(12e) Structure-Based Prediction and Design of Polypeptide Substrate Specificities of Glycosyltransferases

Protein glycosylation is primarily mediated by enzymes known as oligosaccharyltransferases (OSTs) and glycosyltransferases (GTs). Polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts) are higher eukaryotic retaining GTs that transfer a GalNAc moiety from uridine diphosphate N-acetylgalactosamine (UDP-GalNAc) onto Ser/Thr residues. This enzyme family initiates the most common type of O-glycosylation and thus affects the structure, stability, and function of many proteins. Here, we present new Rosetta-based computational protocols that provide molecular insight into the peptide substrate specificities of GTs. Our Rosetta-based algorithm docks a fully flexible glycosylated polypeptide substrate to the catalytic site of the GT, extensively sampling polypeptide and glycan conformational space. Using our protocol, we are able to predict relative specificities for polypeptide glycosylation for a number of GTs including enzymes from the ppGalNAc-T family and hOGT. Our predictions match well with experimental data (Kightlinger, W. et al., Nat. Chem. Biol. 14, 627-635 (2018)). Furthermore, molecular insight into the substrate specificity preference allows us to predict enzyme variants with altered preferences. To the best of our knowledge, our protocol is the first example of a structure-based computational protocol for predicting polypeptide substrate specificities of GTs.