Crystal structures of human soluble adenylyl cyclase reveal mechanisms of catalysis and of its activation through bicarbonate
Crystal structures of human soluble adenylyl cyclase reveal mechanisms of catalysis and of its activation through bicarbonate
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DOI：
10.1073/pnas.1322778111
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发表时间：
2014-03-11
影响因子：
11.1
通讯作者：
Steegborn, Clemens
中科院分区：
文献类型：
--
作者：
Kleinboelting, Silke;Diaz, Ana;Steegborn, Clemens
cAMP是一种进化保守的原型第二信使，调节多种细胞功能。在哺乳动物中，cAMP由10种同源腺苷酸环化酶（AC）之一合成：9种跨膜酶和1种可溶性AC（sAC）。其中，只有sAC直接被碳酸氢盐（HCO 3-）激活;因此，它在生理功能中充当HCO 3-、二氧化碳（CO2）和pH的细胞传感器，如精子激活、房水形成和代谢调节。在这里，我们描述了晶体结构的人sAC催化结构域的载脂蛋白状态和复杂的底物类似物，产品和监管机构。活化剂HCO 3-与Arg 176相邻结合，Arg 176充当开关，使得能够形成催化阳离子位点。阴离子抑制剂4，4 '-二异硫氰基芪-2，2'-二磺酸通过结合到活性位点入口来抑制sAC，其通过空间位阻和捕获Arg 176侧链来阻断HCO 3-活化。最后，产物络合物揭示了促进催化的小的局部重排。我们的研究结果提供了sAC催化和细胞HCO 3传感的分子机制，并为靶向该系统的药物提供了基础。
cAMP is an evolutionary conserved, prototypic second messenger regulating numerous cellular functions. In mammals, cAMP is synthesized by one of 10 homologous adenylyl cyclases (ACs): nine transmembrane enzymes and one soluble AC (sAC). Among these, only sAC is directly activated by bicarbonate (HCO3-); it thereby serves as a cellular sensor for HCO3-, carbon dioxide (CO2), and pH in physiological functions, such as sperm activation, aqueous humor formation, and metabolic regulation. Here, we describe crystal structures of human sAC catalytic domains in the apo state and in complex with substrate analog, products, and regulators. The activator HCO3- binds adjacent to Arg176, which acts as a switch that enables formation of the catalytic cation sites. An anionic inhibitor, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, inhibits sAC through binding to the active site entrance, which blocks HCO3- activation through steric hindrance and trapping of the Arg176 side chain. Finally, product complexes reveal small, local rearrangements that facilitate catalysis. Our results provide a molecular mechanism for sAC catalysis and cellular HCO3- sensing and a basis for targeting this system with drugs.