How the terminal glucoside of the N-glycan donor affects the catalytic efficiency of the eukaryotic oligosaccharyltransferase

The eukaryotic oligosaccharyltransferase (OST) is responsible for initiating N-glycosylation of secreted proteins by transferring a pre-assembled lipid-linked oligosaccharide (LLO) donor to target asparagine residues most often found within N-x-S/T consensus sequences, or sequons. OST preferentially selects LLO donors with a distinctive glucoside Glc-α(1-2)-Glc-α(1-3)-Glc-α(1-3)- capping the A-branch. This motif is cleaved right after the N-glycan transfer in a stepwise manner, before the folded glycoprotein exits the endoplasmic reticulum quality control (ERQC) cycle. While the -Glc-α(1-3)-Glc-α(1-3)- disaccharide regulates access and binding to the calreticulin/calnexin chaperones, the terminal Glc-α(1-2)- is removed immediately after OST catalysis, suggesting that its biological function may be directly linked to the OST catalytic efficiency. To understand how and why this capping motif affects the OST N-glycosylation efficiency, we rebuilt 3D models of the yeast OST in complex with an acceptor peptide and LLO substrates with and without terminal Glc-α(1-2)- and analysed their stability with all-atom molecular dynamics (MD) simulations through conventional and Gaussian-accelerated (GaMD) sampling schemes. Our results indicate that the terminal Glc-α(1-2)- is essential to anchor the full-length LLO donor to the OST through a complex network of intermolecular contacts extending from the catalytic site to distal subdomains. We show how this contact network is crucial to preserve the LLO catalytically productive alignment of its reducing end. We also show that the removal of the terminal Glc-α(1-2)- leads to an increased flexibility of the LLO, which displaces the reducing end, leading to less catalytically productive states.