Computationally Guided Directed Evolution of O-GlcNAcase into a Reagent Specific for [beta]-O-GlcNAc
Martin, Joanne C.
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Protein glycosylation with O-linked N-Acetylglucosamine (O-GlcNAc) is a post-translational modification of serine and threonine residues in nucleocytoplasmic proteins. Alterations in the glycosylation pattern of 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, the addition and removal O-GlcNAc 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 acts on all O-GlcNAcylated proteins, seemingly independent of their sequence. Despite the importance of this modification, very little is known about the structural relationship between these enzymes and their substrates. Nor is it clear, how only a single hydrolase is capable of removing O-GlcNAc from all GlcNAcylated proteins, independent of the protein sequence. Here a combination of computational and experimental techniques is employed to develop an understanding of the interactions between an inactive point mutant of OGA and its substrate, in an aim to designing an OGA-based receptor with enhanced affinity for O-GlcNAc. The key interactions involved in binding have been identified computationally, and are separated into residues which are directly responsible for the specificity of the complex (hot residues) and those whose mutation may increase the affinity (tepid residues). An inactive human OGA, which was engineered using site directed mutagenesis to knock out the nucleophile, was used to construct a combinatorial library in bacteriophage by saturation mutagenesis of the tepid residues. This library has been screened leading to the discovery of a single clone showing high affinity and specificity for O-GlcNAc. Reagents of this type are termed Lectenz®, and may be important diagnostic tools for the detection of enzyme substrates, such as O-GlcNAcylated proteins. In addition, five models of O-GlcNAcylated peptides in complex with a bacterial OGA were generated and assessed in terms of conformational dynamics and binding contributions in an aim to understand how OGA recognizes O-GlcNAc in situ. The results show that each of the five glycopeptides bind to OGA in a similar fashion, forming OGA-peptide interactions primarily, but not exclusively, with 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 reagents that could target specific O-GlcNAcylated peptide sequences.