Synergistic computational and experimental studies of a phosphoglycosyl transferase membrane/ligand ensemble

Abstract

Complex glycans serve important functions in all living systems. Many of these intricate and byzantine biomolecules are assembled employing biosynthetic pathways wherein the constituent enzymes are membrane associated. A signature feature of the stepwise assembly processes is the essentiality of unusual linear long-chain polyprenol phosphate-linked substrates, such as un-decaprenol phosphate in bacteria. In this study we focus on a small enzyme, PglC from Campylobacter, structurally characterized for the first time in 2018, as a detergent solubilized construct. PglC is a monotopic phosphoglycosyl transferase (PGT), that embodies the functional core structure of the entire enzyme superfamily and catalyzes the first membrane-committed step in a glycoprotein assembly pathway. The size of the enzyme is significant as it enables high level computation and relatively facile, for a membrane protein, experimental analysis. Our ensemble computational and experimental results reveal a specific interaction of undecaprenol phosphate with PGT cationic residues and suggest a role for critical conformational transitions and electrostatic steering in substrate recognition, overcoming significant energetic barriers to binding.