Conformational dynamics of peptide substrate recognition in polypeptide N-acetylgalactosaminyltransferase 2: Markov...

Polypeptide N-acetylgalactosaminyltransferase 2 (ppGalNAc-T2) governs the post-translational modifications of O-GalNAc glycosylation on a vast array of signaling proteins. Despite of the availability of substrate-bound crystal structures and extensive activity studies on ppGalNAc-T2, the dynamic mechanisms underlying peptide substrate recognition remain poorly understood. Leveraging extensive all-atom molecular dynamics (MD) simulations (totaling ∼20 μs), we constructed a Markov state model (MSM) to elucidate the conformational dynamics of peptide substrate EA2 binding to ppGalNAc-T2. The MSM identified six metastable states along two distinct binding pathways, highlighting the pivotal anchoring and stabilizing roles of the conserved PXP motif in peptide-enzyme recognition. The rate-limiting step was proved to be involved in the establishment of a stable hydrophobic core and a high-occupied hydrogen bonding network (HBN), and desolvation effects within the binding site. Furthermore, the coupling relationship between dynamic behaviors of the peptide and hydrogen bonding interactions was revealed, emphasizing how conformational rearrangements of peptide substrate affect glycosylation site specificity. Notably, a key intermediate state was captured, exhibiting good conformational congruence to the reported Michaelis complex for ppGalNAc-T2. These findings would advance the understanding of peptide substrate recognition in ppGalNAc-T2, and facilitate the fine-tuning of enzymatic activity and therapeutic intervention in glycosylation-related disorders.