Kissing G Domains of MnmE Monitored by X-Ray Crystallography and Pulse Electron Paramagnetic Resonance Spectroscopy
Kissing G Domains of MnmE Monitored by X-Ray Crystallography and Pulse Electron Paramagnetic Resonance Spectroscopy
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DOI：
10.1371/journal.pbio.1000212
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发表时间：
2009-10-01
期刊：
影响因子：
9.8
通讯作者：
Wittinghofer, Alfred
中科院分区：
文献类型：
--
作者：
Meyer, Simon;Boehme, Sabine;Wittinghofer, Alfred
MnmE参与某些trna摆动位置的修饰，属于由核苷酸依赖性二聚化（GADs）激活的扩展类G蛋白。先前的模型表明该蛋白是一个多结构域蛋白，其G结构域以核苷酸依赖的方式相互联系。在这里，我们采用x射线晶体学和脉冲电子顺磁共振（EPR）光谱的联合方法来证明MnmE的大结构域运动与G蛋白循环耦合。x射线结构表明MnmE是一个组成型同二聚体，其中高流动性的G畴在不同的方向上彼此面对，但并不像孤立畴的GDP-AlF(x)结构所表明的那样紧密接触。脉冲双电子-电子共振（DEER）光谱距离测量表明，G域在无核苷酸/ gdp结合状态下为开放构象，在gtp结合状态下为开/闭两态平衡，最大距离变化为18埃。GDP和AlF(x)模拟了磷酰转移反应的过渡态，只观察到封闭的构象。活性位点与GDP-AlF(x)的二聚化需要特定单价阳离子的存在，从而反映了MnmE GTPase反应的要求。我们的研究结果直接证明了MnmE在其GTPase周期中所经历的构象变化的性质。它们显示了G结构域相对于蛋白质其余部分围绕两个旋转位置的核苷酸依赖的动态运动，它们对于理解这种广泛性遗传疾病的机制原理至关重要。
MnmE, which is involved in the modification of the wobble position of certain tRNAs, belongs to the expanding class of G proteins activated by nucleotide-dependent dimerization (GADs). Previous models suggested the protein to be a multidomain protein whose G domains contact each other in a nucleotide dependent manner. Here we employ a combined approach of X-ray crystallography and pulse electron paramagnetic resonance (EPR) spectroscopy to show that large domain movements are coupled to the G protein cycle of MnmE. The X-ray structures show MnmE to be a constitutive homodimer where the highly mobile G domains face each other in various orientations but are not in close contact as suggested by the GDP-AlF(x) structure of the isolated domains. Distance measurements by pulse double electron-electron resonance (DEER) spectroscopy show that the G domains adopt an open conformation in the nucleotide free/GDP-bound and an open/closed two-state equilibrium in the GTP-bound state, with maximal distance variations of 18 angstrom. With GDP and AlF(x), which mimic the transition state of the phosphoryl transfer reaction, only the closed conformation is observed. Dimerization of the active sites with GDP-AlF(x) requires the presence of specific monovalent cations, thus reflecting the requirements for the GTPase reaction of MnmE. Our results directly demonstrate the nature of the conformational changes MnmE was previously suggested to undergo during its GTPase cycle. They show the nucleotide-dependent dynamic movements of the G domains around two swivel positions relative to the rest of the protein, and they are of crucial importance for understanding the mechanistic principles of this GAD.