Grant: Discovery Grants Program - Individual

This proposal is focused (1) on the exploring antibody (Ab) response to oligosaccharide antigens and (2) on the synthesis and binding of secondary cell wall polymers (SCWPs) using a structure-function approach, primarily X-ray crystallography.    First, unlike proteins and peptides, a carbohydrate immunogen cannot generally induce affinity maturation and class switching, which results in the Ab germline experiencing great evolutionary pressure to recognize carbohydrates directly. It has been shown that the diversity of antigenic determinants far exceeds the combinatorial potential of the Ab germline, which requires at least some germline Abs to display polyspecificity.    We are studying several groups of Abs. In one, in a powerful demonstration of polyspecificity, we are exploring anti-idiotypic Abs that mimic the original carbohydrate antigens. In another, we are exploring the ability of two near-germline Abs to adopt multiple stable conformations through which polyspecific antigen recognition may proceed via conformational selection. In a third, we are studying the interesting cross-reactive potential of an Ab specific for a glycopeptide.    Second, self-assembling protein surface layers (S-layers) are common prokaryotic cell wall features involved in key physiological processes including nutrient breakdown, cell wall assembly and, in the case of infection, host cell adhesion. In Gram-positive bacteria, S-layer proteins are anchored to the cell wall by surface layer homology (SLH) domains linked to specific peptidoglycan-linked SCWPs. In several Gram-positive bacterial strains this interaction relies on pyruvylated SCWPs. Remarkably, despite the ubiquity of pyruvylated SCWPs, surprisingly little is known at the molecular level about their biosynthesis, recognition, or the concomitant insights into therapeutic inhibition such understanding would bring.    The SCWP's from model organism Paenibacillus alvei CCM 2051T consists of pyruvylated GlcNAc-ManNAc disaccharide repeats.  Two steps involved in SCWP extension are TagA-catalyzed transfer of a N-acetylmannosaminyl group from UDP-ManNAc to the terminal GlcNAc, and CsaB-catalyzed transfer of a pyruvyl group from phosphoenolpyruvate to the terminal ManNAc-GlcNAc acceptor. We have made significant progress on TagA (which belongs to CAZy family 26 with only one structurally characterized example) and CsaB (for which there is currently no suitable model), and aim to determine their structures unliganded and in complex with  synthetic donor and acceptor analogues.    Finally, we are extending structural studies of the SLH domain SpaA. Our recent publication of the structure of SpaA in complex with the terminal SCWP monosaccharide reported in Nature Commun. revealed an unexpected conformational shift in the protein which we demonstrated had biological significance. We are moving toward characterizing SpaA SLH interactions using longer and more biologically relevant synthetic SCWP fragments.