An auto-inhibited state of protein kinase G and implications for selective activation.
An auto-inhibited state of protein kinase G and implications for selective activation.
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DOI：
10.7554/elife.79530
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发表时间：
2022-08-05
期刊：
影响因子：
7.7
通讯作者：
Kim, Choel
中科院分区：
文献类型：
--
作者：
Sharma, Rajesh;Kim, Jeong Joo;Qin, Liying;Henning, Philipp;Akimoto, Madoka;VanSchouwen, Bryan;Kaur, Gundeep;Sankaran, Banumathi;MacKenzie, Kevin R.;Melacini, Giuseppe;Casteel, Darren E.;Herberg, Friedrich W.;Kim, Choel
关键词：
环GMP依赖性蛋白激酶（PKG）是一氧化氮/环鸟苷一磷酸（cGMP）信号通路的关键介质，其调节如平滑肌收缩、心脏功能和轴突引导等多种生物功能。了解cGMP如何差异触发哺乳动物PKG亚型可能导致抑制或激活PKG的新疗法，补充靶向一氧化氮合酶和环核苷酸磷酸二酯酶的药物。PRKG 1转录物的选择性剪接赋予PKG Iα和Iβ不同的亮氨酸拉链、接头和自抑制（AI）假底物序列，导致亚型特异性激活特性，但酶自抑制的机制及其通过cGMP的缓解作用尚不清楚。在这里，我们展示了PKG Iβ的晶体结构，其中AI序列和环核苷酸结合（CNB）结构域与催化结构域结合，提供了自抑制状态的快照。PKG Iβ AI序列和酶活性位点之间的特异性接触有助于解释亚型特异性激活常数和连接子中磷酸化的影响。我们还展示了PKG I CNB结构域的晶体结构，该结构域具有与胸主动脉瘤和夹层相关的激活突变。该结构与野生型cGMP结合结构域的相似性和与自抑制酶的差异为组成性激活提供了机制基础。我们发现PKG Iβ自身抑制是由天然全长二聚体蛋白的每个单体内的接触介导的，并且使用可用的结构和生化数据，我们开发了PKG的调节和协同激活的模型。
Cyclic GMP-dependent protein kinases (PKGs) are key mediators of the nitric oxide/cyclic guanosine monophosphate (cGMP) signaling pathway that regulates biological functions as diverse as smooth muscle contraction, cardiac function, and axon guidance. Understanding how cGMP differentially triggers mammalian PKG isoforms could lead to new therapeutics that inhibit or activate PKGs, complementing drugs that target nitric oxide synthases and cyclic nucleotide phosphodiesterases in this signaling axis. Alternate splicing of PRKG1 transcripts confers distinct leucine zippers, linkers, and auto-inhibitory (AI) pseudo-substrate sequences to PKG Iα and Iβ that result in isoform-specific activation properties, but the mechanism of enzyme auto-inhibition and its alleviation by cGMP is not well understood. Here, we present a crystal structure of PKG Iβ in which the AI sequence and the cyclic nucleotide-binding (CNB) domains are bound to the catalytic domain, providing a snapshot of the auto-inhibited state. Specific contacts between the PKG Iβ AI sequence and the enzyme active site help explain isoform-specific activation constants and the effects of phosphorylation in the linker. We also present a crystal structure of a PKG I CNB domain with an activating mutation linked to Thoracic Aortic Aneurysms and Dissections. Similarity of this structure to wildtype cGMP-bound domains and differences with the auto-inhibited enzyme provide a mechanistic basis for constitutive activation. We show that PKG Iβ auto-inhibition is mediated by contacts within each monomer of the native full-length dimeric protein, and using the available structural and biochemical data we develop a model for the regulation and cooperative activation of PKGs.