Mechanism of Intracellular cAMP Sensor Epac2 Activation cAMP-INDUCED CONFORMATIONAL CHANGES IDENTIFIED BY AMIDE HYDROGEN/DEUTERIUM EXCHANGE MASS SPECTROMETRY (DXMS)
Mechanism of Intracellular cAMP Sensor Epac2 Activation cAMP-INDUCED CONFORMATIONAL CHANGES IDENTIFIED BY AMIDE HYDROGEN/DEUTERIUM EXCHANGE MASS SPECTROMETRY (DXMS)
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DOI：
10.1074/jbc.m111.224535
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发表时间：
2011-05-20
影响因子：
4.8
通讯作者：
Cheng, Xiaodong
中科院分区：
文献类型：
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作者：
Li, Sheng;Tsalkova, Tamara;Cheng, Xiaodong
Epac 2是一种鸟嘌呤核苷酸交换因子，响应第二信使cAMP调节多种细胞内过程。在这项研究中，我们已经使用肽酰胺氢/氘交换质谱探测的解决方案的结构和构象动力学的全长Epac 2在cAMP的存在和不存在下。结果支持cAMP诱导的Epac 2激活的机制，其中介导的主要铰链运动集中在第二个cAMP结合域的C末端。这种构象变化使Epac 2的调节组分重新排列远离催化核心，使后者可用于效应物结合。此外，第一和第二cAMP结合结构域之间的界面是高度动态的，提供了cAMP如何进入配体结合位点的解释，在晶体结构中，所述配体结合位点被认为是由另一个cAMP结合结构域相互封闭的。此外，cAMP还诱导离子锁/发夹结构的构象变化，其直接参与RAP 1结合。这些结果表明，除了减轻调节叶对催化叶施加的空间位阻外，cAMP也可能是直接影响Epac 2和RAP 1之间相互作用的变构调节剂。最后，cAMP结合还诱导在凌乱的/Egl/普列克底物蛋白（DEP）结构域中的显著构象变化，所述DEP结构域是保守的结构基序，尽管从活性Epac 2晶体结构中缺失，但对于Epac亚细胞靶向和体内功能是重要的。
Epac2, a guanine nucleotide exchange factor, regulates a wide variety of intracellular processes in response to second messenger cAMP. In this study, we have used peptide amide hydrogen/deuterium exchange mass spectrometry to probe the solution structural and conformational dynamics of full-length Epac2 in the presence and absence of cAMP. The results support a mechanism in which cAMP-induced Epac2 activation is mediated by a major hinge motion centered on the C terminus of the second cAMP binding domain. This conformational change realigns the regulatory components of Epac2 away from the catalytic core, making the later available for effector binding. Furthermore, the interface between the first and second cAMP binding domains is highly dynamic, providing an explanation of how cAMP gains access to the ligand binding sites that, in the crystal structure, are seen to be mutually occluded by the other cAMP binding domain. Moreover, cAMP also induces conformational changes at the ionic latch/hairpin structure, which is directly involved in RAP1 binding. These results suggest that in addition to relieving the steric hindrance imposed upon the catalytic lobe by the regulatory lobe, cAMP may also be an allosteric modulator directly affecting the interaction between Epac2 and RAP1. Finally, cAMP binding also induces significant conformational changes in the dishevelled/Egl/pleckstrin (DEP) domain, a conserved structural motif that, although missing from the active Epac2 crystal structure, is important for Epac subcellular targeting and in vivo functions.