Bacterial glycoconjugates, including N-linked glycoproteins, are a diverse group of macromolecules that provide mechanical stability to microorganisms in challenging environments and mediate interactions among bacteria and between bacterial pathogens and their hosts. These interactions are often critical to bacterial viability and virulence in humans. These intricate pathways for glycoconjugate biosynthesis draw, in early steps, on substrates found in the bacterial cytoplasm to ultimately afford products that are localized to the periplasm or cell surface. Despite their great structural diversity, many glycoconjugates are biosynthesized using a common biosynthetic strategy involving en bloc transfer of glycan to proteins, lipids, or other glycans. The glycan to be transferred is assembled on a polyprenol-linked carrier at the membrane interface. The pathways start with a “commitment to membrane” step catalyzed by a polyprenol phosphate-phosphoglycosyl transferase (PGT). This step is followed by sequential glycan-assembly steps mediated by glycosyl transferases (GTs), each acting on membrane-resident PrenPP-derivatives, to complete glycan assembly on the lipid-linked carrier.
The goal of our studies is to uncover the determinants of specificity and mechanisms by which these enzymes catalyze their reactions on membrane-embedded and soluble substrates. Biochemical studies and the X-ray crystal structure of the PGT from Campylobacter concisus, PglC at 2.74 Å resolution, show that the monoPGTs include a reentrant membrane helix that penetrates only one leaflet of the bilayer, then re-emerges.
Subsequent molecular dynamics (MD) simulations show the undecaprenol phosphate (UndP) carrier mirrors this occupancy of a single leaflet with frequent transitions between stretched, coiled, and unstructured conformations of the polyprenyl tail. These simulations also allow a first view of UndP binding to PglC, corroborated by bioinformatic and mutagenesis studies. Moreover, a loop closure motion of PglC in the MD simulation matches the motion inferred from X-ray crystallographic data, consistant with an induced-fit model. Sequence-similarity networks and phylogenetic analysis of the monotopic PGT superfamily uncovered extensive numbers of fusions with other pathway enzymes and provide evidence that the enzymes in glycoconjucate synthesis are structured to gather rare substrates.
We have recently determined the X-ray crystal structure of the enzyme that carries out the next step in assembly, C. concisus PglA, in complex with the donor-sugar substrate UDP-GalNAc at 2.5 Å resolution. The structure of PglA has remarkable similarity to the GT PglH (rmsd 1.9 Å) which catalyzes the precessive addition of three GalNAc moieties in the penultimate assembly step of the pathway. Comparative analysis of membrane-docked structures highlights significant differences between PglA and PglH in the relative orientation of the active site with the membrane interface. We posit that acceptor-substrate positioning in the membrane may play an integral part in specificity in the GT enzymes.
This work is funded by NIH R01GM131627.