Lipid bilayer nanodisc platform for investigating polyprenol-dependent enzyme interactions and activities

Significance Linear polyprenols are recurring molecular components in the biosynthetic pathways responsible for the assembly of essential glycoconjugates, including peptidoglycan and N-linked glycoproteins. Despite their highly conserved presence in all domains of life, the role of the extended linear polyprenyl groups in the dynamics of membrane-bound glycan assembly pathways remains a mystery. Here we apply the nanodisc model membrane platform to simultaneously assess the interactions and activities of the polyprenyl-linked substrates, enzymes, and lipid bilayer by investigating initial steps from the Campylobacter jejuni N-linked glycosylation pathway. This work represents a proof-of-concept demonstrating that nanodiscs can be used for the precise manipulation and study of polyprenol-dependent pathways. Membrane-bound polyprenol-dependent pathways are important for the assembly of essential glycoconjugates in all domains of life. However, despite their prevalence, the functional significance of the extended linear polyprenyl groups in the interactions of the glycan substrates, the biosynthetic enzymes that act upon them, and the membrane bilayer in which they are embedded remains a mystery. These interactions are investigated simultaneously and uniquely through application of the nanodisc membrane technology. The Campylobacter jejuni N-linked glycosylation pathway has been chosen as a model pathway in which all of the enzymes and substrates are biochemically accessible. We present the functional reconstitution of two enzymes responsible for the early membrane-committed steps in glycan assembly. Protein stoichiometry analysis, fluorescence-based approaches, and biochemical activity assays are used to demonstrate the colocalization of the two enzymes in nanodiscs. Isotopic labeling of the substrates reveals that undecaprenyl-phosphate is coincorporated into discs with the two enzymes, and furthermore, that both enzymes are functionally reconstituted and can sequentially convert the coembedded undecaprenyl-phosphate into undecaprenyl-diphosphate-linked disaccharide. These studies provide a proof-of-concept demonstrating that the nanodisc model membrane system represents a promising experimental platform for analyzing the multifaceted interactions among the enzymes involved in polyprenol-dependent glycan assembly pathways, the membrane-associated substrates, and the lipid bilayer. The stage is now set for exploration of the roles of the conserved polyprenols in promoting protein–protein interactions among pathway enzymes and processing of substrates through sequential steps in membrane-associated glycan assembly.