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.
【저자키워드】 conformational changes, disulphide bond, O-antigen polymerase, Wzy, mPEG.,