Biochemical and Structural Analysis of Bacterial O-antigen Chain Length Regulator Proteins Reveals a Conserved Quaternary Structure

Matthew S Kimber,Chris Whitfield,Kane Larue,Robert Ford

doi:10.1074/jbc.m809068200

Matthew S Kimber, Chris Whitfield + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m809068200

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Abstract

Lipopolysaccharide (LPS) is a major component of the Gram-negative outer membrane and is an important virulence determinant. The O-antigen polysaccharide of the LPS molecule provides protection from host defenses, and the length of O-antigen chains plays a pivotal role. In the Wzy-dependent O-antigen biosynthesis pathway, the integral inner membrane protein Wzz determines the O-antigen chain length. How these proteins function is currently unknown, but the hypothesis includes activities such as a "molecular ruler" or a "molecular stopwatch," and other possibilities may exist. Wzz homologs are membrane proteins with two transmembrane helices that flank a large periplasmic domain. Recent x-ray crystallographic studies of the periplasmic portions of Wzz proteins found multiple oligomeric forms, with quaternary structures favoring the "molecular ruler" interpretation. Here, we have studied full-length Wzz proteins with the transmembrane portions embedded in lipid membranes. Using electron microscopy and image analysis we find a unique hexameric state rather than differing oligomeric forms. The data suggest that in vivo Wzz proteins determine O-antigen chain length via subtle structure-function relationships at the level of primary, secondary, or tertiary structure within the context of a hexameric complex.

Highlights

LPSs are complex glycoconjugates comprising a hydrophobic lipid A that is essential for outer membrane integrity, a core oligosaccharide, and a long-chain polysaccharide known as the O-antigen [1]
Each preparation contains molecules consisting of lipid A and all or part of the core region, known as rough LPS (R-LPS), as well smooth LPS (S-LPS), where the lipid A-core is substituted with varying lengths of O-antigen
These proteins have a regulatory role in polymerization, and, in the case of capsular polysaccharides (CPSs) systems, the polysaccharide co-polymerase (PCP) proteins are required for both polymerization and surface export [14, 15]

Summary

Introduction

LPSs are complex glycoconjugates comprising a hydrophobic lipid A that is essential for outer membrane integrity, a core oligosaccharide, and (in many bacterial genera) a long-chain polysaccharide known as the O-antigen [1]. Homologs of the PCP-1a protein, Wzz, regulate the polymerization of LPS O-antigens to generate the characteristic strain-specific modality evident in silver-stained SDS-PAGE profiles. A double mutant (CWG860) lacking both wzzST and wzzFepE was constructed, and the LPS from this strain displays an SDS-PAGE profile typical of Wzz-deficient mutants, where lower molecular weight forms (Ͻ15 repeat units) predominate and modal clusters are absent.

Results

Conclusion