Allosteric inhibition of Aurora-A kinase by a synthetic vNAR domain.
Allosteric inhibition of Aurora-A kinase by a synthetic vNAR domain.
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DOI：
10.1098/rsob.160089
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发表时间：
2016-07
期刊：
影响因子：
5.8
通讯作者：
Bayliss R
中科院分区：
文献类型：
--
作者：
Burgess SG;Oleksy A;Cavazza T;Richards MW;Vernos I;Matthews D;Bayliss R
绝大多数临床批准的蛋白激酶抑制剂直接靶向atp结合口袋。因此，许多抑制剂具有广泛的选择性，并且大多数具有显著的脱靶效应。变构抑制剂通常更具选择性，但很难识别，因为变构结合位点通常是未知的或特征不明确。Aurora-A通过TPX2与激酶催化结构域上的变构位点结合而被激活，这一知识可以用来产生抑制剂。在这里，我们基于合成的vNAR单域支架vNAR- d01生成了Aurora-A激酶的变构抑制剂。生化研究和Aurora-A/vNAR- d01复合物的晶体结构表明，vNAR结构域与TPX2结合位点重叠。与TPX2的结合稳定了激酶的活性构象相反，vNAR结构域的结合稳定了激酶的非活性构象，其中α c -螺旋扭曲，典型的Lys-Glu盐桥断裂，调节（R-）脊柱被激活环的额外疏水侧链破坏。这些研究说明了单域抗体可以用来表征激酶的调节机制，并为结构导向设计变构性Aurora-A激酶抑制剂提供了合理的基础。
The vast majority of clinically approved protein kinase inhibitors target the ATP-binding pocket directly. Consequently, many inhibitors have broad selectivity profiles and most have significant off-target effects. Allosteric inhibitors are generally more selective, but are difficult to identify because allosteric binding sites are often unknown or poorly characterized. Aurora-A is activated through binding of TPX2 to an allosteric site on the kinase catalytic domain, and this knowledge could be exploited to generate an inhibitor. Here, we generated an allosteric inhibitor of Aurora-A kinase based on a synthetic, vNAR single domain scaffold, vNAR-D01. Biochemical studies and a crystal structure of the Aurora-A/vNAR-D01 complex show that the vNAR domain overlaps with the TPX2 binding site. In contrast with the binding of TPX2, which stabilizes an active conformation of the kinase, binding of the vNAR domain stabilizes an inactive conformation, in which the αC-helix is distorted, the canonical Lys-Glu salt bridge is broken and the regulatory (R-) spine is disrupted by an additional hydrophobic side chain from the activation loop. These studies illustrate how single domain antibodies can be used to characterize the regulatory mechanisms of kinases and provide a rational basis for structure-guided design of allosteric Aurora-A kinase inhibitors.