Academic Journal
Detection of a disulphide bond and conformational changes in Shigella flexneri Wzy, and the role of cysteine residues in polymerase activity
| العنوان: | Detection of a disulphide bond and conformational changes in Shigella flexneri Wzy, and the role of cysteine residues in polymerase activity |
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| المؤلفون: | Ascari, A., Tran, E.N.H., Eijkelkamp, B.A., Morona, R. |
| المصدر: | http://dx.doi.org/10.1016/j.bbamem.2022.183871. |
| بيانات النشر: | Elsevier BV |
| سنة النشر: | 2022 |
| المجموعة: | The University of Adelaide: Digital Library |
| مصطلحات موضوعية: | Wzy, O-antigen polymerase, Conformational changes, Disulphide bond, mPEG, Shigella flexneri, Disulfides, Polyethylene Glycols, Cysteine, Glycosyltransferases, Lipopolysaccharides, O Antigens, Bacterial Proteins, Mutagenesis, Site-Directed, Temperature, Protein Structure, Tertiary, Protein Folding, Serogroup |
| الوصف: | 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, WzzSF and WzzpHS-2, together interact with WzySF to regulate production of short- (S-Oag) and very long- (VL-Oag) Oag modal lengths, respectively. The 2D arrangement of WzySF transmembrane and soluble regions has been previously deciphered, however, attaining information on the 3D structural and conformational arrangement of WzySF, or any homologue, has proven difficult. For the first time, the current study detected insights into the in situ WzySF arrangement. In vitro assays using thiol-reactive PEG-maleimide were used to probe WzySF conformation, which additionally detected novel, unique conformational changes in response to interaction with intrinsic factors, including WzzSF and WzzpHS-2, and extrinsic factors, such as temperature. Site-directed mutagenesis of WzySF cysteine residues revealed the presence of a putative intramolecular disulphide bond, between cysteine moieties 13 and 60. Subsequent ana- lyses highlighted both the structural and functional importance of WzySF cysteines. Substitution of WzySF 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 WzySF interaction. These data suggest WzySF cysteine substitutions directly impair the interaction between Wzy/WzzpHS-2, without altering the Wzy/WzzSF interplay, and in combination with structural data, we propose that the N- and C-termini of WzySF are arranged in close proximity, and together may form the unique WzzpHS-2 interaction site. ; Alice Ascari, Elizabeth Ngoc Hoa Tran, Bart A. Eijkelkamp, Renato Morona |
| نوع الوثيقة: | article in journal/newspaper |
| اللغة: | English |
| تدمد: | 0005-2736 1879-2642 |
| Relation: | http://purl.org/au-research/grants/arc/160103903; BBA: Biomembranes, 2022; 1864(5):183871-1-183871-12; https://hdl.handle.net/2440/135066; Ascari, A. [0000-0002-5626-7973]; Tran, E.N.H. [0000-0003-1644-2287]; Morona, R. [0000-0001-7009-7440] |
| DOI: | 10.1016/j.bbamem.2022.183871 |
| الاتاحة: | https://hdl.handle.net/2440/135066 https://doi.org/10.1016/j.bbamem.2022.183871 |
| Rights: | © 2022 Elsevier B.V. All rights reserved |
| رقم الانضمام: | edsbas.A20E9E31 |
| قاعدة البيانات: | BASE |
Full Text Finder | تدمد: | 00052736 18792642 |
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| DOI: | 10.1016/j.bbamem.2022.183871 |