The Single Transmembrane Segment Drives Self-assembly of OutC and the Formation of a Functional Type II Secretion System in Erwinia chrysanthemi

Abstract

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, crosslinking, 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.

Solution Structure of Homology Region (HR) Domain of Type II Secretion System

“…This was confirmed by dynamic light scattering. For NMR spectroscopy, uniformly 15 N-and 13 C-labeled proteins were produced by growing cell cultures in M9 minimal medium that contained 1 g/liter 15 N-ammonium chloride and 2 g/liter 13 C-D-glucose (Cambridge Isotope Laboratories, Inc.) as the sources of nitrogen and carbon, respectively.…”

“…Three fractions from the HPLC column were characterized by mass spectroscopy, HRF1, -2, and -3; HRF3 was the shortest of these nested fragments and was therefore selected for further study. N0, N1, and N1-N2 domains of OutD were produced in E. coli and purified by nickel-affinity chromatography as described previously (13).…”

“…GspC is a bitopic inner membrane protein. The single trans-membrane segment drives dimerization of the protein (13). The periplasmic region possesses a so-called homology region (HR) and, in most known GspCs, including OutC, a PDZ domain (see Fig.…”

On the path to uncover the bacterial type II secretion system

“…Proteins of this family are organized into several domains including an N-terminal cytoplasmic region, a transmembrane (TM) domain, a highly conserved central periplasmic domain (homology region, HR) and a C-terminal part containing specific secondary structures such as coiled-coil domains (in P. aeruginosa and Pseudomonas alcaligenes, for instance) or PDZ domains (in K. oxytoca and E. chrysanthemi) (figure 4) [71]. GspC P was shown to be active as a dimer and self-associates by its TM domain, which is not a simple membrane anchor but plays an active role in the function of the protein [72]. The HR domain of V. cholerae GspC P (EpsC P ) was shown to interact directly with the periplasmic N 0 domain of the secretin EpsD Q [44,73].…”

The Crystal Structure of Pectate Lyase PelI from Soft Rot Pathogen Erwinia chrysanthemi in Complex with Its Substrate

“…PelI possesses a 19-residue signal peptide that is cleaved during export by the Sec system, following which the protein is secreted into the external medium by the type 2 secretion system (20). The two structural modules are separated in planta by E. chrysanthemi proteases (6).…”