Mapping of the auto-inhibitory interactions of protein kinase R by nuclear magnetic resonance
Mapping of the auto-inhibitory interactions of protein kinase R by nuclear magnetic resonance
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DOI：
10.1016/j.jmb.2006.08.077
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发表时间：
2006-12-01
影响因子：
5.6
通讯作者：
Wagner, Gerhard
中科院分区：
文献类型：
--
作者：
Gelev, Vladimir;Aktas, Huseyin;Wagner, Gerhard
dsRNA依赖性蛋白激酶（PKR）是干扰素抗病毒和抗增殖作用的关键介质。非磷酸化PKR的特征在于激酶和RNA结合结构域（RBD）之间的抑制性相互作用，但是潜伏状态的结构细节及其在激活期间的解开还没有很好地理解。为了通过NMR研究PKR调节，我们分配了催化失活的K296 R激酶结构域的大部分骨架共振，并使用RBD对该激酶结构域进行了N-15-杂原子量子相干（HSQC）滴定。激酶中的化学位移扰动表明RBD 2结合激酶C-叶中的底物eIF 2 α对接位点。与这些结果一致，eIF 2 α对接位点F495 A的突变显示与RBD的相互作用较弱。全长RBD 1 + 2比单独的RBD 2更强地结合激酶结构域。所观察到的化学位移变化从eIF 2 α结合位点延伸到激酶N-叶和活性位点内部，与RBD的N-末端部分和激酶之间的弱相互作用一致。(c)2006爱思唯尔有限公司保留所有权利。
The dsRNA-dependent protein kinase (PKR) is a key mediator of the antiviral and anti-proliferative effects of interferon. Unphosphorylated PKR is characterized by inhibitory interactions between the kinase and RNA binding domains (RBDs), but the structural details of the latent state and its unraveling during activation are not well understood. To study PKR regulation by NMR we assigned a large portion of the backbone resonances of the catalytically inactive K296R kinase domain, and performed N-15-heteronuclear sin le quantum coherence (HSQC) titrations; of this kinase domain with the RBDs. Chemical shift perturbations in the kinase indicate that RBD2 binds to the substrate eIF2 alpha docking site in the kinase C-lobe. Consistent with these results, a mutation in the eIF2 alpha docking site, F495A, displays weaker interactions with the RBD. The full-length RBD1 + 2 binds more strongly to the kinase domain than RBD2 alone. The observed chemical shift changes extend from the eIF2 alpha binding site into the kinase N-lobe and inside the active site, consistent with weak interactions between the N-terminal part of the RBD and the kinase. (c) 2006 Elsevier Ltd. All rights reserved.