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; &gt;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 <i>Enterobacteriaceae</i>, that produce dual Oag chain length determinants. <b>IMPORTANCE</b> 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.