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

Lookup NU author(s): Dr Christine Basmadjian, Dr Celine Cano

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Abstract

© 2020 The Author(s).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.


Publication metadata

Author(s): Byrne MJ, Nasir N, Basmadjian C, Bhatia C, Cunnison RF, Carr KH, Mas-Droux C, Yeoh S, Cano C, Bayliss R

Publication type: Article

Publication status: Published

Journal: Biochemical Journal

Year: 2020

Volume: 477

Issue: 8

Pages: 1525-1539

Print publication date: 29/04/2020

Online publication date: 03/04/2020

Acceptance date: 03/04/2020

ISSN (print): 0264-6021

ISSN (electronic): 1470-8728

Publisher: Portland Press Ltd

URL: https://doi.org/10.1042/BCJ20200128

DOI: 10.1042/BCJ20200128


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