Structural Superimposition

Abstact

The Helicobacter pylori Hom group of outer-membrane proteins (OMPs) has been implicated in virulence-related processes. Here, we present a structural and functional characterization of HomA from H. pylori strain 26695. Comparative in vivo analyses of the wild-type strain and a homA deletion mutant demonstrate a role for HomA in focal adhesion kinase phosphorylation at Y(397) and in bacterial adhesion to human leukaemia-derived MEC-1 cells. Recombinant production of several HomA variants identified a soluble ectodomain ((ECD)HomA) that harbours the conserved zinc-binding motif of metzincin metallopeptidases (MPs) and displays dimerization capacity, acid resistance, and immunomodulatory activity by triggering a pro-inflammatory response in Raji human B-lymphoblastoid cancer cells. Site-directed mutagenesis of the zinc-binding motif confirmed its contribution to zinc coordination and thermal stability. Structural analysis of (ECD)HomA revealed a two-domain organization comprising an N-terminal accessory beta-sandwich domain (ND) and a C-terminal catalytic domain (CD), in which a non-canonical 'Thr-turn' replaces the hallmark metzincin 'Met-turn'. Integrating bioinformatics with AI-based structure predictions uncovered putative HomA homologues across 44 proteomes, predominantly from Gram-negative bacteria. Structural and biochemical characterization of the closest non-Helicobacter homologue from the oral pathogen Capnocytophaga canimorsus, dubbed 'canilysin', revealed an architecture equivalent to (ECD)HomA and highly specific MP activity. Together, these findings define the Hom group and canilysin as helicolysins, a previously uncharacterized metzincin family distinguished by a conserved Thr-turn and an accessory ND, and implicates these proteins in host-pathogen interactions, adhesion, and immunomodulation.