Structure and Glycan-Binding Properties of the Vibrio Vulnificus Hemolysin B-Trefoil Lectin

Abstract

Pore forming toxins (PFTs) are secreted by bacterial pathogens as water-soluble monomers that oligomerize on the cell surface to form transmembrane channels. To increase their affinity for the membrane, many PFTs utilize proteins, carbohydrates and/or lipids as receptors. Previous studies suggest that a PFT secreted by Vibrio cholerae, V. cholerae cytolysin (VCC), recognizes cell surface glycans in its mode of action. However, while VCC contains two domains whose structures resemble known carbohydrate binding motifs, a β-trefoil and β-prism fold, only the β-prism domain actively binds carbohydrate ligands. This presents the question of why an organism would make a protein with an inactive lectin domain. Sequence analysis identified that orthologs of VCC contain either both of the lectin-like domains, or only the β-trefoil domain. Here, we utilize functional and structural approaches to investigate the carbohydrate-binding activities of a VCC ortholog, Vibrio vulnificus hemolysin (VVH), which contains only a β-trefoil lectin-like domain. Our studies identify that the VVH β-trefoil lectin domain binds a broad range of branched sugars containing multiple N-acetyllactosamine (LacNAc) repeats, with micromolar affinity. This is in contrast to the β-prism domain of VCC, which binds a narrower range of complex N-glycans with nanomolar affinity. We solved the X-ray crystal structure of the VVH β-trefoil domain identifying two interesting traits. First, the β-trefoil lectin domain alone forms a heptameric ring characteristic of many PFTs. Second, VVH contains a unique loop and coordinating residues absent in VCC. We hypothesize an evolutionary relationship between VVH and VCC such that upon gaining the more specific β-prism domain, activity of the β-trefoil domain was lost.