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.
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