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Structural studies of a surface‐entropy reduction mutant of O‐GlcNAcase
Author(s) -
Males Alexandra,
Davies Gideon J.
Publication year - 2019
Publication title -
acta crystallographica section d
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.374
H-Index - 138
ISSN - 2059-7983
DOI - 10.1107/s2059798318016595
Subject(s) - chemistry , stereochemistry , conformational entropy , monomer , enzyme , active site , mutant , hydrolase , crystallography , molecule , biochemistry , organic chemistry , gene , polymer
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.