The Influenza M2 Ectodomain Regulates the Conformational Equilibria of the Transmembrane Proton Channel: Insights from Solid-State Nuclear Magnetic Resonance.
The Influenza M2 Ectodomain Regulates the Conformational Equilibria of the Transmembrane Proton Channel: Insights from Solid-State Nuclear Magnetic Resonance.
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流感 M2 胞外域调节跨膜质子通道的构象平衡：来自固态核磁共振的见解。
DOI：
10.1021/acs.biochem.6b00727
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发表时间：
2016
期刊：
影响因子：
2.9
通讯作者：
Hong,Mei
中科院分区：
文献类型：
--
作者：
Kwon,Byungsu;Hong,Mei
流感病毒M2蛋白是金刚烷胺家族抗病毒药物的靶标，其跨膜（TM）结构域结构和动力学已被广泛研究。然而，很少有人知道的高度保守的N-末端胞外域，其中包含流感疫苗靶向表位的结构。在这项研究中，我们合成了一个M2的构建体包含N-末端的胞外域和TM结构域，了解特定位点的构象和动力学的胞外域和研究的影响TM结构。我们将13 C-和15 N-标记的残基插入到两个结构域中，并使用固态核磁共振测量它们的化学位移和线宽。数据表明，整个胞外域是非结构化的和动态的，但运动是较慢的残基更接近TM域。13 C线形表明，这种外TM构建经历快速单轴旋转扩散，如分离的TM肽，但药物结合增加的运动速率的TM螺旋，同时减慢的局部运动的胞外域残基接近TM域。此外，13 C和15 N化学位移表明，即使在不存在金刚烷胺的情况下，胞外域也将TM残基的构象平衡向药物结合状态转移，从而为全长M2的抑制浓度低于胞外域截短的M2提供了分子结构基础。我们提出，这种构象选择可能是由于四聚体蛋白中带负电荷的胞外域残基之间的静电排斥。连同最近对M2胞质结构域的研究，这些结果表明，膜蛋白中内在无序的膜外结构域可以调节结构有序的TM结构域的功能相关的构象和动力学。
The influenza M2 protein is the target of the amantadine family of antiviral drugs, and its transmembrane (TM) domain structure and dynamics have been extensively studied. However, little is known about the structure of the highly conserved N-terminal ectodomain, which contains epitopes targeted by influenza vaccines. In this study, we synthesized an M2 construct containing the N-terminal ectodomain and the TM domain, to understand the site-specific conformation and dynamics of the ectodomain and to investigate the effect of the ectodomain on the TM structure. We incorporated13C- and15N-labeled residues into both domains and measured their chemical shifts and line widths using solid-state nuclear magnetic resonance. The data indicate that the entire ectodomain is unstructured and dynamic, but the motion is slower for residues closer to the TM domain.13C line shapes indicate that this ecto-TM construct undergoes fast uniaxial rotational diffusion, like the isolated TM peptide, but drug binding increases the motional rates of the TM helix while slowing the local motion of the ectodomain residues that are close to the TM domain. Moreover,13C and15N chemical shifts indicate that the ectodomain shifts the conformational equilibria of the TM residues toward the drug-bound state even in the absence of amantadine, thus providing a molecular structural basis for the lower inhibitory concentration of full-length M2 compared to that of the ectodomain-truncated M2. We propose that this conformational selection may result from electrostatic repulsion between negatively charged ectodomain residues in the tetrameric protein. Together with the recent study of the M2 cytoplasmic domain, these results show that intrinsically disordered extramembrane domains in membrane proteins can regulate the functionally relevant conformation and dynamics of the structurally ordered TM domains.
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