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

Autor: Ascari A; School of Biological Sciences, Department of Molecular and Biomedical Science, Research Centre for Infectious Diseases, University of Adelaide, Adelaide 5005, Australia. Electronic address: alice.ascari@adelaide.edu.au., Tran ENH; School of Biological Sciences, Department of Molecular and Biomedical Science, Research Centre for Infectious Diseases, University of Adelaide, Adelaide 5005, Australia. Electronic address: elizabeth.tran@adelaide.edu.au., Eijkelkamp BA; Molecular Sciences and Technology, College of Science and Engineering, Flinders University, Adelaide 5042, South Australia, Australia. Electronic address: bart.eijkelkamp@flinders.edu.au., Morona R; School of Biological Sciences, Department of Molecular and Biomedical Science, Research Centre for Infectious Diseases, University of Adelaide, Adelaide 5005, Australia. Electronic address: renato.morona@adelaide.edu.au.
Jazyk: angličtina
Zdroj: Biochimica et biophysica acta. Biomembranes [Biochim Biophys Acta Biomembr] 2022 May 01; Vol. 1864 (5), pp. 183871. Date of Electronic Publication: 2022 Jan 26.
DOI: 10.1016/j.bbamem.2022.183871
Abstrakt: 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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Databáze: MEDLINE
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