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

JOURNAL OF MOLECULAR BIOLOGY

卷:429

Article

Santos-Moreno, Javier^1,2,3,4,5  East, Alexandra⁶  Guilvout, Ingrid^7,8,9  Nadeau, Nathalie^7,8  Bond, Peter J.^10,11  Guy Tran Van Nhieu^2,3,4,5  Francetic, Olivera^7,8,9 

[1] Univ Paris Diderot Paris 7, Sorbonne Paris Cite, 11 Pl Marcelin Berthelot, F-75231 Paris, France

[2] Coll France, Lab Intercellular Commun & Microbial Infect, CIRB, F-75231 Paris, France

[3] INSERM, U1050, F-75231 Paris, France

[4] CNRS, UMR7241, F-75231 Paris, France

[5] MEMOLIFE Lab Excellence & Paris Sci & Lettres, F-75231 Paris, France

[6] Univ Cambridge, Dept Chem, Ctr Mol Informat, Lensfield Rd, Cambridge CB2 1EW, England

[7] Inst Pasteur, Lab Macromol Syst & Signalling, Dept Microbiol, 25 Rue Dr Roux, F-75724 Paris 15, France

[8] CNRS, ERL6002, 25 Rue Dr Roux, F-75724 Paris 15, France

[9] Inst Pasteur, Biochem Macromol Interact Unit, Dept Struct Biol & Chem, 28 Rue Dr Roux, F-75724 Paris 15, France

[10] ASTAR, Bioinformat Inst, 30 Biopolis Str 07-01 Matrix, Singapore 138671, Singapore

[11] Natl Univ Singapore, Fac Sci, Dept Biol Sci, 5A Engn Dr 1, Singapore 117411, Singapore

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

DOI  :  10.1016/j.jmb.2017.04.005

来源: Elsevier

【 摘 要 】

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 PuIG remains N-methylated but is affected in interaction with the T2SS component PuIM. Here, biochemical and functional analyses showed that the PuIM interaction defect only partly accounts for the PulG(E5A) assembly defect. First, PulG(T2A) variant, equally defective in PuIM 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 PuIG, we propose that PuIM acts as chaperone to promote pseudopilin recruitment and coordinate its membrane extraction with subsequent steps of the fibre assembly process. (C) 2017 Elsevier Ltd. All rights reserved.

【 授权许可】

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