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Nek7 conformational flexibility and inhibitor binding probed through protein engineering of the R-spine

DOI: 10.1042/BCJ20200128 DOI Help

Authors: Matthew J. Byrne (University of Leeds) , Nazia Nasir (University of Leeds) , Christine Basmadjian (Newcastle University) , Chitra Bhatia (University of Leicester) , Rory F. Cunnison (University of Leicester) , Katherine H. Carr (University of Leicester) , Corine Mas-droux (The Institute of Cancer Research) , Sharon Yeoh (University of Leeds) , Céline Cano (Newcastle University) , Richard Bayliss (University of Leeds)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Biochemical Journal , VOL 477 , PAGES 1525 - 1539

State: Published (Approved)
Published: April 2020
Diamond Proposal Number(s): 307 , 10369 , 19248

Open Access Open Access

Abstract: Nek7 is a serine/threonine-protein kinase required for proper spindle formation and cytokinesis. Elevated Nek7 levels have been observed in several cancers, and inhibition of Nek7 might provide a route to the development of cancer therapeutics. To date, no selective and potent Nek7 inhibitors have been identified. Nek7 crystal structures exhibit an improperly formed regulatory-spine (R-spine), characteristic of an inactive kinase. We reasoned that the preference of Nek7 to crystallise in this inactive conformation might hinder attempts to capture Nek7 in complex with Type I inhibitors. Here, we have introduced aromatic residues into the R-spine of Nek7 with the aim to stabilise the active conformation of the kinase through R-spine stacking. The strong R-spine mutant Nek7SRS retained catalytic activity and was crystallised in complex with compound 51, an ATP-competitive inhibitor of Nek2 and Nek7. Subsequently, we obtained the same crystal form for wild-type Nek7WT in apo form and bound to compound 51. The R-spines of the three well-ordered Nek7WT molecules exhibit variable conformations while the R-spines of the Nek7SRS molecules all have the same, partially stacked configuration. Compound 51 bound to Nek2 and Nek7 in similar modes, but differences in the precise orientation of a substituent highlights features that could be exploited in designing inhibitors that are selective for particular Nek family members. Although the SRS mutations are not required to obtain a Nek7–inhibitor structure, we conclude that it is a useful strategy for restraining the conformation of a kinase in order to promote crystallogenesis.

Journal Keywords: kinases; protein engineering; small molecules

Subject Areas: Biology and Bio-materials

Instruments: I04-Macromolecular Crystallography , I24-Microfocus Macromolecular Crystallography

Other Facilities: ID23-1 at ESRF