Structure of M2 Proton Channel from Influenza A Virus

描述（由申请人提供）：甲型流感病毒的M2质子通道蛋白是抗流感药物金刚烷胺的靶点。M2包含一个单一的跨膜结构域，形成该通道的均匀四聚体孔。M2的小尺寸和简单的结构使其成为理解膜蛋白的电荷稳定和质子传导机制的一个有吸引力的模型。在此之前，通过高分辨率核磁共振和晶体结构来阐明质子传导的机制。该通道具有一个长而充满水的孔，导致由His37和Trp41定义的选择性过滤器。质子扩散通过这个水孔结合在His37四分体上并打开Trp41门。为了了解这一过程的结构基础，结构将在下一个资助期得到解决。甲型流感病毒是对人类健康的重大威胁。目前已批准的抗流感药物有两类：金刚烷胺和金刚乙胺靶向M2质子通道，而达菲（奥司他韦）及相关化合物靶向神经氨酸酶。对这两类药物的耐药性构成了一个主要问题，大多数甲型流感病毒分离株现在对金刚烷胺具有耐药性。因此，我们正在解决金刚烷胺和金刚乙胺配合物与M2和耐药突变体的结构。药物通过将其氨靶向于沿通道轴排列的三个低能热点中的一个而与M2结合。在可传播的病毒中，只有少数突变在这些位点是可耐受的。在这里，假设导向和基于结构的方法被用于设计这组耐药突变的新抑制剂。这一努力不仅将为抗流感药物提供新的线索，而且还将推进基于结构的靶向膜蛋白药物设计——随着具有重要医学意义的膜蛋白结构数量的增加，这是一项越来越重要的努力。在Aim 1中，使用假设导向的结构方法来发现抑制相关M2突变的小分子。在Aim 2中，EPR，晶体学和核磁共振研究将探讨传导和药物抑制的机制。位点定向自旋标记将重点关注磷脂囊泡中的全长蛋白，并评估由于pH和药物结合变化而引起的构象变化。这些研究将促进对更高分辨率晶体结构和核磁共振结构的理解。M2突变体的晶体结构将在有和没有结合药物的情况下解决，为Aim 1的药物设计提供信息，并探索质子传导的机制。我们早期的晶体学工作是在胶束中孤立的跨膜区域进行的；膜环境以及缺失的结构域可能会影响结构。因此，我们现在结晶更长的结构，从双胞体和脂质立方相。为了帮助这些结构稳定在它们的天然构象中，我们正在用已知的治疗性抗体和噬菌体上选择的抗体来解决结构。最后，我们将对乙型流感病毒BM2的质子通道进行结构表征，该通道除了具有His-X3-Trp基序外，与M2没有序列相似性。这些研究也将使未来基于结构的BM2抑制剂药物设计成为可能。同时，我们将在胶束、单束和纳米盘中进行溶液核磁共振研究。我们结合了生物合成和合成标记策略，以方便结构确定和提高溶液核磁共振结构的分辨率。这些研究将为质子通过M2传导的机制提供新的见解，并为设计新的抑制剂奠定基础。

DESCRIPTION (provided by applicant): The M2 proton channel protein of the influenza A virus is the target of the anti-influenza drug, amantadine. M2 contains a single transmembrane domain that forms the homo-tetrameric pore of this channel. M2's small size and simple structure makes it an attractive model for understanding the mechanism of charge-stabilization and proton conduction through membrane proteins. In the previous period, high-resolution NMR and crystal structures were solved to elucidate the mechanism of proton conduction. The channel has a long, water-filled pore that leads to a selectivity filter defined by His37 and Trp41 Protons diffuse through this aqueous pore to bind at the His37 tetrad and open the Trp41 gate. To understand the structural basis for this process, structures will be solved in the next funding period. Influenza A virus is a major threat to human health. There are two different classes of approved anti-influenza drugs: amantadine and rimantadine target the M2 proton channel, while Tamiflu (oseltamivir) and related compounds target neuraminidase. Resistance to both classes of drugs poses a major problem, and most isolates of influenza A virus are now amantadine-resistant. We therefore are solving structures of amantadine and rimantadine complexes with M2, and drug-resistant mutants. Drug binds to M2 by targeting its ammonium to one of the three low energy hotspot sites aligned along the channel axis. Only a handful of mutations are tolerated at these sites in transmissible viruses. Here, hypothesis-directed and structure-based approaches are used to design new inhibitors of this set of resistant mutants. This endeavor will not only provide new leads for anti-influenza medications, but it should also advance structure-based design of drugs targeting membrane proteins - an increasingly important endeavor as the number of medicinally important membrane protein structures grows. In Aim 1, hypothesis-directed structural approaches are used to discover small molecules that inhibit relevant mutants of M2. In Aim 2, EPR, crystallographic, and NMR investigations will probe the mechanism of conduction and drug inhibition. Site-directed spin labeling will focus on the full-length protein in phospholipid vesicles, and evaluate conformational changes due to variations in pH and drug-binding. These studies will facilitate understanding of more high-resolution crystal structures and NMR structures. Crystallographic structures of M2 mutants will be solved with and without bound drugs to inform drug design in Aim 1 and also probe the mechanism of proton conduction. Our early crystallographic work was conducted with the isolated transmembrane domain in micelles; the membrane environment as well as missing domains might influence the structure. Thus, we are now crystallizing longer constructs from both bicelles and lipidic cubic phases. To help stabilize these constructs in their native conformations, we are solving structures with known therapeutic antibodies and antibodies selected on phage. Finally we will structurally characterize the proton channel of influenza B virus, BM2, which shows no sequence similarity to M2 aside from having a His-X3-Trp motif. These studies will also enable future structure-based drug design of BM2 inhibitors. In parallel we will conduct solution NMR studies in micelles, bicelles, and nanodisks. We combine biosynthetic and synthetic labeling strategies to facilitate structure determination and to increase the resolution of solution NMR structures. These studies will provide new insight into the mechanism of proton conduction through M2 and lay the groundwork for the design of new inhibitors.