Membrane protein conformational dynamics and ligand-binding interactions in bacterial glycoconjugate biosynthesis

Abstract

Membrane associated proteins are an essential component of the complex biochemistry that is carried out at the membrane interface and perform essential functions for cellular life. Biophysical characterization of protein structure-function relationships faces a unique set of challenges due to the constraints of phospholipid bilayer chemistry and geometry. Advances in x-ray crystallography and cryo electron microscopy have made progress in this regard, but dynamic structural features remain difficult to study. Small membrane proteins, such as those responsible for bacterial glycosylation, remain challenging to structurally characterize at all. Bacterial glycan synthesis pathways are essential for cell function yet highly variable between strains, making them promising systems for targeted antibiotic development. Many pathways have initiating SmPGTs that show incredible specificity for minute changes in glycan chemistry despite being small enough to streamline many computational methods, which makes them ideal model systems for developing multidisciplinary strategies to study membrane protein dynamics. This thesis presents a strategy that employs structural bioinformatics in Chapter 2, molecular dynamics simulation (MD) in Chapter 3, and single-molecule FRET microscopy (smFRET) in Chapter 4 to observe the ligand-dependent conformational dynamics of integral membrane proteins in situ. It focuses on representative members of the small monotopic phosphoglycosyl transferase (SmPGT) superfamily, which catalyze transfer of a phosphosugar from a soluble nucleotide-sugar donor to a membrane-embedded polyprenol phosphate acceptor in the initiating step of glycoconjugate biosynthesis in prokaryotes. The pipeline is employed to confirm the role of SmPGT conformational dynamics in substrate binding and informs the design of non-hydrolyzable substratemimetic inhibitors. Chapter 5 further sets the stage for the use of structural bioinformatics and molecular simulation to characterize subsequent glycosyl transferase (GT) enzymes down pathway and presents initial results characterizing inter-protein cooperative interactions. The integrated approach to incorporate computational and experimental characterization methods has significantly contributed to the understanding of SmPGT structure-function relationships and opened up new directions of inquiry into specific PGTligand interactions, the development of new inhibitory compounds, and the role of interprotein interactions in bacterial glycan synthesis.