title: Cysteine scanning mutagenesis and disulfide mapping analysis of arrangement of GspC and GspD protomers within the type 2 secretion system.
abstract: The type II secretion system (T2SS) secretes enzymes and toxins across the outer membrane of Gram-negative bacteria. The precise assembly of T2SS, which consists of at least 12 core-components called Gsp, remains unclear. The outer membrane secretin, GspD, forms the channels, through which folded proteins are secreted, and interacts with the inner membrane component, GspC. The periplasmic regions of GspC and GspD consist of several structural domains, HR(GspC) and PDZ(GspC), and N0(GspD) to N3(GspD), respectively, and recent structural and functional studies have proposed several interaction sites between these domains. We used cysteine mutagenesis and disulfide bonding analysis to investigate the organization of GspC and GspD protomers and to map their interaction sites within the secretion machinery of the plant pathogen Dickeya dadantii. At least three distinct GspC-GspD interactions were detected, and they involve two sites in HR(GspC), two in N0(GspD), and one in N2(GspD). None of these interactions occurs through static interfaces because the same sites are also involved in self-interactions with equivalent neighboring domains. Disulfide self-bonding of critical interaction sites halts secretion, indicating the transient nature of these interactions. The secretion substrate diminishes certain interactions and provokes an important rearrangement of the HR(GspC) structure. The T2SS components OutE/L/M affect various interaction sites differently, reinforcing some but diminishing the others, suggesting a possible switching mechanism of these interactions during secretion. Disulfide mapping shows that the organization of GspD and GspC subunits within the T2SS could be compatible with a hexamer of dimers arrangement rather than an organization with 12-fold rotational symmetry.
authors.affiliation: Université de Lyon, F-69003, Université Lyon 1, INSA-Lyon, Villeurbanne F-69621, CNRS, UMR5240, Microbiologie Adaptation et Pathogénie, Lyon F-69622, France.
authors.forename: Xiaohui
authors.initials: X
authors.lastname: Wang
authors.forename: Camille
authors.initials: C
authors.lastname: Pineau
authors.forename: Shuang
authors.initials: S
authors.lastname: Gu
authors.forename: Natalia
authors.initials: N
authors.lastname: Guschinskaya
authors.forename: Richard W
authors.initials: RW
authors.lastname: Pickersgill
authors.forename: Vladimir E
authors.initials: VE
authors.lastname: Shevchik
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D058947:Bacterial Secretion Systems / Q000502:physiology*; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D016974:Dickeya chrysanthemi / Q000235:genetics / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D016296:Mutagenesis; D010449:Peptide Mapping / Q000379:methods; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary
chemical_list: D001426:Bacterial Proteins; D058947:Bacterial Secretion Systems; D004220:Disulfides; D008565:Membrane Proteins; D003545:Cysteine