Nek7 conformational flexibility and inhibitor binding probed through protein engineering of the R-spine
Nek7 conformational flexibility and inhibitor binding probed through protein engineering of the R-spine
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DOI：
10.1042/bcj20200128
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发表时间：
2020-04-01
影响因子：
4.1
通讯作者：
Bayliss, Richard
中科院分区：
文献类型：
--
作者：
Byrne, Matthew J.;Nasir, Nazia;Bayliss, Richard
Nek7是一种丝氨酸/苏氨酸蛋白激酶，是形成纺锤体和胞质分裂所必需的。在几种癌症中观察到了Nek7水平的升高，抑制Nek7可能为癌症治疗的发展提供了一条途径。到目前为止，还没有发现选择性和有效的Nek7抑制剂。Nek7晶体结构表现出一种不正确形成的调节-脊柱(R-脊柱)，这是一种不活跃的激酶的特征。我们推测，Nek7倾向于在这种不活跃的构象中结晶，可能会阻碍在与I型抑制剂的复合体中捕获Nek7的尝试。在这里，我们将芳香族残基引入到Nek7的R-骨架中，目的是通过R-骨架的堆积来稳定激酶的活性构象。强R-Spin突变体Nek7(SRS)保持了催化活性，并与化合物51形成络合物，化合物51是Nek2和Nek7的ATP竞争性抑制剂。随后，我们得到了与化合物51结合的apo形式的野生型Nek7(WT)的相同晶型。三个有序的Nek7(WT)分子的R-刺呈现不同的构象，而Nek7SRS分子的R-刺都具有相同的部分堆积构型。化合物51以类似的方式与Nek2和Nek7结合，但取代基精确定位的差异突出了可用于设计对特定Nek家族成员具有选择性的抑制剂的特征。虽然SRS突变并不是获得Nek7-抑制剂结构所必需的，但我们得出结论，这是一种有用的策略，可以抑制激酶的构象，以促进晶体形成。
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 Nek7(SRS) 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 Nek7(WT) in apo form and bound to compound 51. The R-spines of the three well-ordered Nek7(WT) 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.