Detection of a disulphide bond and conformational changes in Shigella flexneri Wzy, and the role of cysteine residues in polymerase activity

Shigella flexneri utilises the Wzy-dependent pathway for the production of a plethora of complex polysaccharides, including the lipopolysaccharide O-antigen (Oag) component. The inner membrane protein WzySF polymerises Oag repeat units, whilst two co-polymerase proteins, Wzz(SF) and Wzz(pHS-2), together interact with Wzy(SF) to regulate production of short-(S-Oag) and very long-(VL-Oag) Oag modal lengths, respectively. The 2D arrangement of Wzy(SF) transmembrane and soluble regions has been previously deciphered, however, attaining information on the 3D structural and conformational arrangement of Wzy(SF), or any homologue, has proven difficult. For the first time, the current study detected insights into the in situ Wzy(SF) arrangement. In vitro assays using thiol-reactive PEG-maleimide were used to probe Wzy(SF) conformation, which additionally detected novel, unique conformational changes in response to interaction with intrinsic factors, including Wzz(SF) and Wzz(pHS-2), and extrinsic factors, such as temperature. Site-directed mutagenesis of Wzy(SF) cysteine residues revealed the presence of a putative intramolecular disulphide bond, between cysteine moieties 13 and 60. Subsequent analyses highlighted both the structural and functional importance of Wzy(SF) cysteines. Substitution of Wzy(SF) cysteine residues significantly decreased biosynthesis of the VL-Oag modal length, without disruption to S-Oag production. This phenotype was corroborated in the absence of co-polymerase competition for Wzy(SF) interaction. These data suggest Wzy(SF) cysteine substitutions directly impair the interaction between Wzy/Wzz(pHS-2), without altering the Wzy/Wzz(SF) interplay, and in combination with structural data, we propose that the N-and C-termini of Wzy(SF) are arranged in close proximity, and together may form the unique Wzz(pHS-2) interaction site.