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

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 crystallize 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 stabilize the active conformation of the kinase through R-spine stacking. The strong R-spine mutant Nek7SRSretained catalytic activity and was crystallized in complex with compound 51, an ATP-competitive inhibitor of Nek2 and Nek7. Subsequently, we obtained the same crystal form for wild-type Nek7WTin apo form and bound to compound 51. The R-spines of the three well-ordered Nek7WTmolecules exhibit variable conformations while the R-spines of the Nek7SRSmolecules 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