Department of Chemistry, Iowa State University, Ames, Iowa 50011, USA.
J Am Chem Soc. 2012 Feb 29;134(8):3703-13. doi: 10.1021/ja2081185. Epub 2011 Oct 21.
The acid-activated proton channel formed by the influenza M2 protein is important for the life cycle of the virus. A single histidine, His37, in the M2 transmembrane domain (M2TM) is responsible for pH activation and proton selectivity of the channel. Recent studies suggested three models for how His37 mediates proton transport: a shuttle mechanism involving His37 protonation and deprotonation, a H-bonded imidazole-imidazolium dimer model, and a transporter model involving large protein conformational changes in synchrony with proton conduction. Using magic-angle-spinning (MAS) solid-state NMR spectroscopy, we examined the proton exchange and backbone conformational dynamics of M2TM in a virus-envelope-mimetic membrane. At physiological temperature and pH, (15)N NMR spectra show fast exchange of the imidazole (15)N between protonated and unprotonated states. To quantify the proton exchange rates, we measured the (15)N T(2) relaxation times and simulated them for chemical-shift exchange and fluctuating N-H dipolar fields under (1)H decoupling and MAS. The exchange rate is 4.5 × 10(5) s(-1) for Nδ1 and 1.0 × 10(5) s(-1) for Nε2, which are approximately synchronized with the recently reported imidazole reorientation. Binding of the antiviral drug amantadine suppressed both proton exchange and ring motion, thus interfering with the proton transfer mechanism. By measuring the relative concentrations of neutral and cationic His as a function of pH, we determined the four pK(a) values of the His37 tetrad in the viral membrane. Fitting the proton current curve using the charge-state populations from these pK(a)'s, we obtained the relative conductance of the five charge states, which showed that the +3 channel has the highest time-averaged unitary conductance. At physiologically relevant pH, 2D correlation spectra indicated that the neutral and cationic histidines do not have close contacts, ruling out the H-bonded dimer model. Moreover, a narrowly distributed nonideal helical structure coexists with a broadly distributed ideal helical conformation without interchange on the sub-10 ms time scale, thus excluding the transporter model in the viral membrane. These data support the shuttle mechanism of proton conduction, whose essential steps involve His-water proton exchange facilitated by imidazole ring reorientations.
流感 M2 蛋白形成的酸激活质子通道对于病毒的生命周期至关重要。M2 跨膜域（M2TM）中的单个组氨酸残基 His37 负责通道的 pH 激活和质子选择性。最近的研究提出了 His37 介导质子运输的三种模型：涉及 His37 质子化和去质子化的穿梭机制、氢键咪唑-咪唑二聚体模型和涉及与质子传导同步的大蛋白构象变化的转运体模型。使用魔角旋转（MAS）固态 NMR 光谱法，我们在病毒包膜模拟膜中研究了 M2TM 的质子交换和骨架构象动力学。在生理温度和 pH 下，（15）N NMR 谱显示咪唑（15）N 在质子化和非质子化状态之间快速交换。为了定量质子交换速率，我们测量了（15）N T2 弛豫时间，并在（1）H 去耦和 MAS 下对其进行了化学位移交换和波动 N-H 偶极场的模拟。Nδ1 的交换速率为 4.5×10(5) s(-1)，Nε2 的交换速率为 1.0×10(5) s(-1)，这与最近报道的咪唑重排大致同步。抗病毒药物金刚烷胺的结合抑制了质子交换和环运动，从而干扰了质子转移机制。通过测量中性和阳离子 His 的相对浓度作为 pH 的函数，我们确定了病毒膜中 His37 四联体的四个 pK(a) 值。使用这些 pK(a) 的电荷状态种群拟合质子电流曲线，我们得到了五个荷电状态的相对电导率，表明+3 通道具有最高的时间平均单位电导。在生理相关的 pH 下，二维相关谱表明中性和阳离子组氨酸没有紧密接触，排除了氢键二聚体模型。此外，在亚 10ms 时间尺度上，非理想的窄分布螺旋结构与广泛分布的理想螺旋构象共存，没有相互交换，从而排除了病毒膜中的转运体模型。这些数据支持质子传导的穿梭机制，其基本步骤涉及 His-水质子交换，由咪唑环重排促进。
