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

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.

• Authors: Matthew J Byrne, Nazia Nasir, Christine Basmadjian, Chitra Bhatia, Rory F Cunnison, Katherine H Carr, Corine Mas-Droux, Sharon Yeoh, Céline Cano, Richard Bayliss
• Journal: The Biochemical journal (Biochem J) Vol. 477 Issue 8 Pg. 1525-1539 (04 30 2020) ISSN: 1470-8728 [Electronic] England
• PMID: 32242624 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: © 2020 The Author(s).

Chemical References

• Enzyme Inhibitors
• NEK2 protein, human
• NEK7 protein, human
• NIMA-Related Kinases

Topics

• Catalysis
• Enzyme Inhibitors (chemistry, metabolism)
• Humans
• Kinetics
• Mutation
• NIMA-Related Kinases (chemistry, genetics, metabolism)
• Protein Binding
• Protein Conformation
• Protein Engineering