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