Defining the structural origin of the substrate sequence independence of O-GlcNAcase using a combination of molecular docking and dynamics simulation

Abstract

Protein glycosylation with O-linked N-acetylglucosamine (O-GlcNAc) is a post-translational modification of serine/threonine residues in nucleocytoplasmic proteins. O-GlcNAc has been shown to play a role in many different cellular processes and O-GlcNAcylation is often found at sites that are also known to be phosphorylated. Unlike phosphorylation, O-GlcNAc levels are regulated by only two enzymes, O-GlcNAc transferase (OGT) and O-GlcNAc hydrolase (O-GlcNAcase or OGA). So far, no obvious consensus sequence has been found for sites of O-GlcNAcylation. Additionally, O-GlcNAcase recognizes and cleaves all O-GlcNAcylated proteins, independent of their sequence. In this work, we generate and analyze five models of O-GlcNAcylated peptides in complex with a bacterial OGA. Each of the five glycopeptides bind to OGA in a similar fashion, with OGA–peptide interactions primarily, but not exclusively, involving the peptide backbone atoms, thus explaining the lack of sensitivity to peptide sequence. Nonetheless, differences in peptide sequences, particularly at the −1 to −4 positions, lead to variations in predicted affinity, consistent with observed experimental variations in enzyme kinetics. The potential exists, therefore, to employ the present analysis to guide the development glycopeptide-specific inhibitors, or conversely, the conversion of OGA into a reagent that could target specific O-GlcNAcylated peptide sequences.

Protein O-GlcNAcylation: emerging mechanisms and functions

“…Similar results were obtained from molecular docking and dynamics simulations analyzing models of various O -GlcNAcylated peptides in complex with Bacteroides thetaiotaomicron GH84 ( Bt OGA). These findings suggest that OGA substrate recognition generally lacks sequence sensitivity, though variations in sequence near the O -GlcNAcylation site are predicted to have some effect on OGA-glycopeptide binding affinity 47 . While structural features and adaptor proteins could also confer substrate specificity to OGA, evidence for these mechanisms is limited at this time.…”

O‐GlcNAcase: Emerging Mechanism, Substrate Recognition and Small‐Molecule Inhibitors

“…It was also shown that OGA bounds to the peptide backbone through intramolecular hydrogen bonds and hydrophobic interactions . Molecular docking and dynamics simulations on models of Bt OGA in complex with O ‐GlcNAcylated peptides also gave similar results . From these observations, OGA substrate recognition was believed to lack sequence sensitivity, although differences in peptide sequences have led to small variations in predicting enzyme binding affinity …”

“…[43] Molecular docking and dynamics simulations on models of BtOGA in complex with O-GlcNAcylated peptides also gave similar results. [44] From these observations, OGA substrate recognition was believed to lack sequence sensitivity, although differences in peptide sequences have led to small variations in predicting enzyme binding affinity. [44] Recently, the partial structure of human OGA containing the catalytic and stalk domains revealed that OGA is present in a dimerized form creating a potential substrate-binding pocket (Figure 3).…”

“…[44] From these observations, OGA substrate recognition was believed to lack sequence sensitivity, although differences in peptide sequences have led to small variations in predicting enzyme binding affinity. [44] Recently, the partial structure of human OGA containing the catalytic and stalk domains revealed that OGA is present in a dimerized form creating a potential substrate-binding pocket (Figure 3). [38][39][40] Roth et al removed the putative disordered regions to generate hOGA in a suitable form for crystallization while retaining activity.…”

O-GlcNAcase: Promiscuous Hexosaminidase or Key Regulator of O-GlcNAc Signaling?

“…A molecular view of the binding of short O -GlcNAc peptides in the active site of a catalytically impaired Cp OGA mutant, obtained through crystallographic studies, showed that interactions were limited to the sequence-independent stacking of the peptide backbone to a surface-exposed, conserved, aromatic residue in OGA ( 45 ). Conversely, a combination of molecular docking and molecular dynamics simulation suggests that although sequence-independent interactions with the peptide component do contribute, the affinity for OGA substrates varies as a function of the peptide sequence ( 46 ). It should be noted that peptide sequence and length may have entropic in addition to enthalpic effects on binding and that their precise conformations in the active site may affect not only binding but also turnover.…”