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资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。在现代生物体中，跨细胞壁传输电荷的蛋白质结构复杂，这使得剖析其作用的分子机制变得极其困难。因此，希望有一种蛋白质模型，它是小的，并具有众所周知的结构基序，但操作的效率和控制更复杂的蛋白质。这导致了对甲型流感M2蛋白的研究-一种小型的同源四聚体电压门控离子通道，其在脂质双层中自组装并以高效率和选择性运输质子。每个单体由97个氨基酸组成，含有一个跨膜结构域。此外，活性通道已经从仅含有25个氨基酸的子集（包括跨膜区）的合成肽重建，而没有特异性或效率的损失。该肽的氨基酸序列为Ser-Ser-Asp-Pro-Leu- Val-Val-Ala-Ala-Ser-Ile-Ile-Gly-Ile-Leu-His-Leu-Ile-Leu-Trp Ile-Leu-Asp-Arg-Leu。与grimicidin A相比，也许是最广泛研究的质子通道模型，截短的M2通道的质子传输速率快1000倍以上。这种简单性和效率的显著结合使得M2不仅是理解简单肽如何实现高效率质子传输的极好模型，而且是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结构是已知的，但四聚体束的结构尚未确定。与短杆菌肽中质子转运的实验和理论研究一致，已经提出质子转运通过沿着跨越通道孔的水分子的瞬时链易位而发生。通道由四个组氨酸残基门控，这些组氨酸残基封闭管腔。这种门控机制可以解释为什么M2对碱金属离子是不可渗透的。然而，了解质子传导通过通道的完整过程需要更多的研究。半胱氨酸扫描诱变表明，孔衬残基的替换导致通道性质的大扰动，表明这些残基对通道效率是必不可少的。其他残基的身份起较小的作用。孔衬残基如何影响质子运输尚不清楚，因为这些残基都不是高极性的，也不能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下，它似乎是封闭的。低于pH 5.5（也是组氨酸的pKa）时，通道打开，观察到质子转运。因此，有人认为，四个中性组氨酸残基从门，并打开通道涉及质子化一个（或多个组氨酸残基）。Cross及其同事最近的NMR工作表明色氨酸残基接近组氨酸残基，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名称1 NYJ）。此外，他们新的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，当三个（或四个）组氨酸在较低pH下被质子化时，门打开。