Solid-state NMR studies of the dynamic interactions of the influenza A M2 membrane protein with water, antiviral drugs, and the M1 protein

摘要 季节性流感病毒每年感染5%-10%的成年人和20%-30%的儿童，但只有两个班级 一种是针对A型流感M2(AM2)蛋白的，另一种是针对A型流感M2(AM2)蛋白的 神经氨酸酶(NA)。AM2在病毒包膜中形成一个对病毒生命周期至关重要的质子通道； AM2毛孔的堵塞可抑制病毒感染。不幸的是，近年来99%的流感病毒株已经成为 抵抗FDA批准的AM2通道阻滞剂金刚烷胺和金刚乙胺。最近，新的抗病毒药物 已经报道了有效对抗金刚烷胺耐药AM2蛋白的药物，但它们的作用机制 仍然难以捉摸。水在通过AM2孔的质子传导中起着重要的作用，但它不是 了解新的AM2抑制剂如何影响渠道中的水动力学。解说航道水 动力学应该为抗流感药物的作用机制提供洞察力。我建议发展和发展 用固体核磁共振(SS核磁共振)谱研究动态相互作用 AM2与水、药物和另一种病毒蛋白--基质蛋白1(AM1)的相互作用。具体地说，我将发展 将动态敏感的~2H四极耦合与~(13)C化学位移关联起来的SS核磁共振技术 提供有关小分子动力学和蛋白质动力学的现场解析信息。在目标1中，我将测量 抗病毒药物对通道水化和AM2孔内水动力学的影响。1H-13C和2H- 将进行~(13)C关联实验，以探测质子化和氢化通道的动力学 水分别提供了补充信息。结果将提供详细的分子 了解新药的作用机制，为今后设计更好的抗病毒药物提供帮助 化合物。在目标2中，我将测量药物对AM2的构象动力学的影响，以解决 跨膜(TM)结构域动力学如何影响抗病毒药物活性以及蛋白质动力学是否 与河道水动力有关。这些研究将揭示最近未知的结合部位。 研发了抗病毒药物。本研究将使用2H，13C标记的蛋白质。其次，我将描述AM2的特征 与AM1的交互。生化证据表明，这种相互作用对病毒组装和 因此，阐明这种相互作用的结构基础可能有助于设计替代的抗病毒药物。 我将测量AM2细胞质结构域与AM1结合时的动力学和化学位移的变化，以 了解结合是否使AM2固定并改变蛋白质结构。我已经获得了广泛的 初步数据证明了2H-13C关联技术的可行性，并已成功 表达和纯化13C、2H、15N标记蛋白。它还将提供有关重要性的第一次信息 分子运动对重要生物医学靶标功能的影响。

Abstract Seasonal influenza viruses infect 5-10% of adults and 20-30% of children each year, yet only two classes of antiviral drugs are available so far, one targeting the influenza A M2 (AM2) protein and the other targeting neuraminidase (NA). AM2 forms a proton channel in the virus envelope that is crucial for the virus lifecycle; blockage of the AM2 pore curbs virus infection. Unfortunately, in recent years 99% of flu strains have become resistant to the FDA-approved AM2 channel blockers, amantadine and rimantadine. Recently, new antiviral drugs effective against amantadine-resistant AM2 proteins have been reported, but their mechanisms of action have remained elusive. Water plays an important role in proton conduction through the AM2 pore, but it is not understood how the new AM2 inhibitors affect water dynamics in the channel. Elucidating channel water dynamics should provide insights into the mechanism of action of anti-influenza drugs. I propose to develop and employ solid-state nuclear magnetic resonance (SSNMR) spectroscopy to investigate the dynamic interactions of AM2 with water, with drugs, and with another virus protein, matrix protein 1 (AM1). Specifically, I will develop SSNMR techniques that correlate the dynamically sensitive 2H quadrupolar coupling with 13C chemical shifts to provide site-resolved information about small-molecule dynamics and protein dynamics. In Aim 1, I will measure the effects of antiviral drugs on channel hydration and on water dynamics in the AM2 pore. Both 1H-13C and 2H- 13C correlation experiments will be conducted to detect the dynamics of protonated and deuterated channel water, respectively, giving complementary information. The results will provide a detailed molecular understanding of the mechanism of action of the novel drugs and facilitate future design of better antiviral compounds. In Aim 2, I will measure the conformational dynamics of AM2 as influenced by drugs, to address how the transmembrane (TM) domain dynamics affect antiviral drug activity and whether the protein dynamics is related to channel-water dynamics. These studies will shed light on the yet unkown binding site of recently developed antiviral drugs. 2H, 13C-labeled proteins will be used in this study. Secondly, I will characterize AM2 interaction with AM1. Biochemical evidence suggests that this interaction is important for virus assembly and budding, thus elucidating the structural basis for this interaction may help the design of alternative antiviral drugs. I will measure changes in the AM2 cytoplasmic domain’s dynamics and chemical shifts upon binding to AM1, to understand whether binding immobilizes AM2 and changes protein structure. I have obtained extensive preliminary data that demonstrate the feasibility of the 2H-13C correlation techniques and have successfully expressed and purified 13C,2H,15N-labeled proteins. It will also provide first-time information about the importance of molecular motion on the function of an important biomedical target.