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

  • Byrne, Matthew J.1
  • Nasir, Nazia1
  • Basmadjian, Christine2
  • Bhatia, Chitra3
  • Cunnison, Rory F.3
  • Carr, Katherine H.3
  • Mas-Droux, Corine4
  • Yeoh, Sharon1
  • Cano, Céline2
  • Bayliss, Richard1
  • 1 Astbury Centre for Structural Molecular Biology, School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, U.K.
  • 2 Newcastle University Centre for Cancer, School of Natural and Environmental Sciences, Newcastle University, Newcastle Upon Tyne, U.K.
  • 3 Department of Molecular and Cell Biology, University of Leicester, Leicester, U.K.
  • 4 Section of Structural Biology, The Institute of Cancer Research, London, U.K.
Published Article
Biochemical Journal
Portland Press
Publication Date
Apr 29, 2020
DOI: 10.1042/BCJ20200128
PMID: 32242624
PMCID: PMC7200626
PubMed Central


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.

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