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 Wzy_SF 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.