McKenna Sean A, Lindhout Darrin A, Kim Insil, Liu Corey W, Gelev Vladimir M, Wagner Gerhard, Puglisi Joseph D
Department of Structural Biology and Stanford Magnetic Resonance Laboratory, Stanford University School of Medicine, Stanford, California 94305-5126, USA.
J Biol Chem. 2007 Apr 13;282(15):11474-86. doi: 10.1074/jbc.M700301200. Epub 2007 Feb 6.
The RNA-dependent protein kinase (PKR) plays an integral role in the antiviral response to cellular infection. PKR contains three distinct domains consisting of two conserved N-terminal double-stranded RNA (dsRNA)-binding domains, a C-terminal Ser-Thr kinase domain, and a central 80-residue linker. Despite rich structural and biochemical data, a detailed mechanistic explanation of PKR activation remains unclear. Here we provide a framework for understanding dsRNA-dependent activation of PKR using nuclear magnetic resonance spectroscopy, dynamic light scattering, gel filtration, and autophosphorylation kinetics. In the latent state, PKR exists as an extended monomer, with an increase in self-affinity upon dsRNA association. Subsequent phosphorylation leads to efficient release of dsRNA followed by a greater increase in self-affinity. Activated PKR displays extensive conformational perturbations within the kinase domain. We propose an updated model for PKR activation in which the communication between RNA binding, central linker, and kinase domains is critical in the propagation of the activation signal and for PKR dimerization.
RNA依赖性蛋白激酶（PKR）在细胞感染的抗病毒反应中发挥着不可或缺的作用。PKR包含三个不同的结构域，由两个保守的N端双链RNA（dsRNA）结合结构域、一个C端丝氨酸-苏氨酸激酶结构域和一个中间的80个残基的连接区组成。尽管有丰富的结构和生化数据，但PKR激活的详细机制解释仍不清楚。在这里，我们利用核磁共振光谱、动态光散射、凝胶过滤和自磷酸化动力学，提供了一个理解PKR的dsRNA依赖性激活的框架。在潜伏状态下，PKR以伸展的单体形式存在，与dsRNA结合后自亲和力增加。随后的磷酸化导致dsRNA的有效释放，接着自亲和力进一步增加。活化的PKR在激酶结构域内表现出广泛的构象扰动。我们提出了一个更新的PKR激活模型，其中RNA结合、中间连接区和激酶结构域之间的通讯对于激活信号的传播和PKR二聚化至关重要。
