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Structural studies of a surface-entropy reduction mutant of O-GlcNAcase

DOI: 10.1107/S2059798318016595 DOI Help

Authors: Alexandra Males (University of York) , Gideon J. Davies (University of York)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Acta Crystallographica Section D Structural Biology , VOL 75 , PAGES 70 - 78

State: Published (Approved)
Published: January 2019
Diamond Proposal Number(s): 13587

Open Access Open Access

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[Roth, C., Chan, S., Offen, W. A., Hemsworth, G. R., Willems, L. I., King, D. T., Varghese, V., Britton, R., Vocadlo, D. J. & Davies, G. J. (2017). Nature Chem. Biol. 13, 610-612.]), 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.

Journal Keywords: O-GlcNAc; O-GlcNAcase; neurodegeneration; surface-entropy reduction; crystallization

Diamond Keywords: Enzymes

Subject Areas: Biology and Bio-materials

Instruments: I04-1-Macromolecular Crystallography (fixed wavelength)

Added On: 23/01/2019 14:22


Discipline Tags:

Life Sciences & Biotech Structural biology

Technical Tags:

Diffraction Macromolecular Crystallography (MX)