In prokaryotes, protein glycosylation can be a determinant of pathogenicity as it plays a role in host adherence, invasion, and colonization. Impairment of glycosylation in some organisms, for example N-linked glycosylation in Campylobacter jejuni, leads to decreased pathogenicity; thus, opening new avenues for the development of antivirulence agents. A member of the protein glycosylation (pgl) gene locus in C. jejuni, PglC, is predicted single-pass transmembrane (TM) protein, that catalyzes the phosphoglycosyl transferase (PGT) reaction in the first membrane-committed step of the N-linked glycosylation pathway. The small size of PglC (201 aa) compared to homologous PGTs suggests it may represent the minimal catalytic unit for the monotopic PGT superfamily.

Herein, the structure of C. concisus PglC including its putative TM domain has been solved to 2.74 A resolution to reveal a novel protein fold with a unique alpha-helix-associated beta-hairpin (AHABh) motif and largely solvent-exposed structure. There is noted a parsimony of fold in the form of short-range motifs underpinning the structural basis for critical functions of PglC: membrane association and active-site geometry. Biochemical and bioinformatics studies support structural evidence suggesting the crystallographically-observed, kinked TM helix is re-entrant on the cytoplasmic face of the membrane rather than membrane spanning. Thus, PglC represents a first-in-class structure of a novel membrane interaction mode for monotopic membrane proteins. Additionally, the AHABh-motif and active-site helical geometry establishes co-facial positioning of the catalytic-dyad. Molecular docking of PglC substrates, undecaprenyl phosphate (UndP) and UDP-N,N-diacetylbacillosamine (UDP-diNAcBac), within the active-site reveals co-incident binding sites, consistent with the proposed ping-pong enzymatic mechanism.

Loading of PglC into membrane-bilayer nanodiscs (ND) allows for the investigation of PglC structure and function within a native-like membrane environment by small-angle x-ray scattering (SAXS). Observation of PglC in ND via SAXS confirms the application of the method for studying small, integral, monotopic membrane proteins in a membrane environment. Moreover, development of a mathematical approach by which resident-protein: ND stoichiometry can be deduced from measured scattering intensity enables independent confirmation of loading stoichiometry.

Overall, the membrane-interaction modality observed for PglC is the first structurally characterized example of a new membrane association mode for monotopic proteins with the membrane. These studies provide insight into the structural determinants of the chemical mechanism and substrate-binding for C. concisus PglC and for the extensive homologous monotopic PGT superfamily, thus allow homology modeling and enabling future inhibitor design.