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

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.

The influence of N-linked glycans on the molecular dynamics of the HIV-1 gp120 V3 loop.

N-linked glycans attached to specific amino acids of the gp120 envelope trimer of a HIV virion can modulate the binding affinity of gp120 to CD4, influence coreceptor tropism, and play an important role in neutralising antibody responses. Because of the challenges associated with crystallising fully glycosylated proteins, most structural investigations have focused on describing the features of a non-glycosylated HIV-1 gp120 protein. Here, we use a computational approach to determine the influence of N-linked glycans on the dynamics of the HIV-1 gp120 protein and, in particular, the V3 loop. We compare the conformational dynamics of a non-glycosylated gp120 structure to that of two glycosylated gp120 structures, one with a single, and a second with five, covalently linked high-mannose glycans. Our findings provide a clear illustration of the significant effect that N-linked glycosylation has on the temporal and spatial properties of the underlying protein structure. We find that glycans surrounding the V3 loop modulate its dynamics, conferring to the loop a marked propensity towards a more narrow conformation relative to its non-glycosylated counterpart. The conformational effect on the V3 loop provides further support for the suggestion that N-linked glycosylation plays a role in determining HIV-1 coreceptor tropism.