Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, United States.
J Am Chem Soc. 2010 Dec 22;132(50):17695-7. doi: 10.1021/ja108458u. Epub 2010 Nov 23.
The M2 protein of the flu virus forms a proton selective channel that is necessary for viral replication. The channel has a slow rate of conduction but attains near perfect selectivity for protons. Many models have been proposed to explain the mechanism of proton conduction based on whole cell channel recordings and molecular dynamics simulations, but a detailed kinetic analysis of the channel activity has not yet been performed. We obtained detailed conduction vs pH measurements for M2 and a number of its variants using a sensitive and reproducible liposome proton flux assay. The proton transport follows Michaelis-Menten-like kinetics with two saturation steps: one pseudosaturation at pH ∼5.5, and another full saturation at pH ∼4. The heart of the mechanism is the pore-lining His37 and Trp41. NMR measurements suggest that histidine and tryptophan act in unison to transport protons down the concentration gradient. The log of apparent K(m) derived from the kinetics data matches closely to the histidine pK(a) and correlates with chemical shift perturbation of the Trp41 gate, indicating that histidine protonation and opening of the channel gate are synchronized events. Finally, mutagenesis and structural analysis identified key residues that affect the rate of conduction.
流感病毒的 M2 蛋白形成质子选择性通道，这对于病毒复制是必要的。该通道的传导速度较慢，但对质子具有近乎完美的选择性。许多模型已被提出，以解释基于全细胞通道记录和分子动力学模拟的质子传导机制，但对通道活性的详细动力学分析尚未进行。我们使用灵敏且可重复的脂质体质子通量测定法，获得了 M2 及其多种变体的详细传导与 pH 值关系的测量结果。质子传输遵循米氏动力学，具有两个饱和步骤：一个在 pH 值约为 5.5 时的伪饱和，另一个在 pH 值约为 4 时的完全饱和。该机制的核心是位于孔道内的 His37 和 Trp41。NMR 测量表明，组氨酸和色氨酸协同作用，沿着浓度梯度运输质子。从动力学数据得出的表观 Km 的对数与组氨酸 pKa 非常吻合，并与 Trp41 门控的化学位移扰动相关，表明组氨酸质子化和通道门控的开启是同步事件。最后，突变和结构分析确定了影响传导速度的关键残基。
