Contributors: Centre interdisciplinaire de recherche en biologie (CIRB); Labex MemoLife; École normale supérieure - Paris (ENS-PSL); Université Paris Sciences et Lettres (PSL)-Université Paris Sciences et Lettres (PSL)-Collège de France (CdF (institution))-Ecole Superieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI Paris); Université Paris Sciences et Lettres (PSL)-École normale supérieure - Paris (ENS-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 Diderot - Paris 7 (UPD7); Université Paris Sciences et Lettres (PSL); University of Cambridge UK (CAM); Biochimie des Interactions Macromoléculaires / Biochemistry of Macromolecular Interactions; Institut Pasteur Paris (IP)-Centre National de la Recherche Scientifique (CNRS); Systèmes macromoléculaires et Signalisation; Agency for science, technology and research Singapore (A*STAR); National University of Singapore (NUS); This work was funded by the ANR FiberSpace grant N°ANR-14-CE09-0004. Javier Santos-Moreno was funded by a fellowship from the Basque Government, and Alexandra East was supported by an EPSRC PhD studentship. The computational work benefited from use of the Darwin Supercomputer of the University of Cambridge High Performance Computing Service (http://www.hpc.cam.ac.uk/), provided by Dell Inc. using Strategic Research, Infrastructure Funding from the Higher Education Funding Council for England.; We thank Evelyne Richet and Jenny-Lee Thomassin for insightful comments and critical reading of the manuscript. We thank Cesar Valencia and Jenny-Lee Thomassin for help with IF data processing and statistical analysis, Mariette Bonnet for help and advice, and Stéphane Romero for stimulating discussions about biological fibres. We are grateful to all members of the Laboratory of Intercellular Communication and Microbial Infections, the Laboratory for Macromolecular Systems and Signalling and of the Biochemstry of Macromolecular Interactions Unit for helpful discussions and friendly support. We thank Gouzel Karimova and Daniel Ladant for the strains, plasmids, and advice concerning the BAC2H analysis.; ANR-14-CE09-0004,FiberSpace,Pili de type IV et pseudopili: structure, dynamique, assemblage et fonction moléculaire(2014)
نبذة مختصرة : International audience ; Bacterial type 2 secretion systems (T2SS), type 4 pili, and archaeal flagella assemble fibres from initially membrane-embedded pseudopilin and pilin subunits. Fibre subunits are made as precursors with positively charged N-terminal anchors, whose cleavage via the prepilin peptidase, essential for pilin membrane extraction and assembly, is followed by N-methylation of the mature (pseudo)pilin N terminus. The conserved Glu residue at position 5 (E5) of mature (pseudo)pilins is essential for assembly. Unlike T4 pilins, where E5 residue substitutions also abolish N-methylation, the E5A variant of T2SS pseudopilin PulG remains N-methylated but is affected in interaction with the T2SS component PulM. Here, biochemical and functional analyses showed that the PulM interaction defect only partly accounts for the PulG(E5A) assembly defect. First, PulG(T2A) variant, equally defective in PulM interaction, remained partially functional. Furthermore, pseudopilus assembly defect of pulG(E5A) mutant was stronger than that of the pulM deletion mutant. To understand the dominant effect of E5A mutation, we used molecular dynamics simulations of PulG(E5A), methylated PulG(WT) (MePulG(WT)), and MePulG(E5A) variant in a model membrane. These simulations pointed to a key role for an intramolecular interaction between the pseudopilin N-terminal amine and E5 to limit polar interactions with membrane phospholipids. N-methylation of the N-terminal amine further limited its interactions with phospholipid head-groups to facilitate pseudopilin membrane escape. By binding to polar residues in the conserved N-terminal region of PulG, we propose that PulM acts as chaperone to promote pseudopilin recruitment and coordinate its membrane extraction with subsequent steps of the fibre assembly process.
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