Solid-state NMR of the influenza M2 protein in lipid bilayers

描述(由申请人提供)：流感M2蛋白形成一个pH激活的质子通道，这是病毒生命周期所必需的。金刚烷胺类抗病毒药物对H+通道活性的抑制因M2跨膜区的突变而无效。因此，M2的高分辨结构测定对于开发针对金刚烷胺耐药M2突变体的新的抗病毒药物至关重要。小M2蛋白包含pH激活、H+选择性和门控所需的所有机制，因此也为理解更大、更复杂的电压门控H+通道和其他pH门控离子通道提供了一个很好的模型系统。由这项研究计划资助的工作已经1)导致阐明M2中与药物相关的药物结合部位和脂质双层中的药物络合高分辨结构，以及2)揭示质子选择性残基His37的新的pH依赖动力学。然而，与此同时，新的替代H+传导模型也被提出，而Trp41通道选通的结构基础还没有得到研究。这一建议的第一个目的是通过研究通道首次激活时在中等酸性pH下的His37结构来阐明M2的H+传导机制。将测量侧链氢键，质子化/去质子化动力学，以及抑制剂对His37结构的影响。金刚烷胺和Cu2+都将被用作抑制剂，并将探索Cu2+的顺磁驰豫增强效应来确定结构。第二个目标是阐明Trp41的结构以及与His37的相互作用作为pH的函数，以了解这两个残基如何一致作用来实现通道门控，同样是在双层环境中。除了H+通道活性外，M2还通过以胆固醇依赖的方式引起细胞膜弯曲来调节病毒的萌发。我们将通过距离和松弛核磁共振测量来研究M2膜和M2与胆固醇的相互作用。M2优先定位于膜的高度弯曲区域的假设将得到检验。最后，在病毒组装和萌发过程中，M2通过其细胞质尾巴与基质蛋白M1相互作用。到目前为止，还没有关于细胞质区域的结构信息。我们将利用多维魔角旋转固体核磁共振技术确定脂质双层中全长M2的三维结构，为未来研究M2-M1相互作用奠定基础。

DESCRIPTION (provided by applicant): The influenza M2 protein forms a pH-activated proton channel that is essential for the virus lifecycle. Inhibition of the H+ channel activity by the amantadine class of antiviral drugs has been made ineffective by mutations in the M2 transmembrane domain. High-resolution structure determination of M2 is thus paramount for developing new antiviral drugs to target amantadine-resistant M2 mutants. The small M2 protein contains all the machinery necessary for pH activation, H+ selectivity, and gating, and thus also provides an excellent model system for understanding larger and more complex voltage-gated H+ channels and other pH-gated ion channels. Work funded by this research proposal has already 1) led to the elucidation of the pharmacologically relevant drug binding site in M2 and the drug-complexed high-resolution structure in the lipid bilayer, and 2) revealed novel pH-dependent dynamics of the proton-selective residue, His37. However, new alternative H+ conduction models have been proposed in the meantime, and the structure basis for channel gating by Trp41 has not been studied. The first aim of this proposal is to elucidate the H+ conduction mechanism of M2 by examining His37 structure at mildly acidic pH when the channel is first activated. Sidechain H-bonding, protonation/deprotonation dynamics, and the effects of inhibitors on His37 structure will be measured. Both amantadine and Cu2+ will be used as inhibitors, and Cu2+ paramagnetic relaxation enhancement effects will be explored for structure determination. The second aim is to elucidate Trp41 structure and interaction with His37 as a function of pH, to understand how these two residues act in unison to achieve channel gating, again in a bilayer environment. In addition to the H+ channel activity, M2 also mediates virus budding by causing membrane curvature in a cholesterol-dependent fashion. We will investigate M2-membrane and M2-cholesterol interactions by distance and relaxation NMR measurements. The hypothesis that M2 preferentially localizes to highly curved regions of the membrane will be tested. Finally, M2 interacts with matrix protein M1 through its cytoplasmic tail during virus assembly and budding. No structural information is available so far for the cytoplasmic domain. We will determine the three-dimensional structure of full-length M2 in lipid bilayers using multidimensional magic-angle-spinning solid-state NMR techniques, to lay the ground for future investigations of the M2-M1 interactions important for the influenza life cycle.