Nek7 conformational flexibility and inhibitor binding probed through protein engineering of the R-spine
Matthew J Byrne1, Nazia Nasir1, Christine Basmadjian2
1Astbury Centre for Structural Molecular Biology, School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, U.K.
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.
Insights
Researchers engineered a Nek7 kinase mutant to stabilize its active form, aiding in the development of novel cancer therapeutics by facilitating inhibitor complex crystallization.
Area of Science:
- Biochemistry
- Structural Biology
- Cancer Therapeutics
Background:
- Nek7 (Never in mitosis gene A-related kinase 7) is a kinase crucial for cell division.
- Elevated Nek7 levels are linked to various cancers, making it a potential therapeutic target.
- Lack of selective and potent Nek7 inhibitors hinders drug development.
Purpose of the Study:
- To develop a strategy for obtaining high-resolution crystal structures of Nek7 in complex with inhibitors.
- To stabilize the active conformation of Nek7 for structural studies.
- To identify key structural features for designing selective Nek family inhibitors.
Main Methods:
- Introduction of aromatic residues into the Nek7 regulatory-spine (R-spine) to create a 'strong R-spine' mutant (Nek7SRS).
- Crystallization of Nek7SRS and wild-type Nek7 (Nek7WT) in apo and inhibitor-bound forms.
- X-ray crystallography to determine the structures of Nek7 complexes.
- Comparative analysis of inhibitor binding modes across Nek family members.
Main Results:
- The Nek7SRS mutant retained catalytic activity and facilitated crystallization of Nek7-inhibitor complexes.
- Crystal structures revealed stabilized R-spine conformations in Nek7SRS compared to Nek7WT.
- Compound 51, an ATP-competitive inhibitor, bound to both Nek7 and Nek2.
- Structural analysis identified subtle differences in inhibitor binding that could be exploited for selectivity.
Conclusions:
- Engineering the R-spine is a viable strategy to stabilize active kinase conformations and promote crystallogenesis.
- This approach aids in obtaining structures of challenging targets like Nek7.
- Structural insights provide a foundation for designing selective Nek family kinase inhibitors for cancer therapy.
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