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ph激活的质子通道，这对病毒生命周期至关重要。金刚烷胺类抗病毒药物对H+通道活性的抑制作用由于M2跨膜结构域的突变而失效。因此，高分辨率的M2结构测定对于开发针对金刚烷胺耐药M2突变体的新抗病毒药物至关重要。小的M2蛋白包含pH活化、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。由该研究计划资助的工作已经1)导致了M2中药理学相关的药物结合位点和脂质双分子层中药物络合的高分辨率结构的阐明，2)揭示了质子选择残基His37的新ph依赖动力学。然而，与此同时，新的替代H+传导模型被提出，Trp41通道门控的结构基础尚未研究。本提案的第一个目的是通过检测His37通道首次激活时在温和酸性pH下的结构来阐明M2的H+传导机制。将测量侧链h键、质子化/去质子化动力学以及抑制剂对His37结构的影响。将金刚烷胺和Cu2+作为抑制剂，探索Cu2+的顺磁弛豫增强效应，以确定其结构。第二个目标是阐明Trp41的结构和与His37的相互作用作为pH的函数，以了解这两个残基如何在双层环境中协同作用以实现通道门控。除了H+通道活性外，M2还通过引起膜弯曲以胆固醇依赖的方式介导病毒出芽。我们将通过距离和弛豫核磁共振测量研究m2 -膜和m2 -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被验证。最后，在病毒组装和出芽过程中，M2通过其细胞质尾部与基质蛋白M1相互作用。到目前为止还没有细胞质结构域的结构信息。我们将使用多维魔角旋转固态核磁共振技术确定脂质双层中全长M2的三维结构，为未来研究对流感生命周期重要的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.