cGMP Binding Domain D Mediates a Unique Activation Mechanism in Plasmodium falciparum PKG
cGMP Binding Domain D Mediates a Unique Activation Mechanism in Plasmodium falciparum PKG
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DOI：
10.1021/acsinfecdis.7b00222
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发表时间：
2018-03-01
影响因子：
5.3
通讯作者：
Herberg, Friedrich W.
中科院分区：
文献类型：
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作者：
Franz, Eugen;Knape, Matthias J.;Herberg, Friedrich W.
来自恶性疟原虫 (PfPKG) 的 cGMP 依赖性蛋白激酶在这种引起人类疟疾的寄生虫的有性和无性增殖中发挥着至关重要的作用。然而，PfPKG 的功能和调节很大程度上未知。先前的研究表明，PfPKG 的结构域组织与人类 PKG (hPKG) 显着不同，并表明环核苷酸结合结构域 D (CNB-D) 的关键作用。我们发现了一种新的机制，其中 CNB-D 控制寄生虫特异性蛋白激酶的激活和调节。此处，激酶活性不依赖于人 PKG 报道的假底物自抑制序列 (IS)。缺乏假定的 IS 且仅包含 CNB-D 和催化结构域的构建体在没有 cGMP 的情况下是无活性的，并且可以被 cGMP 有效地激活。在结构证据的基础上，我们描述了一种调节机制，cGMP 与 CNB-D 结合诱导涉及 CNB-D 的 α C 螺旋的构象变化。非活性状态由催化结构域的 Asp597 和 aC 螺旋的 Arg528 之间的独特相互作用定义。然而，相同的精氨酸 (R528) 通过与磷酸盐结合盒 (PBC) 的 Tyr480 相互作用来稳定 cGMP 结合。这代表 PfPKG 的活跃状态。我们的结果揭示了 PfPKG 和 hPKG 激活机制的根本差异，为制定抗击疟疾的靶向药物设计策略奠定了基础。
cGMP-dependent protein kinase from Plasmodium falciparum (PfPKG) plays a crucial role in the sexual as well as the asexual proliferation of this human malaria causing parasite. However, function and regulation of PfPKG are largely unknown. Previous studies showed that the domain organization of PfPKG significantly differs from human PKG (hPKG) and indicated a critical role of the cyclic nucleotide binding domain D (CNB-D). We identified a novel mechanism, where the CNB-D controls activation and regulation of the parasite specific protein kinase. Here, kinase activity is not dependent on a pseudosubstrate autoinhibitory sequence (IS), as reported for human PKG. A construct lacking the putative IS and containing only the CNB-D and the catalytic domain is inactive in the absence of cGMP and can efficiently be activated with cGMP. On the basis of structural evidence, we describe a regulatory mechanism, whereby cGMP binding to CNB-D induces a conformational change involving the alpha C-helix of the CNB-D. The inactive state is defined by a unique interaction between Asp597 of the catalytic domain and Arg528 of the aC-helix. The same arginine (R528), however, stabilizes cGMP binding by interacting with Tyr480 of the phosphate binding cassette (PBC). This represents the active state of PfPKG. Our results unveil fundamental differences in the activation mechanism between PfPKG and hPKG, building the basis for the development of strategies for targeted drug design in fighting malaria.