COMPUTER SIMULATION OF THE INFLUENZA M2 CHANNEL

M2 流感通道的计算机模拟

• 批准号: 7367788
• 负责人: ANDREW POHORILLE
• 金额: $ 0.77万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7367788
• 关键词: COMPUTER; SIMULATION; INFLUENZA; M2; CHANNEL

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The structural complexity of proteins that transport charge across cell walls in contemporary organisms makes it extremely difficult to dissect the molecular mechanisms of their action. It is therefore desirable to have a protein model which is small and has a well known structural motif, yet operates with the efficiency and control of more complex proteins. This has led to the study of the Influenza A M2 protein -- a small, homotetrameric, voltage-gated ion channel which self-assembles in lipid bilayers and transports protons with high efficiency and selectivity. Each monomer is built of 97 amino acids and contains a single transmembrane domain. Additionally, active channels have been reconstituted from a synthetic peptide containing only a subset of 25 amino acids, including the transmembrane region, with no loss in specificity or efficiency. The sequence of amino acids in the peptide is Ser-Ser-Asp-Pro-Leu- Val-Val-Ala-Ala-Ser-Ile-Ile-Gly-Ile-Leu-His-Leu-Ile-Leu-Trp Ile-Leu-Asp-Arg-Leu. Compared with grimicidin A, perhaps the most extensively studied model of a proton channel, the rate of proton transport across the truncated M2 channel is over 1000-fold faster. This remarkable combination of simplicity and efficiency makes M2 not only an excellent model for understanding how simple peptides can achieve high efficiency of proton transport but also an attractive, potential target for re-engineering a simple proton pump. While a high resolution NMR structure for a single helix is known, the structure of the tetrameric bundle has not been determined. In line with experimental and theoretical studies of proton transport in gramicidin, it has been suggested that proton transport occurs via translocation along a transient chain of water molecules that span the pore of the channel. The channel is gated by four histidine residues which occlude the lumen. This mechanism of gating can explain why M2 is impermeable to alkali ions. However, understanding the complete process of proton conductance through the channel requires additional studies. Cysteine scanning mutagenesis has shown that replacement of the pore-lining residues results in a large perturbation of the properties of the channel, indicating that these residues are essential for channel efficiency. The identities of other residues play a smaller role. How the pore-lining residues influence proton transport is not known, as none of these residues is highly polar or capable of forming particularly strong hydrogen bonds with the hydronium ion. The M2 channel is pH gated. At basic and neutral pH, it appears to be closed. Below a pH of 5.5 (which is also the pKa of histidine), the channel opens and proton transport is observed. It has therefore been argued that four neutral histidine residues from the gate, and that opening the channel involves protonating one (or more of the histidine residues). Recent NMR work by Cross and co-workers has suggested that the tryptophan residues are close to the histidine residues, and might also participate in channel gating. They constructed a model structure based on these results (PDB designation 1NYJ). Additionally, their newer NMR results indicate that the actual gate might consist of two His-His+ hydrogen bonding pairs. Based on this result, they have argued that at neultral pH, two of the histidine residues are protonated, and that the gate opens when three (or four) histidines become protonated at lower pH.

该子项目是利用 NIH/NCRR 资助的中心拨款提供的资源的众多研究子项目之一。子项目和研究者 (PI) 可能已从另一个 NIH 来源获得主要资金，因此可以在其他 CRISP 条目中出现。列出的机构是中心的机构，不一定是研究者的机构。在当代生物体中跨细胞壁传输电荷的蛋白质的结构复杂性使得剖析其作用的分子机制变得极其困难。因此，需要一种小型且具有众所周知的结构基序、但能够以更复杂的蛋白质的效率和控制进行操作的蛋白质模型。这引发了对甲型流感 M2 蛋白的研究，这是一种小型同源四聚体电压门控离子通道，可在脂质双层中自组装并高效、选择性地传输质子。每个单体由 97 个氨基酸组成，并包含一个跨膜结构域。此外，活性通道是由仅包含 25 个氨基酸子集（包括跨膜区域）的合成肽重建而成，而不会损失特异性或效率。肽中的氨基酸序列为Ser-Ser-Asp-Pro-Leu-Val-Val-Ala-Ala-Ser-Ile-Ile-Gly-Ile-Leu-His-Leu-Ile-Leu-Trp Ile-亮氨酸-天冬氨酸-精氨酸-亮氨酸。与 Grimicidin A（也许是研究最广泛的质子通道模型）相比，质子穿过截短的 M2 通道的传输速率快了 1000 倍以上。这种简单性和效率的卓越结合使 M2 不仅成为了解简单肽如何实现高效质子运输的优秀模型，而且成为重新设计简单质子泵的有吸引力的潜在目标。虽然单螺旋的高分辨率 NMR 结构已知，但四聚束的结构尚未确定。根据短杆菌肽中质子传输的实验和理论研究，有人认为质子传输是通过沿着跨越通道孔隙的水分子瞬时链易位而发生的。该通道由封闭管腔的四个组氨酸残基门控。这种门控机制可以解释为什么 M2 不能渗透碱离子。然而，了解质子通过通道传导的完整过程需要额外的研究。半胱氨酸扫描诱变表明，孔衬里残基的替换会导致通道特性的巨大扰动，表明这些残基对于通道效率至关重要。其他残基的特性发挥较小的作用。孔衬里残基如何影响质子传输尚不清楚，因为这些残基都不具有高极性或能够与水合氢离子形成特别强的氢键。 M2 通道是 pH 门控的。在碱性和中性 pH 值下，它似乎是封闭的。 pH 值低于 5.5（也是组氨酸的 pKa）时，通道打开并观察到质子传输。因此，有人认为来自门的四个中性组氨酸残基，并且打开通道涉及质子化一个（或多个组氨酸残基）。 Cross 和同事最近的 NMR 工作表明，色氨酸残基与组氨酸残基接近，并且也可能参与通道门控。他们根据这些结果构建了一个模型结构（PDB 编号为 1NYJ）。此外，他们最新的 NMR 结果表明，实际的门可能由两个 His-His+ 氢键对组成。基于这一结果，他们认为在中性 pH 下，两个组氨酸残基被质子化，并且当三个（或四个）组氨酸在较低 pH 下质子化时，门打开。