The single transmembrane segment drives self-assembly of OutC and the formation of a functional type II secretion system in Erwinia chrysanthemi.
Many pathogenic Gram-negative bacteria secrete toxins and lytic enzymes via a multiprotein complex called the type II secretion system. This system, named Out in Erwinia chrysanthemi, consists of 14 proteins integrated or associated with the two bacterial membranes. OutC, a key player in this process, is probably implicated in the recognition of secreted proteins and signal transduction. OutC possesses a short cytoplasmic sequence, a single transmembrane segment (TMS), and a large periplasmic region carrying a putative PDZ domain. A hydrodynamic study revealed that OutC forms stable dimers of an elongated shape, whereas the PDZ domain adopts a globular shape. Bacterial two-hybrid, cross-linking, and pulldown assays revealed that the self-association of OutC is driven by the TMS, whereas the periplasmic region is dispensable for self-association. Site-directed mutagenesis of the TMS revealed that cooperative interactions between three polar residues located at the same helical face provide adequate stability for OutC self-assembly. An interhelical H-bonding mediated by Gln(29) appears to be the main driving force, and two Arg residues located at the TMS boundaries are essential for the stabilization of OutC oligomers. Stepwise mutagenesis of these residues gradually diminished OutC functionality and self-association ability. The triple OutC mutant R15V/Q29L/R36A became monomeric and nonfunctional. Self-association and functionality of the triple mutant were partially restored by the introduction of a polar residue at an alternative position in the interhelical interface. Thus, the OutC TMS is more than just a membrane anchor; it drives the protein self-association that is essential for formation of a functional secretion system.
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Frédéric H
FH
Unité de Microbiologie et Génétique, UMR 5122 CNRS, INSA de Lyon, Université Lyon 1, 69622 Villeurbanne, France.
Shevchik
Vladimir E
VE
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000737:chemistry; D003545:Cysteine / Q000378:metabolism; D016974:Dickeya chrysanthemi / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D004220:Disulfides / Q000378:metabolism; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010447:Peptide Hydrolases / Q000378:metabolism; D013379:Substrate Specificity; D020798:Two-Hybrid System Techniques
D001425:Bacterial Outer Membrane Proteins; D003432:Cross-Linking Reagents; D004220:Disulfides; C105759:OutC protein, bacteria; D010447:Peptide Hydrolases; D003545:Cysteine