Structure-function relationships in monotopic phosphoglycosyl transferases

Abstract

Complex glycans play essential roles in prokaryotic and eukaryotic biology. While this ubiquitous post-translational modification takes a diversity of forms, many glycoconjugate biosynthesis pathway across domains of life follows a common logic. Glycan assembly is initiated by a phosphoglycosyl transferase (PGT) that transfers a phosphosugar from a nucleotide donor to a polyprenol phosphate (PrenP) chain embedded in the membrane. The PrenPP-sugar product is elaborated by downstream glycosyltransferases, transferred across the membrane and ultimately appended to various acceptor molecules. The PGTs initiating glycan assembly adopt diverse membrane architectures. An extensive superfamily of PGTs, elucidated in part by this thesis, is exemplified by PglC from the Gramnegative pathogen, Campylobacterjejuni. PglC comprises a globular cytosolic domain and an N-terminal membrane-resident domain.

Recent structural and biochemical analyses determined that this domain forms a helix-break-helix motif, termed the reentrant membrane helix (RMH), that enters and exits on the same face of the membrane, resulting in a monotopic topology. The RMH anchors the PglC fold in the membrane in a manner not previously observed among other monotopic membrane proteins. This thesis focuses on structure-function relationships in the RMH and associated domains. Two conserved motifs are shown to drive formation of a reentrant topology for PglC, and to exemplify common principles of topology determination among diverse monotopic proteins. These principles are further applied to the identification of reentrant domains in an extensive superfamily of monotopic lipid A acyltransferases previously thought to be membrane-spanning. The next section of the thesis explores the highly conserved role of PrenP in complex glycan biosynthesis.

The significance of PrenP geometry in mediating substrate binding and modulating the local membrane environment is presented. Additionally, a conserved proline residue in the PglC RMH is determined to drive PrenP binding and specificity. Molecular insights from this study shed new light on the roles of PrenP in facilitating diverse glycoconjugate biosynthesis pathways. Finally, a cell-free methodology for expression of PglC directly into model membrane lipid Nanodiscs is described. This system has valuable applications for the study of interactions between PglC and downstream glycosyltransferase enzymes, and for further structural characterization of PglC in a membrane environment.