The main determinants in toxin cell specificity are often interactions with surface receptors. It hasrecently been determined that the membrane phospholipid sphingomyelin (SM) is the primary receptorfor the Helicobacter pylori vacuolating cytotoxin (VacA). While the cellular receptor has been identified,the molecular basis of SM recognition remains unknown. Molecular modeling of unrelated SM-bindingproteins suggests that specific binding occurs through tryptophan rich aromatic clusters located in solventexposed peptide loops between β-sheet domains. Structural comparison of these proteins with VacA ledto the identification of a similar tryptophan containing aromatic cluster in the mid-region of the p55subunit of VacA. Additionally, sequence alignment of this putative binding site exhibited sequencehomology with the phosphorylcholine binding site of another SM-binding toxin. In this study we testedthe effects of mutating each of the 11 tryptophan residues in VacA. While mutating each of the 8tryptophan residues present in the auto-transporter p33 domain did not cause substantial attenuation intoxin activity, alanine substitution of 2 tryptophan residues within the putative binding site led tosignificant attenuation. Further, tryptophan to alanine mutation of W603, a residue present in the center ofthe putative binding site, was found to be attenuated in cell binding to human-derived AZ-521 gastricepithelial cells. Interestingly, a W603A mutation is also present within a vacA allele associated with alessened propensity for gastric disease, decreased toxin binding, and differences in cell specificity. Thisthesis proposes a novel VacA-SM binding site and identifies a possible cause of allele associateddifferences in VacA pathogenicity.
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Molecular characterization of a sphingomyelin binding site in the vacuolating cytotoxin of H. pylori
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