Rémy A. Le Meur, Marc Delarue, Didier Flament, Mart Krupovic, Pierre Legrand, and Ludovic Sauguet

Abstract

Replication Protein A (RPA) is a heterotrimeric single stranded DNA-binding protein with essential roles in DNA replication, recombination and repair, in both eukaryotic and archaeal cells. By using an integrative approach that combines three crystal structures, four cryo-EM structures in complex with single-stranded DNA (ssDNA) of different lengths, we extensively characterized RPA from Pyrococcus abyssi in different states. These structures show two essential features conserved in eukaryotes: a trimeric core and a module that promotes cooperative binding to ssDNA, as well as a newly identified archaeal-specific domain. These structures reveal for the first time how ssDNA is handed over from one RPA complex to the other, and uncover an unanticipated mechanism of self-association on ssDNA tracts. This work constitutes a significant step forward in the molecular understanding of the structure and DNA-binding mechanism of RPA, with far-reaching implications for the evolution of this primordial replication factor in Archaea and Eukarya.

Published
2022

31. Essential dynamic interdependence of FtsZ and SepF for Z-ring and septum formation in Corynebacterium glutamicum

Author

Quentin Gaday, Anne Marie Wehenkel, Adrià Sogues, Sylvain Trépout, Rosario Durán, Alexandre Chenal, Mathilde Ben Assaya, Martín Graña, Pedro M. Alzari, Mariano A. Martinez, Alexis Voegele, Patrick England, Ahmed Haouz, Michael S. VanNieuwenhze, Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Institut Pasteur [Paris] (IP)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Bioinformatics / Bioinformática [Montevideo], Institut Pasteur de Montevideo, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), Biochimie des Interactions Macromoléculaires / Biochemistry of Macromolecular Interactions, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Department of Chemistry, Indiana University, Indiana University [Bloomington], Indiana University System-Indiana University System, Biophysique Moléculaire (Plate-forme), Cristallographie (Plateforme) - Crystallography (Platform), Institut Curie [Paris], Chimie et modélisation pour la biologie du cancer (CMBC), Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Analytical Biochemistry and Proteomics / Bioquímica y Proteómica Analíticas [Montevideo], This work was partially supported by grants from the Institut Pasteur (Paris), the CNRS (France) and the Agence Nationale de la Recherche (PhoCellDiv, ANR-18-CE11-0017-01). A.S. is part of the Pasteur-Paris University (PPU) International Ph.D Program, funded by the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No 665807. Q.G. was funded by MTCI Ph.D school (ED 563), A.V. was supported by a DIM MalInf (infectious diseases) grant. M.G. acknowledges support from Programa de Desarrollo de las Ciencias Básicas and Sistema Nacional de Investigación e Innovación, Uruguay., We thank A. Ducret for help with MicrobeJ, F. Gubellini for help with electron microscopy, M. Bott and M. Baumgart for the pk19-P3323-lcpA plasmid and help with corynebacterial genetics, and H. Gramajo for the pTGR5 plasmid. We gratefully acknowledge the core facilities at the Institut Pasteur C2RT, in particular G. Pehau-Arnaudet (UBI), B. Raynal, S. Brule (PFBMI), P. Weber, C. Pissis (PFC), and J. Fernandes (UtechS PBI/Imagopole, supported by France BioImaging, ANR-10–INSB–04, Investments for the Future). We thank the staff of ESRF and of EMBL-Grenoble for assistance and support in using beamlines ID30B and ID23-1, and the staff of SOLEIL Synchrotron for assistance in using the beamline Disco. We acknowledge the PICT-IBISA for providing access to the cryo-EM facility at Orsay. Finally, we would like to thank the reviewers for their coments and suggestions, which have helped us to improve the quality of the manuscript., ANR-18-CE11-0017,PhoCellDiv,Mécanismes moléculaires phospho-dépendants de l'assemblage et de la régulation du divisome bactérien(2018), European Project: 665807,H2020,H2020-MSCA-COFUND-2014,PASTEURDOC(2015), BENEDIC, Bénédicte, APPEL À PROJETS GÉNÉRIQUE 2018 - Mécanismes moléculaires phospho-dépendants de l'assemblage et de la régulation du divisome bactérien - - PhoCellDiv2018 - ANR-18-CE11-0017 - AAPG2018 - VALID, Institut Pasteur International Docotal Program - PASTEURDOC - - H20202015-10-01 - 2020-10-01 - 665807 - VALID, Institut Pasteur [Paris]-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), and Université Paris Diderot - Paris 7 (UPD7)-Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, Cell division, [SDV]Life Sciences [q-bio], ved/biology.organism_classification_rank.species, Cell, General Physics and Astronomy, Plasma protein binding, physiological processes, Corynebacterium glutamicum, lcsh:Science, Peptide sequence, 0303 health sciences, Multidisciplinary, biology, Chemistry, Cell biology, [SDV] Life Sciences [q-bio], medicine.anatomical_structure, biological phenomena, cell phenomena, and immunity, Dimerization, Cell division site, Protein Binding, Science, 030106 microbiology, Sequence alignment, macromolecular substances, Bacterial physiology, General Biochemistry, Genetics and Molecular Biology, Article, 03 medical and health sciences, Bacterial Proteins, medicine, Amino Acid Sequence, Model organism, Mode of action, FtsZ, X-ray crystallography, 030304 developmental biology, 030306 microbiology, ved/biology, General Chemistry, Gene Expression Regulation, Bacterial, biology.organism_classification, Cytoskeletal proteins, 030104 developmental biology, biology.protein, bacteria, lcsh:Q, Sequence Alignment, Function (biology), Cytokinesis, Bacteria
Abstract

The mechanisms of Z-ring assembly and regulation in bacteria are poorly understood, particularly in non-model organisms. Actinobacteria, a large bacterial phylum that includes the pathogen Mycobacterium tuberculosis, lack the canonical FtsZ-membrane anchors and Z-ring regulators described for E. coli. Here we investigate the physiological function of Corynebacterium glutamicum SepF, the only cell division-associated protein from Actinobacteria known to interact with the conserved C-terminal tail of FtsZ. We show an essential interdependence of FtsZ and SepF for formation of a functional Z-ring in C. glutamicum. The crystal structure of the SepF–FtsZ complex reveals a hydrophobic FtsZ-binding pocket, which defines the SepF homodimer as the functional unit, and suggests a reversible oligomerization interface. FtsZ filaments and lipid membranes have opposing effects on SepF polymerization, indicating that SepF has multiple roles at the cell division site, involving FtsZ bundling, Z-ring tethering and membrane reshaping activities that are needed for proper Z-ring assembly and function., The mechanisms of Z-ring assembly and regulation in bacteria are poorly understood, particularly in non-model organisms. Here, Sogues et al. study the interaction between FtsZ and SepF in Corynebacterium glutamicum, showing an essential interdependence of these proteins for formation of a functional Z-ring.

Published
2020

32. Dynamics and structural changes of calmodulin upon interaction with its potent antagonist calmidazolium

Author

Corentin Léger, Irène Pitard, Mirko Sadi, Nicolas Carvalho, Sébastien Brier, Ariel Mechaly, Dorothée Raoux-Barbot, Maryline Davi, Sylviane Hoos, Patrick Weber, Patrice Vachette, Dominique Durand, Ahmed Haouz, J. Iñaki Guijarro, Daniel Ladant, and Alexandre Chenal

Abstract

Calmodulin (CaM) is a eukaryotic multifunctional, calcium-modulated protein that regulates the activity of numerous effector proteins involved in a variety of physiological processes. Calmidazolium (CDZ) is a potent small molecule antagonist of CaM and one the most widely used inhibitors of CaM in cell biology. Here, we report the structural characterization of CaM:CDZ complexes using combined SAXS, X-ray crystallography, HDX-MS and NMR approaches. Our results provide molecular insights into the CDZ-induced dynamics and structural changes of CaM leading to its inhibition. CDZ-binding induces an open-to-closed conformational change of CaM and results in a strong stabilization of its structural elements associated with a reduction of protein dynamics over a large time range. These CDZ-triggered CaM changes mimic those induced by CaM-binding peptides derived from protein targets, despite their distant chemical nature. CaM residues in close contact with CDZ and involved in the stabilization of the CaM:CDZ complex have been identified. These results open the way to rationally design new CaM-selective drugs.Figure and text for the Table of Contents (ToC)Calmidazolium is a potent and widely used inhibitor of calmodulin, a major mediator of calcium-signaling in eukaryotic cells. Structural characterization of calmidazolium-binding to calmodulin reveals that it triggers open-to-closed conformational changes similar to those induced by calmodulin-binding peptides derived from enzyme targets. These results open the way to rationally design new and more selective inhibitors of calmodulin.

Published
2022

33. Structural basis of synergistic neutralization of Crimean-Congo hemorrhagic fever virus by human antibodies

Author

Akaash K. Mishra, Jan Hellert, Natalia Freitas, Pablo Guardado-Calvo, Ahmed Haouz, J. Maximilian Fels, Daniel P. Maurer, Dafna M. Abelson, Zachary A. Bornholdt, Laura M. Walker, Kartik Chandran, François-Loïc Cosset, Jason S. McLellan, Felix A. Rey, University of Texas at Austin [Austin], Virologie Structurale - Structural Virology, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Université Paris Cité (UPCité), Centre International de Recherche en Infectiologie (CIRI), École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Université Jean Monnet - Saint-Étienne (UJM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Cristallographie (Plateforme) - Crystallography (Platform), Albert Einstein College of Medicine [New York], Adimab LLC, Mapp Biopharmaceutical Inc., This work was supported by National Institutes of Health award U19 AI142777 to J.S.M., Z.A.B., K.C., and L.M.W., as well as by Institut Pasteur, CNRS and grant ANR-10-LABX-62-10 IBEID to F.A.R. and by the LabEx Ecofect (ANR-11-LABX-0048) of the 'Université de Lyon,' within the program 'Investissements d’Avenir' (ANR-11-IDEX-0007) operated by the French National Research Agency (ANR), to F.-L.C. Research was funded in part by Welch Foundation grant F-0003-19620604 awarded to J.S.M. The Pasteur-Cantarini 24-month fellowship was granted to J.H., who was further supported by the Région Ile de France (Domaine d’intérêt majeur - innovative technologies for life sciences, DIM 1HEALTH)., ANR-11-LABX-0048,ECOFECT,Dynamiques eco-évolutives des maladies infectieuses(2011), ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), ANR-11-IDEX-0007,Avenir L.S.E.,PROJET AVENIR LYON SAINT-ETIENNE(2011), Centre International de Recherche en Infectiologie - UMR (CIRI), École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Ribierre, Hélène, Dynamiques eco-évolutives des maladies infectieuses - - ECOFECT2011 - ANR-11-LABX-0048 - LABX - VALID, Integrative Biology of Emerging Infectious Diseases - - IBEID2010 - ANR-10-LABX-0062 - LABX - VALID, and PROJET AVENIR LYON SAINT-ETIENNE - - Avenir L.S.E.2011 - ANR-11-IDEX-0007 - IDEX - VALID

Subjects
Models, Molecular, Protein Folding, Multidisciplinary, Protein Conformation, [SDV]Life Sciences [q-bio], Virus Internalization, Antibodies, Viral, Crystallography, X-Ray, Antibodies, Neutralizing, Article, [SDV] Life Sciences [q-bio], Epitopes, Immunoglobulin Fab Fragments, Protein Domains, Neutralization Tests, Hemorrhagic Fever Virus, Crimean-Congo, Humans, Protein Multimerization, Viral Fusion Proteins, Protein Binding
Abstract

International audience; Crimean-Congo hemorrhagic fever virus (CCHFV) is the most widespread tick-borne zoonotic virus, with a 30% case fatality rate in humans. Structural information is lacking in regard to the CCHFV membrane fusion glycoprotein Gc-the main target of the host neutralizing antibody response-as well as antibody-mediated neutralization mechanisms. We describe the structure of prefusion Gc bound to the antigen-binding fragments (Fabs) of two neutralizing antibodies that display synergy when combined, as well as the structure of trimeric, postfusion Gc. The structures show the two Fabs acting in concert to block membrane fusion, with one targeting the fusion loops and the other blocking Gc trimer formation. The structures also revealed the neutralization mechanism of previously reported antibodies against CCHFV, providing the molecular underpinnings essential for developing CCHFVspecific medical countermeasures for epidemic preparedness.

Published
2022

34. Integrative Study of the Structural and Dynamical Properties of a KirBac3.1 Mutant: Functional Implication of a Highly Conserved Tryptophan in the Transmembrane Domain

Author

Charline Fagnen, Ludovic Bannwarth, Iman Oubella, Dania Zuniga, Ahmed Haouz, Eric Forest, Rosa Scala, Saïd Bendahhou, Rita De Zorzi, David Perahia, Catherine Vénien-Bryan, Institut de minéralogie, de physique des matériaux et de cosmochimie (IMPMC), Muséum national d'Histoire naturelle (MNHN)-Institut de recherche pour le développement [IRD] : UR206-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de biologie et pharmacologie appliquée (LBPA), Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Ecole Normale Supérieure Paris-Saclay (ENS Paris Saclay), Cristallographie (Plateforme) - Crystallography (Platform), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Université Grenoble Alpes (UGA), Laboratoire de PhysioMédecine Moléculaire (LP2M), Université Nice Sophia Antipolis (1965 - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA), Università degli studi di Trieste = University of Trieste, This research was funded by SERVIER and ANR-CNRS CACSICE (CVB), AFM-téléthongrant (#19928 and #23207, SB and CVB) and Ph.D. research funding, MESRI (CF and DZ). Thiswork was performed using HPC resources from the Mésocentre computing center of Centrale-Supélec and ENS Paris-Saclay supported by CNRS and the Région Île-de-France (http://mesocentre.centralesupelec.fr/, accessed on 1 March 2021). It was granted access to the HPC resources of IDRISunder the allocation 100512 made by GENCI/IDRIS (DP)., and ANR-11-EQPX-0008,CACSICE,Centre d'analyse de systèmes complexes dans les environnements complexes(2011)

Subjects
QH301-705.5, [SDV]Life Sciences [q-bio], Hydrogen Deuterium Exchange-Mass Spectrometry, Crystallography, X-Ray, Catalysis, Article, Protein Structure, Secondary, Inorganic Chemistry, Protein Domains, neonatal diabetes mellitus, [CHIM]Chemical Sciences, Amino Acid Sequence, Protein Interaction Maps, Physical and Theoretical Chemistry, Potassium Channels, Inwardly Rectifying, Biology (General), Molecular Biology, QD1-999, Spectroscopy, Conserved Sequence, HDX-mass spectrometry, Organic Chemistry, crystal structure of KirBac3.1 W46R, molecular dynamics, normal modes, electrophysiology, gain of function Kir, Tryptophan, General Medicine, Computer Science Applications, Chemistry, Mutation, Mutant Proteins, Ion Channel Gating
Abstract

International audience; ATP-sensitive potassium (K-ATP) channels are ubiquitously expressed on the plasma membrane of cells in several organs, including the heart, pancreas, and brain, and they govern a wide range of physiological processes. In pancreatic β-cells, K-ATP channels composed of Kir6.2 and SUR1 play a key role in coupling blood glucose and insulin secretion. A tryptophan residue located at the cytosolic end of the transmembrane helix is highly conserved in eukaryote and prokaryote Kir channels. Any mutation on this amino acid causes a gain of function and neonatal diabetes mellitus. In this study, we have investigated the effect of mutation on this highly conserved residue on a KirBac channel (prokaryotic homolog of mammalian Kir6.2). We provide the crystal structure of the mutant KirBac3.1 W46R (equivalent to W68R in Kir6.2) and its conformational flexibility properties using HDX-MS. In addition, the detailed dynamical view of the mutant during the gating was investigated using the in silico method. Finally, functional assays have been performed. A comparison of important structural determinants for the gating mechanism between the wild type KirBac and the mutant W46R suggests interesting structural and dynamical clues and a mechanism of action of the mutation that leads to the gain of function.

Published
2022

36. The epitope arrangement on flavivirus particles contributes to Mab C10’s extraordinary neutralization breadth across Zika and dengue viruses

Author

Gavin R. Screaton, Pablo Guardado-Calvo, Félix A. Rey, Xinghong Dai, Danyang Gong, Patrick England, Alexander Rouvinski, Marie-Christine Vaney, Ahmed Haouz, Z. Hong Zhou, Wanwisa Dejnirattisai, Stéphane Duquerroy, Ren Sun, Arvind Sharma, Xiaokang Zhang, Wiyada Wongwiwat, Juthathip Mongkolsapaya, Virologie Structurale - Structural Virology, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Université Paris Cité (UPCité), Institut Pasteur de Shanghai, Académie des Sciences de Chine - Chinese Academy of Sciences (IPS-CAS), Réseau International des Instituts Pasteur (RIIP), University of Oxford, University of California [Los Angeles] (UCLA), University of California (UC), Université Paris-Saclay, Centre de Ressources et de Recherche Technologique - Center for Technological Resources and Research (C2RT), Institut Pasteur [Paris] (IP)-Université Paris Cité (UPCité), Cristallographie (Plateforme) - Crystallography (Platform), Siriraj Hospital, Mahidol University, Mahidol University [Bangkok], This project was supported by a Wellcome Trust collaborative grant (UNS22082 to G.R.S., J.M., and F.A.R.), by the National Institute for Health Research Biomedical Research Centre Funding Scheme (to G.R.S.), and by grants from the National Institutes of Health (DE028583, AI094386, and DE025567 to Z.H.Z. and R.S.)., and We thank Fabrice Agou and the staff of the Chemogenomic and Biological Screening Platform for access to the MALS equipment as well as the staff of the Crytallography core facility at the Institut Pasteur for robot-driven crystallization screens. We acknowledge the use of synchrotron beam lines PX1 and PX2 at SOLEIL (St Aubin, France), ID29 and ID23-1 at ESRF (Grenoble, France) and X06SA at SLS (Villigen, Switzerland), and we thank their staff for assistance. We also acknowledge the use of the resources at the Electron Imaging Center for Nanomachines, supported in part by 1S10RR23057, 1S10OD018111, 1U24GM116792 and NSF DBI-1338135 and DMR-1548924 at UCLA. G.R.S. is supported as a Wellcome Trust Senior Investigator (095541/A/11/Z). F.A.R. is supported by Institut Pasteur and the CNRS.

Subjects
medicine.drug_class, [SDV]Life Sciences [q-bio], MESH: Vero Cells, MESH: Zika Virus, MESH: Dengue, Biology, Dengue virus, Monoclonal antibody, medicine.disease_cause, MESH: Dengue Virus, complex mixtures, General Biochemistry, Genetics and Molecular Biology, Epitope, Neutralization, Zika virus, MESH: Antibodies, Monoclonal, MESH: Antibodies, Neutralizing, MESH: Drosophila melanogaster, MESH: Cross Reactions, MESH: Protein Conformation, MESH: Zika Virus Infection, MESH: Chlorocebus aethiops, medicine, MESH: Protein Binding, MESH: Animals, X-ray crystallography, MESH: Humans, Flaviviruses, broadly neutralizing antibodies, biology.organism_classification, Virology, MESH: Cell Line, Flavivirus, MESH: HEK293 Cells, MESH: Viral Envelope Proteins, biology.protein, vaccine design, cryo-EM, Paratope, lipids (amino acids, peptides, and proteins), Antibody, MESH: Antibodies, Viral
Abstract

International audience; The human monoclonal antibody C10 exhibits extraordinary cross-reactivity, potently neutralizing Zika virus (ZIKV) and the four serotypes of dengue virus (DENV1-DENV4). Here we describe a comparative structure-function analysis of C10 bound to the envelope (E) protein dimers of the five viruses it neutralizes. We demonstrate that the C10 Fab has high affinity for ZIKV and DENV1 but not for DENV2, DENV3, and DENV4. We further show that the C10 interaction with the latter viruses requires an E protein conformational landscape that limits binding to only one of the three independent epitopes per virion. This limited affinity is nevertheless counterbalanced by the particle's icosahedral organization, which allows two different dimers to be reached by both Fab arms of a C10 immunoglobulin. The epitopes' geometric distribution thus confers C10 its exceptional neutralization breadth. Our results highlight the importance not only of paratope/epitope complementarity but also the topological distribution for epitope-focused vaccine design.

Published
2021

37. Interactions of SARS-CoV-2 protein E with cell junctions and polarity PDZ-containing proteins

Author

Yanlei Zhu, Flavio Alvarez, Nicolas Wolff, Ariel Mechaly, Sébastien Brûlé, Benoit Neitthoffer, Sandrine Etienne-Manneville, Ahmed Haouz, Batiste Boëda, and Célia Caillet-Saguy

Subjects
fungi, chemical and pharmacologic phenomena
Abstract

The C-terminus of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) protein E contains a PBM (PDZ binding motif) targeting PDZ (PSD-95/Dlg/ZO-1) domains identical to the PBM of SARS-CoV. The latter is involved in the pathogenicity of the virus. Recently, we identified ten human PDZ-containing proteins showing significant interactions with SARS-CoV-2 protein E PBM. We selected several of them involved in cellular junctions and cell polarity (TJP1, PARD3, MLLT4, LNX2) and MPP5/Pals1 previously shown to interact with SARS-CoV E PBM. Targeting cellular junctions and polarity components is a common strategy by viruses to hijack cell machinery to their advantage. In this study, we showed that these host PDZ domains TJP1, PARD3, MLLT4, LNX2 and MPP5/PALS1 interact in a PBM-dependent manner in vitro and colocalize with the full-length E protein in cellulo, sequestrating the PDZ domains to the Golgi compartment. We solved three crystal structures of complexes between human LNX2, MLLT4 and MPP5 PDZs and SARS-CoV-2 E PBM highlighting its binding preferences for several cellular targets. Finally, we showed different affinities for the PDZ domains with the original SARS-CoV-2 C-terminal sequence containing the PBM and the one of the beta variant that contains a mutation close to the PBM. The acquired mutations in E protein localized near the PBM might have important effects both on the structure and the ion-channel activity of the E protein and on the host machinery targeted by the variants during the infection.

Published
2021

38. Structural characterization of a pathogenic mutant of human protein tyrosine phosphatase PTPN2 (Cys216Gly) that causes very early onset autoimmune enteropathy

Author

Ahmed Haouz, Rongxing Liu, Ariel E. Mechaly, Jean-Marie Dupret, Marianna Parlato, Fernando Rodrigues Lima, Nadine Cerf-Bensussan, Qing Nian, Jérémy Berthelet, Unité de Biologie Fonctionnelle et Adaptative (BFA (UMR_8251 / U1133)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), University of Electronic Science and Technology of China [Chengdu] (UESTC), Imagine - Institut des maladies génétiques (IHU) (Imagine - U1163), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), Cristallographie (Plateforme) - Crystallography (Platform), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), This work was supported by institutional grants from Université de Paris, Institut Pasteur and CNRS. Q. N. and R. L. were supported by a PhD fellowship from the China Scholarship Council (CSC). J. B. was supported by a PhD fellowship from Région Ile de France (Cancéropole)., and The authors thank the staff of the crystallography platform at Institut Pasteur for carrying out robot-driven crystallization screening. The authors acknowledge synchroton SOLEIL (Saint-Aubin, France) for granting access to their facility and the staff of Proxima1 for helpful assistance during the data collection. The authors also thank the technical platform 'Bioprofiler' from the BFA Unit for provision of UFLC facilities.

Subjects
crystal structure, animal structures, Protein Conformation, [SDV]Life Sciences [q-bio], Mutant, Inflammation, Protein tyrosine phosphatase, Biology, Autoimmune enteropathy, pathogenic mutation, protein tyrosine phosphatase, environment and public health, Biochemistry, medicine, Humans, Polyendocrinopathies, Autoimmune, Molecular Biology, chemistry.chemical_classification, Protein Tyrosine Phosphatase, Non-Receptor Type 2, Pathogenic mutation, medicine.disease, Very early onset, Cell biology, enzymes and coenzymes (carbohydrates), Haematopoiesis, Enzyme, chemistry, PTPN2, medicine.symptom, Protein Structure Reports, Signal Transduction
Abstract

PTPN2 is an important protein tyrosine phosphatase (PTP) that plays a key role in cell signaling. Deletions or inactivating mutations of PTPN2 has been described in different pathologies and underline its critical role in hematopoiesis, auto-immunity and inflammation. Surprisingly, despite the major pathophysiological implications of PTPN2, the structural analysis of this PTP and notably of its pathogenic mutants remain poorly documented. Contrary to other human PTP enzymes, to date, only one structure of PTPN2 (wild-type form) has been reported. Here, we report the first crystal structure of a pathogenic mutant of PTPN2 (Cys216Gly) that causes an autoimmune enteropathy. We show in particular that this mutant adopts a classical PTP fold. More importantly, albeit inactive, the mutant retains its ability to bind substrates and to adopt the characteristic catalytically competent closed form of PTP enzymes. This novel PTPN2 structure may serve as a new tool to better understand PTP structures and the structural impacts of pathogenic mutations. Moreover, the C216G PTPN2 structure could also be helpful to design specific ligands/inhibitors. This article is protected by copyright. All rights reserved.

Published
2021

39. A High‐Affinity Calmodulin‐Binding Site in the CyaA Toxin Translocation Domain is Essential for Invasion of Eukaryotic Cells

Author

Nicolas Rodriguez, Daniel Ladant, Maryline Davi, Ahmed Haouz, Bertrand Raynal, Darragh P. O'Brien, Alexis Voegele, Ariel E. Mechaly, Patrick Weber, Sébastien Brûlé, Pauline Gehan, Dominique Durand, Mirko Sadi, Sylviane Hoos, Sébastien Brier, Alexandre Chenal, Dorothée Raoux-Barbot, Patrice Vachette, Biochimie des Interactions Macromoléculaires / Biochemistry of Macromolecular Interactions, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Université de Paris (UP), Laboratoire des biomolécules (LBM UMR 7203), Centre National de la Recherche Scientifique (CNRS)-Sorbonne Université (SU)-Département de Chimie - ENS Paris, École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Chimie Moléculaire de Paris Centre (FR 2769), Institut de Chimie du CNRS (INC)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS)-Ecole Nationale Supérieure de Chimie de Paris - Chimie ParisTech-PSL (ENSCP), Université Paris sciences et lettres (PSL)-Ecole Superieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI Paris), Université Paris sciences et lettres (PSL)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Ecole Nationale Supérieure de Chimie de Paris - Chimie ParisTech-PSL (ENSCP), Université Paris sciences et lettres (PSL)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM), Biophysique Moléculaire (plateforme) - Molecular Biophysics (platform), Cristallographie (Plateforme) - Crystallography (Platform), Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Plateforme technologique de RMN biologique - Biological NMR Technological Platform, A.V. was supported by a DIM MalInf grant from the region Ile‐de‐France. M.S. was supported by the Pasteur – Paris University (PPU) International PhD Program. D.P.O.B. was supported by Institut Pasteur (grants PasteurInnoV15006‐01A and PTR451). P.G. was supported by Sorbonne Université. Funding is acknowledged from Agence Nationale de la Recherche (grant number CACSICE Equipex ANR‐11‐EQPX‐0008). Region Ile de France (grant number DIM MalInf 2016), CNRS (UMR 3528), Institut Pasteur (grant numbers PasteurInnoV15006‐01A, PTR451 and PTR166‐19, PPUIP program). The funders have no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript., The authors acknowledge SOLEIL and ESRF for provision of synchrotron radiation facilities. The authors thank the staffs of the DISCO, PROXIMA‐1, and SWING beamlines for constant support and help during data collection (Synchrotron SOLEIL, St Aubin, France) and MASSIF (Synchrotron ESRF, Grenoble, France) beamlines for assistance during the X‐ray diffraction data collection., ANR-11-EQPX-0008,CACSICE,Centre d'analyse de systèmes complexes dans les environnements complexes(2011), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Université Paris Cité (UPCité), Chimie Moléculaire de Paris Centre (FR 2769), École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Ecole Nationale Supérieure de Chimie de Paris - Chimie ParisTech-PSL (ENSCP), Université Paris sciences et lettres (PSL)-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Département de Chimie - ENS Paris, Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Ecole Nationale Supérieure de Chimie de Paris - Chimie ParisTech-PSL (ENSCP), Université Paris sciences et lettres (PSL)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Ecole Nationale Supérieure de Chimie de Paris - Chimie ParisTech-PSL (ENSCP), Université Paris sciences et lettres (PSL)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM), Chenal, Alexandre, and Equipements d'excellence - Centre d'analyse de systèmes complexes dans les environnements complexes - - CACSICE2011 - ANR-11-EQPX-0008 - EQPX - VALID

Subjects
calmodulin, General Chemical Engineering, General Physics and Astronomy, Medicine (miscellaneous), 02 engineering and technology, [SDV.BBM.BM] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, 01 natural sciences, Bordetella pertussis, protein membrane interaction, General Materials Science, Internalization, Lipid bilayer, [SDV.BBM.BC] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], media_common, biology, Full Paper, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Chemistry, General Engineering, Full Papers, 021001 nanoscience & nanotechnology, [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, Protein Transport, Eukaryotic Cells, Adenylate Cyclase Toxin, 0210 nano-technology, Protein Binding, adenylate cyclase, Calmodulin, [SDV.BBM.BS] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], CyaA toxin, media_common.quotation_subject, Science, Antimicrobial peptides, [SDV.BBM.BP] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, protein–protein interactions, 010402 general chemistry, Biochemistry, Genetics and Molecular Biology (miscellaneous), Protein–protein interaction, Protein Domains, membrane translocation, Binding site, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Binding Sites, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, cyaA, 0104 chemical sciences, Cytosol, Biophysics, biology.protein
Abstract

The molecular mechanisms and forces involved in the translocation of bacterial toxins into host cells are still a matter of intense research. The adenylate cyclase (CyaA) toxin from Bordetella pertussis displays a unique intoxication pathway in which its catalytic domain is directly translocated across target cell membranes. The CyaA translocation region contains a segment, P454 (residues 454–484), which exhibits membrane‐active properties related to antimicrobial peptides. Herein, the results show that this peptide is able to translocate across membranes and to interact with calmodulin (CaM). Structural and biophysical analyses reveal the key residues of P454 involved in membrane destabilization and calmodulin binding. Mutational analysis demonstrates that these residues play a crucial role in CyaA translocation into target cells. In addition, calmidazolium, a calmodulin inhibitor, efficiently blocks CyaA internalization. It is proposed that after CyaA binding to target cells, the P454 segment destabilizes the plasma membrane, translocates across the lipid bilayer and binds calmodulin. Trapping of CyaA by the CaM:P454 interaction in the cytosol may assist the entry of the N‐terminal catalytic domain by converting the stochastic motion of the polypeptide chain through the membrane into an efficient vectorial chain translocation into host cells., The mechanism of cell invasion by bacterial toxins is still poorly understood. The adenylate cyclase (CyaA) toxin from Bordetella pertussis, the causative agent of whooping cough, directly translocates its catalytic domain across plasma membranes. The membrane‐permeabilizing segment (yellow) of CyaA translocates across the plasma membrane and binds calmodulin (red), which assists the entry of the catalytic domain (blue) into host cells while the hydrophobic and acylation domains (green) interact with the membrane and the C‐terminal Repeat‐in‐Toxin domain (grey) remains in the extra‐cellular milieu.

Published
2021

40. Anion exchange effect on structural, antibacterial activity and molecular docking of 1-methyl-3-(4-vinylbenzyl) imidazol-3-ium chloride/bis(trifluoromethylsulfonyl)imide/hexaflurophosphate: Experimental and DFT approach

Author

Abdelkader Tahir, Mohamed Belhocine, Djilali Bassou, Riadh Bourzami, Fayçal Dergal, Louiza Ouksel, Mansour Debdab, Zakaria Benladghem, Abdelkader Ammari, Wafaa Kendil, Somia Bouktab, Ahmed Haouzi, Yassine Chaker, K․L. Chai, and Azizan Ahmad

Subjects
Ionic liquids, NMR, ATG-DSC, DFT calculations, Molecular docking, Antibacterial activity, Chemistry, QD1-999
Abstract

The impact of NTF2− and PF6− anions on the physical, chemical and antibacterial characteristics of ionic liquids (ILs) based 1-methyl-3-(4-vinylbenzyl) imidazol-3-ium VBMIM+ cations is the subject of this work. The molecular structures were compared to the DFT-optimized ones after being investigated experimentally using FTIR and NMR spectroscopy. As a result, the different vibrational modes were identified and assigned. However, DSC and TGA have been used to examine the thermal properties of the ILs. The compounds depicted good thermal stability. Furthermore, different parameters were computed to get insights on the stability and reactivity of the chemical. The ILs were tested for their antibacterial and fungicidal activities against nine reference strains of gram-negative and gram-positive bacteria. Finally, the discovered biological results were supported by the molecular docking analysis.

Published
2024
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41. Conformational plasticity of the response regulator CpxR, a key player in Gammaproteobacteria virulence and drug-resistance

Author

Pedro M. Alzari, Nathalie Sassoon, Ariel E. Mechaly, Ahmed Haouz, Alejandro Buschiazzo, Jean-Michel Betton, Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Institut Pasteur [Paris]-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Université Paris Diderot - Paris 7 (UPD7), Cristallographie (Plateforme) - Crystallography (Platform), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Département de Microbiologie - Department of Microbiology, Institut Pasteur [Paris], Institut Pasteur de Montevideo, Réseau International des Instituts Pasteur (RIIP), Institut Pasteur [Paris] (IP)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP), and Cristallographie (Plate-forme)

Subjects
0301 basic medicine, Virulence, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], biology, Chemistry, Gene Expression Regulation, Bacterial, Drug resistance, Plasticity, biology.organism_classification, medicine.disease_cause, Cell biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], 03 medical and health sciences, Response regulator, 030104 developmental biology, Bacterial Proteins, X-Ray Diffraction, Structural Biology, Transcription (biology), Gammaproteobacteria, Transcriptional regulation, medicine, Escherichia coli
Abstract

International audience; The transcriptional regulator CpxR mediates an adaptive response to envelope stress, tightly linked to virulence and antibiotics resistance in several Gammaproteobacteria pathogens. In this work, we integrated crystallographic and small-angle X-ray scattering data to gain insights into the structure and conformational plasticity of CpxR from Escherichia coli. CpxR dimerizes through two alternative interaction surfaces. Moreover, widely different CpxR conformations coexist in solution, from compact to fully extended ones. The possible functional implications of these structural features are discussed.

Published
2018

43. A high-affinity calmodulin-binding site in the CyaA toxin translocation domain is essential for invasion into eukaryotic cells

Author

Alexandre Chenal, Mirko Sadi, Patrick Weber, Pauline Gehan, Ariel E. Mechaly, Nicolas Rodriguez, Alexis Voegele, Sébastien Brier, Patrice Vachette, Daniel Ladant, Darragh P. O'Brien, Maryline Davi, Dorothée Raoux-Barbot, Sylviane Hoos, Bertrand Raynal, Sébastien Brûlé, Dominique Durand, and Ahmed Haouz

Subjects
Calmodulin, biology, Chemistry, media_common.quotation_subject, Antimicrobial peptides, Chromosomal translocation, cyaA, Cell biology, Cytosol, biology.protein, Binding site, Internalization, Lipid bilayer, media_common
Abstract

The molecular mechanisms and forces involved in the translocation of bacterial toxins into host cells have thus far remained elusive. The adenylate cyclase (CyaA) toxin fromBordetella pertussisdisplays a unique intoxication pathway in which its catalytic domain is directly translocated across target cell membranes. We have previously identified a translocation region in CyaA that contains a segment, P454 (residues 454–484), exhibiting membrane-active properties related to antimicrobial peptides. Herein, we show that this peptide is able to translocate across membranes and interact with calmodulin. Structural and biophysical analyses have revealed the key residues of P454 involved in membrane destabilization and calmodulin binding. Mutational analysis demonstrated that these residues play a crucial role in CyaA translocation into target cells. We have also shown that calmidazolium, a calmodulin inhibitor, efficiently blocks CyaA internalization. We propose that after CyaA binding to target cells, the P454 segment destabilizes the plasma membrane, translocates across the lipid bilayer and binds calmodulin. Trapping of the CyaA polypeptide chain by the CaM:P454 interaction in the cytosol may assist the entry of the N-terminal catalytic domain by converting the stochastic process of protein translocation into an efficient vectorial chain transfer into host cells.

Published
2020

44. A third purine biosynthetic pathway encoded by aminoadenine-based viral DNA genomes

Author

Ahmed Haouz, Simonetta Gribaldo, Pierre Simon Garcia, Najwa Taib, Pascal Röthlisberger, Pierre Alexandre Kaminski, Marion Lagune, Philippe Marliere, Jérôme Loc’h, Dona Sleiman, Biologie des Bactéries pathogènes à Gram-positif - Biology of Gram-Positive Pathogens, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Biologie Evolutive de la Cellule Microbienne - Evolutionary Biology of the Microbial Cell, Adaptation au stress et Métabolisme chez les entérobactéries - Stress adaptation and metabolism in enterobacteria (SAMe), Dynamique structurale des Macromolécules / Structural Dynamics of Macromolecules, Cristallographie (Plateforme) - Crystallography (Platform), Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, Chimie bioorganique des acides nucléiques - Bioorganic chemistry of nucleic acids, Génomique métabolique (UMR 8030), Genoscope - Centre national de séquençage [Evry] (GENOSCOPE), Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université d'Évry-Val-d'Essonne (UEVE)-Centre National de la Recherche Scientifique (CNRS), D.S. and P.S.G. were supported by an ERASynBio 'TNA episome' and by an ERACoBioTech 'Iron Plug’n Play' fellowship, respectively. S.G. acknowledges funding from the French National Agency for research grant Archaevol (ANR-16-CE02-0005-01). This study was funded from the French government’s Investissement d’Avenir program, Laboratoire d’Excellence Integrative Biology of Emerging Infectious Diseases (grant no. ANR-10-LABX-62-IBEID)., ANR-16-CE02-0005,Arch-Evol,Approches phylogenomiques pour étudier l'origine et évolution des Archées(2016), ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université d'Évry-Val-d'Essonne (UEVE)

Subjects
Purine, DNA polymerase, Guanine, Genome, Viral, Viral Nonstructural Proteins, Crystallography, X-Ray, Genome, 03 medical and health sciences, chemistry.chemical_compound, Adenylosuccinate Synthase, MESH: 2-Aminopurine, A-DNA, MESH: Adenylosuccinate Synthase, Bacteriophages, MESH: Bacteriophages, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], MESH: Phylogeny, 2-Aminopurine, Phylogeny, 030304 developmental biology, chemistry.chemical_classification, 0303 health sciences, Multidisciplinary, biology, ATP synthase, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], 030302 biochemistry & molecular biology, MESH: Crystallography, X-Ray, MESH: DNA, Viral, Biosynthetic Pathways, Enzyme, chemistry, Biochemistry, DNA, Viral, biology.protein, MESH: Viral Nonstructural Proteins, MESH: Biosynthetic Pathways, MESH: Genome, Viral, DNA
Abstract

Biosynthesis and replication, from A to Z Four nucleobases. adenine (A), cytosine (C), guanine (G), and thymine (T), are usually thought to be invariable in DNA. In bacterial viruses, however, each of the DNA bases have variations that help them to escape degradation by bacterial restriction enzymes. In the genome of cyanophage S-2L, A is completely replaced by diaminopurine (Z), which forms three hydrogen bonds with T and thus creates non–Watson-Crick base pairing in the DNA of this virus (see the Perspective by Grome and Isaacs). Zhou et al. and Sleiman et al. determined the biochemical pathway that produces Z, which revealed more Z genomes in viruses hosted in bacteria distributed widely in the environment and phylogeny. Pezo et al. identified a DNA polymerase that incorporates Z into DNA while rejecting A. These findings enrich our understanding of biodiversity and expand the genetic palette for synthetic biology. Science , this issue p. 512 , 516 , 520 ; see also p. 460

Published
2020

45. Evolution of abiotic cubane chemistries in a nucleic acid aptamer allows selective recognition of a malaria biomarker

Author

Marcel Hollenstein, Patrick Weber, Ariel E. Mechaly, Ahmed Haouz, Pascal Röthlisberger, Fabienne Levi-Acobas, G. Paul Savage, Young Lo, Alvin W C Wong, Julian A. Tanner, Yee-Wai Cheung, AB Kinghorn, The University of Hong Kong (HKU), Chimie bioorganique des acides nucléiques - Bioorganic chemistry of nucleic acids, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Cristallographie (Plateforme) - Crystallography (Platform), Division of Manufacturing Science and Technology (CSIRO), Commonwealth Scientific and Industrial Research Organisation [Canberra] (CSIRO), and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)

Subjects
M.H, Plasmodium vivax, MESH: Aptamers, Nucleotide, Protozoan Proteins, MESH: Molecular Diagnostic Techniques, 01 natural sciences, malaria diagnosis, Nucleotide, MESH: Molecular Dynamics Simulation, MESH: Protozoan Proteins, chemistry.chemical_classification, modified aptamer, 0303 health sciences, Multidisciplinary, biology, Hydrogen bond, SELEX, Y.W.C. and P.R. were responsible, [CHIM.ORGA]Chemical Sciences/Organic chemistry, Aptamers, Nucleotide, 3. Good health, MESH: Plasmodium vivax, Molecular Diagnostic Techniques, Physical Sciences, Protein Binding, MESH: L-Lactate Dehydrogenase, Aptamer, MESH: Malaria, Computational biology, Molecular Dynamics Simulation, 010402 general chemistry, 03 medical and health sciences, cubane, parasitic diseases, MESH: Protein Binding, [CHIM]Chemical Sciences, Humans, MESH: Hydrogen Bonding, 030304 developmental biology, X-ray crystallography, MESH: Humans, L-Lactate Dehydrogenase, Mechanism (biology), Plasmodium falciparum, Hydrogen Bonding, biology.organism_classification, 0104 chemical sciences, Malaria, chemistry, Nucleic acid, MESH: Biomarkers, malaria diagnosis Author Contributions J.A.T, Systematic evolution of ligands by exponential enrichment, Biomarkers
Abstract

We thank the Shirley Boyde Trust for their generous support through a Shirley Boyde Foundation Grant to J.A.T. and acknowledge Institut Pasteur funding to M.H., A.H., P.R., and F.L.-A.. J.A.T. acknowledges The University of Hong Kong Outstanding Young Researcher Award 2015-16, The University of Hong Kong Outstanding Research Student Supervisor Award 2016-2017, and The University of Hong Kong Seed Fund for Basic Research grants 201711159185 and 201611159269. We thank the staff of the platform for production and purification of recombinant proteins of Institut Pasteur for technical assistance during the expression and purification of the proteins steps and the staff of the crystallography platform at Institut Pasteur for carrying out robot-driven crystallization screening. We acknowledge synchrotron SOLEIL (Saint-Aubin, France) for granting access to their facility and the staff of Proxima1 for helpful assistance during the data collection.; International audience; for the overall study design and data interpretation. Y.W.C. performed all cubamer selections, characterized the cubamers and oversaw all application experiments. A.W.C.W., Y.L., and A.B.K. were involved in aspects of cubamer characterization and in assay design. F.L.-A. expressed and purified the proteins and performed the biochemical characterization of the triphosphate. P.R. performed all chemical syntheses and was involved in aspects of experimental design of cubamer structural analysis. P.R. was also significantly involved in all data interpretation. G.P.S. was involved in steps of synthesis relating to cubanes and advised on study design.

Published
2020

46. Defective lytic transglycosylase disrupts cell morphogenesis by hindering cell wall de-O-acetylation in Neisseria meningitidis

Author

Joseph P. Dillard, Ahmed Haouz, Muhamed-Kheir Taha, William P. Robins, Christian Malosse, Ala-Eddine Deghmane, Julia Chamot-Rooke, Maryse Moya Nilges, Ryan E. Schaub, Ivo G. Boneca, Ignacio Santecchia, Richard J. Wheeler, Allison H. Williams, Samia Hicham, Biologie et Génétique de la Paroi bactérienne - Biology and Genetics of Bacterial Cell Wall, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Immunologie anti-tumorale et immunothérapie des cancers (ITIC), Institut Gustave Roussy (IGR)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris-Saclay, Infections Bactériennes Invasives, Institut Pasteur [Paris], Université Paris Descartes - Paris 5 (UPD5), Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Plateforme BioImagerie Ultrastructurale – Ultrastructural BioImaging Platform (UTechS UBI), Spectrométrie de Masse pour la Biologie – Mass Spectrometry for Biology (UTechS MSBio), Institut Pasteur [Paris]-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Cristallographie (Plateforme) - Crystallography (Platform), Harvard Medical School [Boston] (HMS), European Molecular Biology Organization (ALTF 732-2010), European Research Council (PGN from SHAPE to VIR 202283), Fondation pour la Recherche Médicale (DBF20160635726), Institut Carnot-Pasteur (Maladies Infectious fellowship), Institut Carnot Pasteur Microbes and Santé, Fondation pour la Recherche Médicale (FDT201805005258)AHW was supported by an EMBO long-term fellowship (ALTF 732–2010) and an Institut Carnot-Pasteur Maladies Infectious fellowship. This work was supported by an ERC starting grant (PGN from SHAPE to VIR 202283) and a Fondation pour la recherche médicale (FRM) grant Programme d’Urgence (DBF20160635726) to IGB. This study received funding from the French Government′s Investissement d′Avenir program, Laboratoire d′Excellence ‘Integrative Biology of Emerging Infectious Diseases’ (grant no. ANR- 10-LABX-62-IBEID). IS was supported by the Institut Carnot Pasteur Microbes and Santé given to the Pasteur-Paris University PhD program and the ‘Fin de these de science’ number FDT201805005258 granted by 'Fondation pour la recherche médicale (FRM). The UtechS Photonic BioImaging (Imagopole), C2RT, Institut Pasteur was supported by the French National Research Agency (France BioImaging, ANR-10–INSB–04, Investments for the Future)., We acknowledge the synchrotron beamline staff (PROXIMA-1 at SOLEIL and X06DA at SLS) for their assistance. We are extremely grateful to Frederick Saul, Patrick Weber and Marco Bellinzoni for their constant helpful guidance, advice and assistance. We thank Dr. Antoine Forget for the advice on and help with figure presentations., ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), ANR-10-INBS-0004,France-BioImaging,Développment d'une infrastructure française distribuée coordonnée(2010), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP), and Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, cell division, Cell division, QH301-705.5, Science, infectious disease, 030106 microbiology, Neisseria meningitidis, peptidoglycan, General Biochemistry, Genetics and Molecular Biology, Bacterial cell structure, Cell wall, Lytic transglycosylase, 03 medical and health sciences, chemistry.chemical_compound, Cell Wall, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Catalytic Domain, Morphogenesis, Amino Acid Sequence, Biology (General), X-ray crystallography, chemistry.chemical_classification, Microbiology and Infectious Disease, General Immunology and Microbiology, biology, Cell morphogenesis, General Neuroscience, microbiology, E. coli, Active site, Glycosyltransferases, General Medicine, Cell biology, 030104 developmental biology, Enzyme, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, chemistry, Lytic cycle, cell separation, biology.protein, Medicine, Peptidoglycan, Research Article, Protein Binding
Abstract

Lytic transglycosylases (LT) are enzymes involved in peptidoglycan (PG) remodeling. However, their contribution to cell-wall-modifying complexes and their potential as antimicrobial drug targets remains unclear. Here, we determined a high-resolution structure of the LT, an outer membrane lipoprotein from Neisseria species with a disordered active site helix (alpha helix 30). We show that deletion of the conserved alpha-helix 30 interferes with the integrity of the cell wall, disrupts cell division, cell separation, and impairs the fitness of the human pathogen Neisseria meningitidis during infection. Additionally, deletion of alpha-helix 30 results in hyperacetylated PG, suggesting this LtgA variant affects the function of the PG de-O-acetylase (Ape 1). Our study revealed that Ape 1 requires LtgA for optimal function, demonstrating that LTs can modulate the activity of their protein-binding partner. We show that targeting specific domains in LTs can be lethal, which opens the possibility that LTs are useful drug-targets., eLife digest Bacteria are surrounded by a tough yet flexible wall that protects the cell and serves as an anchor for several of the cell’s structures. This cell wall contains a large mesh-like molecule called peptidoglycan made of many repeated building blocks. When a bacterial cell divides in two, it needs to make more of this material. Making peptidoglycan involves two different sets of enzymes working together: “polymerases” are the enzymes that link the individual building blocks to peptidoglycan, one after the other; while “lytic transglycosylases” are enzymes that modify the peptidoglycan to create space for the addition of new building blocks and for assemblies of proteins that must span the cell wall. Lytic transglycosylases are known to assemble with other proteins and enzymes to form the cell’s peptidoglycan-modifying machinery, but it was not clear exactly what purpose they serve within these “enzyme complexes”. It was also unclear whether these enzymes would be good targets for new antibiotics. To help answer these questions, Williams et al. looked at a lytic transglycoslyase called LtgA. This enzyme is originally from Neisseria meningitidis, a bacterium that can cause meningitis and life-threatening sepsis in humans. Williams et al. discovered that part of the enzyme’s active site – the region of an enzyme where the chemical reaction takes – can switch from an ordered helix to a disordered, flexible loop. Bacteria were then genetically engineered to make a version of the enzyme that lacked this helix. These bacteria had weaker cell walls and were deformed; they were also less able to grow and divide, both in the laboratory and in a mouse model of infection. Further analysis showed that the deletion of the helix from the enzyme resulted in the peptidoglycan being modified much more than normal, which could likely explain their reduced virulence. Williams et al. also found that deleting the helix from LtgA interfered with the activity of a protein that interacts with this enzyme, called Ape1, which also contributed to the fragility of the cell wall. This shows that lytic transglycosylases assembled into enzyme complexes can alter the activities of other proteins in the complex. Together these findings show that researchers could target one enzyme in a complex in bacteria, and disrupt the activity of other proteins in that complex. This highlights the possibility of considering enzyme complexes as useful targets for new drugs, which is important considering the current problem of antibiotic resistance.

Published
2020

47. Structure of the prefusion-locking broadly neutralizing antibody RVC20 bound to the rabies virus glycoprotein

Author

Olivier Schwartz, Amalio Telenti, Guilherme D. Melo, Florence Larrous, Julian Buchrieser, Davide Corti, Leah Soriaga, Félix A. Rey, Ahmed Haouz, Jan Hellert, Patrick England, Hervé Bourhy, Andrea Minola, Virologie Structurale - Structural Virology, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Virus et Immunité - Virus and immunity (CNRS-UMR3569), Lyssavirus, épidémiologie et neuropathologie - Lyssavirus Epidemiology and Neuropathology, Institut Pasteur [Paris] (IP), Humabs BioMed SA, Vir Biotechnology Inc [San Francisco], Biophysique Moléculaire (plateforme) - Molecular Biophysics (platform), Cristallographie (Plateforme) - Crystallography (Platform), This work was funded by Institut Pasteur. F.A.R. received additional funding from the CNRS, the Labex IBEID (ANR-10-IHUB-0002), the GIS IBiSA (Infrastructures en biologie santé et agronomie), grant ANR-13-ISV8-0002-01 and the Région Ile de France in order to support the work of J.H. (Domaine d’intérêt majeur innovative technologies for life sciences, DIM 1HEALTH). O.S. received funding from ANRS, Sidaction, the Vaccine Research Institute (ANR-10-LABX-77), the Labex IBEID (ANR-10-IHUB-0002), the 'TIMTAMDEN' ANR-14-CE14-0029, the 'CHIKV-Viro-Immuno' ANR-14-CE14-0015-01, L’Oréal Sponsorship, the CNRS and the Gilead HIV cure program. H.B., F.L. and G.D.M. received funding from the INFECT-ERA 2016 project ANR 16-IFEC-0006-01 ToRRENT.Part of this work was performed at the UtechS Photonic BioImaging (PBI) platform, member of France Life Imaging network (grant ANR-11-INBS-0006)., ANR-13-ISV8-0002,flavistem,La protéine E des flavivirus : interactions et fusion membranaire(2013), ANR-10-LABX-0077,VRI,Initiative for the creation of a Vaccine Research Institute(2010), ANR-14-CE14-0029,TIMTAMDEN,Rôle des récepteurs TIM et TAM dans l'infection des cellules cibles par le virus de la dengue(2014), ANR-14-CE14-0015,CHIKV-Viro-Immuno,Multiplication et Relation avec l'hôte du virus Chikungunya(2014), ANR-16-IFEC-0006,ToRRENT,Towards a combined post-exposure prophylaxis and successful treatment of rabies in humans(2016), ANR-11-INBS-0006,FLI,France Life Imaging(2011), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Virus et Immunité - Virus and immunity, Institut Pasteur [Paris], and Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris]

Subjects
Viral membrane fusion, 0301 basic medicine, medicine.medical_treatment, [SDV]Life Sciences [q-bio], Antibody Affinity, General Physics and Astronomy, MESH: Amino Acid Sequence, Antibodies, Viral, Crystallography, X-Ray, medicine.disease_cause, MESH: Antibodies, Neutralizing, Epitopes, MESH: Antibody Affinity, MESH: Animals, lcsh:Science, MESH: Mutagenesis, chemistry.chemical_classification, Multidisciplinary, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], 3. Good health, MESH: Rabies virus, Perfusion, Antibody, Encephalitis, MESH: Perfusion, Protein Binding, MESH: Epitopes, medicine.drug_class, Science, 030106 microbiology, MESH: Glycoproteins, Biology, Monoclonal antibody, Article, Antibodies, General Biochemistry, Genetics and Molecular Biology, Cell Line, Viral Proteins, 03 medical and health sciences, medicine, Animals, Humans, MESH: Protein Binding, Amino Acid Sequence, Post-exposure prophylaxis, X-ray crystallography, Glycoproteins, MESH: Humans, Rabies virus, Lipid bilayer fusion, General Chemistry, medicine.disease, MESH: Crystallography, X-Ray, Antibodies, Neutralizing, Virology, MESH: Viral Proteins, MESH: Cell Line, 030104 developmental biology, chemistry, Viral infection, Mutagenesis, biology.protein, Rabies, lcsh:Q, Glycoprotein, MESH: Antibodies, Viral
Abstract

Rabies virus (RABV) causes fatal encephalitis in more than 59,000 people yearly. Upon the bite of an infected animal, the development of clinical disease can be prevented with post-exposure prophylaxis (PEP), which includes the administration of Rabies immunoglobulin (RIG). However, the high cost and limited availability of serum-derived RIG severely hamper its wide use in resource-limited countries. A safe low-cost alternative is provided by using broadly neutralizing monoclonal antibodies (bnAbs). Here we report the X-ray structure of one of the most potent and most broadly reactive human bnAbs, RVC20, in complex with its target domain III of the RABV glycoprotein (G). The structure reveals that the RVC20 binding determinants reside in a highly conserved surface of G, rationalizing its broad reactivity. We further show that RVC20 blocks the acid-induced conformational change required for membrane fusion. Our results may guide the future development of direct antiviral small molecules for Rabies treatment., Rabies virus (RABV) infection of unvaccinated individuals can be treated with post exposure prophylaxis. Here, Hellert et al. analyze the structure of a broadly neutralizing human antibody in complex with its target domain in the RABV glycoprotein and demonstrate that it blocks the acid-induced conformational change required for membrane fusion.

Published
2020

48. PLA2G1B is involved in CD4 anergy and CD4 lymphopenia in HIV-infected patients

Author

Gérard Lambeau, José Alcamí, Jacques Thèze, Ezequiel Ruiz-Mateos, Florence Bugault, Frederick Saul, Louise Jeammet, Thierry Rose, Annalisa Meola, Ahmed Haouz, Luc Teyton, David Geny, Julien Pothlichet, Diaccurate SAS, Centre de Recherche et Innovation Technologique (CITECH), Institut Pasteur [Paris] (IP), Département de Santé Globale - Department Global Health, Cristallographie (Plateforme) - Crystallography (Platform), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Institut de psychiatrie et neurosciences de Paris (IPNP - U1266 Inserm), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), Instituto de Salud Carlos III [Madrid] (ISC), Institut d’investigations Biomèdiques August I Sunyer (IDIBASPS), Institute of Biomedicine of Seville (IBIS), Consejo Superior de Investigaciones Científicas [Spain] (CSIC), The Scripps Research Institute [La Jolla, San Diego], Institut de pharmacologie moléculaire et cellulaire (IPMC), Université Nice Sophia Antipolis (1965 - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA), This work was part of the ANRS programs EP20, EP33, and EP36 (J.F. Delfraissy, O. Lambotte). It was initially supported by the Institut Pasteur (PTR 424) and the Pasteur-Weizmann Foundation., Agence Nationale de la Recherche (France), Institut Pasteur, Lambeau, Gerard, Institut Pasteur [Paris], Cristallographie (Plate-forme), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Paris (UP), University of Seville, Scripps Research Institute, Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015 - 2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015 - 2019) (COMUE UCA)-Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA), Centre National de la Recherche Scientifique (CNRS)-Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Université Côte d'Azur (UCA), and COMUE Université Côte d'Azur (2015 - 2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015 - 2019) (COMUE UCA)-Université Côte d'Azur (UCA)

Subjects
0301 basic medicine, CD4-Positive T-Lymphocytes, Male, T cell, Immunology, T cells, HIV Infections, Gp41, [SDV.IMM.II]Life Sciences [q-bio]/Immunology/Innate immunity, AIDS/HIV, 03 medical and health sciences, 0302 clinical medicine, Immune system, In vivo, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Lymphopenia, medicine, Humans, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Group IB Phospholipases A2, Receptor, Lipid raft, [SDV.IMM.II] Life Sciences [q-bio]/Immunology/Innate immunity, Immunodeficiency, Lipid rafts, Clonal Anergy, Chemistry, General Medicine, medicine.disease, In vitro, 3. Good health, 030104 developmental biology, medicine.anatomical_structure, [SDV.IMM.IA]Life Sciences [q-bio]/Immunology/Adaptive immunology, 030220 oncology & carcinogenesis, [SDV.IMM.IA] Life Sciences [q-bio]/Immunology/Adaptive immunology, Cancer research, [SDV.MHEP.MI] Life Sciences [q-bio]/Human health and pathology/Infectious diseases, HIV-1, Cytokines, Female, Research Article
Abstract

The precise mechanism leading to profound immunodeficiency of HIV-infected patients is still only partially understood. Here, we show that more than 80% of CD4+ T cells from HIV-infected patients have morphological abnormalities. Their membranes exhibited numerous large abnormal membrane microdomains (aMMDs), which trap and inactivate physiological receptors, such as that for IL-7. In patient plasma, we identified phospholipase A2 group IB (PLA2G1B) as the key molecule responsible for the formation of aMMDs. At physiological concentrations, PLA2G1B synergized with the HIV gp41 envelope protein, which appears to be a driver that targets PLA2G1B to the CD4+ T cell surface. The PLA2G1B/gp41 pair induced CD4+ T cell unresponsiveness (anergy). At high concentrations in vitro, PLA2G1B acted alone, independently of gp41, and inhibited the IL-2, IL-4, and IL-7 responses, as well as TCR-mediated activation and proliferation, of CD4+ T cells. PLA2G1B also decreased CD4+ T cell survival in vitro, likely playing a role in CD4 lymphopenia in conjunction with its induced IL-7 receptor defects. The effects on CD4+ T cell anergy could be blocked by a PLA2G1B-specific neutralizing mAb in vitro and in vivo. The PLA2G1B/gp41 pair constitutes what we believe is a new mechanism of immune dysfunction and a compelling target for boosting immune responses in HIV-infected patients., This work was part of the ANRS programs EP20, EP33, and EP36 (J.F. Delfraissy, O. Lambotte). It was initially supported by the Institut Pasteur (PTR 424) and the Pasteur-Weizmann Foundation.

Published
2020

49. Deciphering the Unexpected Binding Capacity of the Third PDZ Domain of Whirlin to Various Cochlear Hair Cell Partners

Author

Baptiste Colcombet-Cazenave, Ariel E. Mechaly, Sylviane Hoos, Nicolas Wolff, Florence Cordier, Célia Caillet-Saguy, Susanne Lüchow, Ahmed Haouz, Ylva Ivarsson, Bertrand Raynal, Amel Bahloul, Yanlei Zhu, Elise Pepermans, Florent Delhommel, Sébastien Brûlé, Candice Gautier, Virginie Girault, Récepteurs Canaux - Channel Receptors, Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], Ecole Doctorale Complexité du Vivant (ED515), Sorbonne Université (SU), Bioinformatique structurale - Structural Bioinformatics, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Uppsala Universitet [Uppsala], Cristallographie (Plateforme) - Crystallography (Platform), Génétique et Physiologie de l'Audition, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), Institut Pasteur, Fondation Cenci Bolognetti - Istituto Pasteur Italia, Fondazione Cenci Bolognetti, Réseau International des Instituts Pasteur (RIIP), Biophysique Moléculaire (plateforme) - Molecular Biophysics (platform), The project has received funding from the European Union's Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement PDZnet No 675341. The authors also acknowledge the Conseil Régional d'Ile de France (France) for financial support through the SESAME 2014 NMRCHR program No. 14014526 (800 MHz spectrometer)., European Project: 675341,H2020-MSCA-ITN-2015,PDZNet(2016), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), and Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)

Subjects
Scaffold protein, Stereocilia (inner ear), [SDV]Life Sciences [q-bio], PDZ domain, PDZ Domains, Cell Cycle Proteins, Myosins, Stereocilia, 03 medical and health sciences, 0302 clinical medicine, Structural Biology, Myosin, Hair Cells, Auditory, medicine, otorhinolaryngologic diseases, Humans, Inner ear, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, CASK, Molecular Biology, Biology, 030304 developmental biology, 0303 health sciences, Stereocilium, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Chemistry, Membrane Proteins, Proteins, Cell biology, [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, Cytoskeletal Proteins, medicine.anatomical_structure, Hair cell, sense organs, Guanylate Kinases, 030217 neurology & neurosurgery, Protein Binding
Abstract

Hearing is a mechanical and neurochemical process, which occurs in the hair cells of inner ear that converts the sound vibrations into electrical signals transmitted to the brain. The multi-PDZ scaffolding protein whirlin plays a critical role in the formation and function of stereocilia exposed at the surface of hair cells. In this article, we reported seven stereociliary proteins that encode PDZ binding motifs (PBM) and interact with whirlin PDZ3, where four of them are first reported. We solved the atomic resolution structures of complexes between whirlin PDZ3 and the PBMs of myosin 15a, CASK, harmonin a1 and taperin. Interestingly, the PBM of CASK and taperin are rare non-canonical PBM, which are not localized at the extreme C terminus. This large capacity to accommodate various partners could be related to the distinct functions of whirlin at different stages of the hair cell development. (C) 2020 Published by Elsevier Ltd.

Published
2020
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50. Structural Basis of Neutralization by Human Antibodies Targeting Crimean-Congo Hemorrhagic Fever Virus Glycoprotein Gc

Author

Laura M. Walker, Dafna M. Abelson, François-Loïc Cosset, Pablo Guardado-Calvo, Natalia Freitas, J. Maximilian Fels, Elisabeth K. Nyakatura, Daniel P. Maurer, Jonathan R. Lai, Jan Hellert, Ariel S. Wirchnianski, Jason S. McLellan, Kartik Chandran, Akaash K. Mishra, Zachary A. Bornholdt, Ahmed Haouz, Olivia Vergnolle, and Félix A. Rey

Subjects
chemistry.chemical_classification, Epitope mapping, biology, chemistry, biology.protein, Antibody, Yeast display, Neutralizing antibody, Glycoprotein, Pathogen, Virology, Crimean Congo hemorrhagic fever virus, Neutralization
Abstract

Crimean-Congo hemorrhagic fever virus (CCHFV), the only endemic biosafety level 4 pathogen in Europe, is the world’s most widely distributed tick-borne zoonotic virus with a 30% fatality rate in humans, underlining the need for specific therapeutics and vaccines. The CCHFV membrane fusion glycoprotein Gc is the main target of the host neutralizing antibody response. Here we describe the structure of pre-fusion Gc in complex with the antigen-binding fragments (Fabs) corresponding to a therapeutically potent bispecific antibody as well as the structure of unbound Gc in its post-fusion conformation. Our findings suggest that one Fab blocks insertion of the fusion loops into the target membrane, whereas the other blocks formation of the post-fusion trimer. Combined with yeast display of mutagenized Gc, the structures allowed epitope mapping of a large panel of neutralizing antibodies. These data provide the essential molecular underpinnings for developing vaccines and therapeutics against CCHFV.

Published
2020

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