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J Mol Biol. 2017 Jun 2;429(11):1746-1765. doi: 10.1016/j.jmb.2017.04.005. Epub 2017 Apr 17.

Polar N-terminal Residues Conserved in Type 2 Secretion Pseudopilins Determine Subunit Targeting and Membrane Extraction Steps during Fibre Assembly.

Author information

1
Université Paris Diderot (Paris 7) Sorbonne Paris Cité, 11 Place Marcelin Berthelot, 75231 Paris, France; Laboratory of Intercellular Communication and Microbial Infections, CIRB, Collège de France, 75231 Paris, France; Institut National de la Santé et de la Recherche Médicale (Inserm) U1050, 75231 Paris, France; Centre National de la Recherche Scientifique (CNRS), UMR7241, 75231 Paris, France; MEMOLIFE Laboratory of Excellence and Paris Sciences et Lettres, 75231 Paris, France.
2
Centre for Molecular Informatics, Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK.
3
Laboratory of Macromolecular Systems and Signalling, Institut Pasteur, Department of Microbiology and CNRS ERL6002, 25 rue du Dr Roux, 75724 Paris, Cedex 15, France; Biochemistry of Macromolecular Interactions Unit, Department of Structural Biology and Chemistry, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris, Cedex 15, France.
4
Laboratory of Macromolecular Systems and Signalling, Institut Pasteur, Department of Microbiology and CNRS ERL6002, 25 rue du Dr Roux, 75724 Paris, Cedex 15, France.
5
Bioinformatics Institute (A*STAR), 30 Biopolis Str, #07-01 Matrix, Singapore 138671, Singapore; Department of Biological Sciences, Faculty of Science, National University of Singapore, 5A Engineering Drive 1, Singapore 117411, Singapore.
6
Laboratory of Intercellular Communication and Microbial Infections, CIRB, Collège de France, 75231 Paris, France; Institut National de la Santé et de la Recherche Médicale (Inserm) U1050, 75231 Paris, France; Centre National de la Recherche Scientifique (CNRS), UMR7241, 75231 Paris, France; MEMOLIFE Laboratory of Excellence and Paris Sciences et Lettres, 75231 Paris, France.
7
Laboratory of Macromolecular Systems and Signalling, Institut Pasteur, Department of Microbiology and CNRS ERL6002, 25 rue du Dr Roux, 75724 Paris, Cedex 15, France; Biochemistry of Macromolecular Interactions Unit, Department of Structural Biology and Chemistry, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris, Cedex 15, France. Electronic address: ofrancet@pasteur.fr.

Abstract

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 PulGE5A assembly defect. First, PulGT2A 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 PulGE5A, methylated PulGWT (MePulGWT), and MePulGE5A 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.

KEYWORDS:

N-methylation; immunofluorescence microscopy; membrane proteins; molecular dynamics simulations; type 4 fibre assembly

PMID:
28427876
DOI:
10.1016/j.jmb.2017.04.005
[Indexed for MEDLINE]
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