Complex glycoconjugates play a pivotal role in bacterial survival, colonization and virulence and contribute to the interactions between symbiotic and pathogenic bacteria and their human hosts. An important mechanism for the assembly of these structures is initiated on the cytoplasmic face of cell membranes, catalyzed by polyprenol phosphate (PrenP) phosphoglycosyl transferases (PGTs). PGTs transfer a C1’- phosphosugar from a soluble nucleoside diphosphate (NDP) activated donor to a PrenP acceptor, yielding a membrane-bound polyprenol diphosphosugar. Our studies focus on a PGT superfamily with a monotopic membrane topology (monoPGTs) for which, until our recent studies, there has been only limited structural and mechanistic information. These enzymes differ from the well-known polytopic PGTs (polyPGTs), which bear many membrane-spanning sequences. Biochemical studies and the structure of Campylobacter concisus PglC, show that the monoPGTs include a reentrant membrane helix (RMH) that penetrates only one leaflet of the bilayer, then re-emerges. This program will pursue synergistic biochemical, bioinformatic, structural and chemical biology studies of the monoPGTs. In Aim 1 structures will be determined via X-ray crystallography with detergent-solubilized protein and, in a membrane environment, by solubilization into lipid nanoparticles and crystallization in the lipidic cubic phase. Cryo-EM in lipid nanoparticles will also be pursued for members of optimal size. Together with substrate and inhibitor liganded structures and activity analysis, we will elucidate the specificity determinants of newly-identified monoPGTs and provide information on their function in the glycoconjugate biosynthetic pathways of various pathogens. In Aim 2, the model that binding of the UDP-sugar substrate triggers the movement of a soluble loop to complete substrate-binding determinants and close the active site for catalysis, will be tested using cross-linking and fluorescence-based approaches in detergent-solubilized and model membrane environments. To provide complementary insight into the binding of the membrane-resident PrenP substrate, the RMH sequences will be analyzed via informatics. This information will be used to develop hidden Markov models to identify RMH segments within the monotopic PGT superfamily and used to predict RMHs in unrelated proteins families across the proteome. Aim 3 will develop nucleoside analogs that will serve as inhibitors and activity-based protein profiling probes of the monotopic PGT superfamily. This analysis will define the contribution of ligand moieties to binding and identify new PGTs and their significance in bacterial metabolism and host infection. Ultimately, the identified proteins can act as targets for the development of new antibacterial and antivirulence agents. Overall, this in-depth study of the structures and binding landscape of the monoPGT superfamily and design of biological probes will establish the fundamental knowledge and tools needed for validating and intervening in the action of potential therapeutic targets.

Narrative The biosynthesis of complex sugars is fundamental to bacterial survival and to the interactions between bacterial pathogens and symbionts and their human hosts. The proposed studies will uncover how a superfamily of enzymes act at bacterial membranes to carry out an initial, critical step in complex sugar assembly. The understanding of how the molecular structure and dynamics lead to function will allow the design of ligands and probes to identify and ultimately yield therapeutic control over pathways that contribute to bacterial survival and virulence.