Abstract
The enzyme O-GlcNAcase catalyses the removal of the O-GlcNAc co/post-translational modification in multicellular eukaryotes. The enzyme has become of acute interest given the intimate role of O-GlcNAcylation in tau modification and stability; small-molecular inhibitors of human O-GlcNAcase are under clinical assessment for the treatment of tauopathies. Given the importance of structure-based and mechanism-based inhibitor design for O-GlcNAcase, it was sought to test whether different crystal forms of the human enzyme could be achieved by surface mutagenesis. Guided by surface-entropy reduction, a Glu602Ala/Glu605Ala variant [on the Gly11-Gln396/Lys535-Tyr715 construct; Roth et al. (2017), Nature Chem. Biol. 13, 610-612] was obtained which led to a new crystal form of the human enzyme. An increase in crystal contacts stabilized disordered regions of the protein, enabling 88% of the structure to be modelled; only 83% was possible for the wild-type construct. Although the binding of the C-terminus was consistent with the wild type, Lys713 in monomer A was bound in the -1 subsite of the symmetry-related monomer A and the active sites of the B monomers were vacant. The new crystal form presents an opportunity for enhanced soaking experiments that are essential to understanding the binding mechanism and substrate specificity of O-GlcNAcase.