Identification of the Shigella flexneri Wzy Domain Modulating Wzz_pHS-2 Interaction and Detection of the Wzy/Wzz/Oag Complex

Shigella flexneri implements the Wzy-dependent pathway to biosynthesize the O antigen (Oag) component of its surface lipopolysaccharide. The inner membrane polymerase Wzy_SF catalyzes the repeat addition of undecaprenol-diphosphate-linked Oag (Und-PP-RUs) to produce a polysaccharide, the length of which is tightly regulated by two competing copolymerase proteins, Wzz_SF (short-type Oag; 10 to 17 RUs) and Wzz_pHS-2 (very-long-type Oag; >90 RUs). The nature of the interaction between Wzy_SF and Wzz_SF/Wzz_pHS-2 in Oag polymerization remains poorly characterized, with the majority of the literature characterizing the individual protein constituents of the Wzy-dependent pathway. Here, we report instead a major investigation into the specific binding interactions of Wzy_SF with its copolymerase counterparts. For the first time, a region of Wzy_SF that forms a unique binding site for Wzz_pHS-2 has been identified. Specifically, this work has elucidated key Wzy_SF moieties at the N- and C-terminal domains (NTD and CTD) that form an intramolecular pocket modulating the Wzz_pHS-2 interaction. Novel copurification data highlight that disruption of residues within this NTD-CTD pocket impairs the interaction with Wzz_pHS-2 without affecting Wzz_SF binding, thereby specifically disrupting polymerization of longer polysaccharide chains. This study provides a novel understanding of the molecular interaction of Wzy_SF with Wzz_SF/Wzz_pHS-2 in the Wzy-dependent pathway and, furthermore, detects the Wzy/Wzz/Und-PP-Oag complex for the first time. Beyond S. flexneri, this work may be extended to provide insight into the interactions between protein homologues expressed by related species, especially members of Enterobacteriaceae, that produce dual Oag chain length determinants. IMPORTANCE Shigella flexneri is a pathogen causing significant morbidity and mortality, predominantly devastating the pediatric age group in developing countries. A major virulence factor contributing to S. flexneri pathogenesis is its surface lipopolysaccharide, which is comprised of three domains: lipid A, core oligosaccharide, and O antigen (Oag). The Wzy-dependent pathway is the most common biosynthetic mechanism implemented for Oag biosynthesis by Gram-negative bacteria, including S. flexneri. The nature of the interaction between the polymerase, Wzy_SF, and the polysaccharide copolymerases, Wzz_SF and Wzz_pHS-2, in Oag polymerization is poorly characterized. This study investigates the molecular interplay between Wzy_SF and its copolymerases, deciphering key interactions in the Wzy-dependent pathway that may be extended beyond S. flexneri, providing insight into Oag biosynthesis in Gram-negative bacteria.