Norwegian Structural Biology Centre, Department of Chemistry, University of Tromsø, N-9037 Tromsø, Norway.
Biochem J. 2011 Nov 15;440(1):85-93. doi: 10.1042/BJ20110592.
We describe in the present paper mutations of the catalytic subunit α of PKA (protein kinase A) that introduce amino acid side chains into the ATP-binding site and progressively transform the pocket to mimic that of Aurora protein kinases. The resultant PKA variants are enzymatically active and exhibit high affinity for ATP site inhibitors that are specific for Aurora kinases. These features make the Aurora-chimaeric PKA a valuable tool for structure-based drug discovery tasks. Analysis of crystal structures of the chimaera reveal the roles for individual amino acid residues in the binding of a variety of inhibitors, offering key insights into selectivity mechanisms. Furthermore, the high affinity for Aurora kinase-specific inhibitors, combined with the favourable crystallizability properties of PKA, allow rapid determination of inhibitor complex structures at an atomic resolution. We demonstrate the utility of the Aurora-chimaeric PKA by measuring binding kinetics for three Aurora kinase-specific inhibitors, and present the X-ray structures of the chimaeric enzyme in complex with VX-680 (MK-0457) and JNJ-7706621 [Aurora kinase/CDK (cyclin-dependent kinase) inhibitor].
我们在本文中描述了蛋白激酶 A（PKA）催化亚基α的突变，这些突变将氨基酸侧链引入 ATP 结合位点，并逐渐将口袋模拟为 Aurora 蛋白激酶的口袋。由此产生的 PKA 变体具有酶活性，并对特异性针对 Aurora 激酶的 ATP 结合位点抑制剂表现出高亲和力。这些特征使 Aurora 嵌合 PKA 成为基于结构的药物发现任务的有价值的工具。对嵌合体晶体结构的分析揭示了单个氨基酸残基在结合各种抑制剂中的作用，为选择性机制提供了关键见解。此外，对 Aurora 激酶特异性抑制剂的高亲和力，结合 PKA 的有利结晶特性，允许以原子分辨率快速确定抑制剂复合物的结构。我们通过测量三种 Aurora 激酶特异性抑制剂的结合动力学来证明 Aurora 嵌合 PKA 的实用性，并展示了嵌合酶与 VX-680（MK-0457）和 JNJ-7706621（Aurora 激酶/CDK（细胞周期蛋白依赖性激酶）抑制剂）复合物的 X 射线结构。
