Department of Chemistry, Chicago Center for Theoretical Chemistry, Institute for Biophysical Dynamics and James Franck Institute, The University of Chicago, Chicago, Illinois 60637, United States.
Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, California 94158, United States.
J Am Chem Soc. 2022 Jan 19;144(2):769-776. doi: 10.1021/jacs.1c09281. Epub 2022 Jan 5.
The influenza A M2 channel, a prototype for viroporins, is an acid-activated viroporin that conducts protons across the viral membrane, a critical step in the viral life cycle. Four central His37 residues control channel activation by binding subsequent protons from the viral exterior, which opens the Trp41 gate and allows proton flux to the interior. Asp44 is essential for maintaining the Trp41 gate in a closed state at high pH, resulting in asymmetric conduction. The prevalent D44N mutant disrupts this gate and opens the C-terminal end of the channel, resulting in increased conduction and a loss of this asymmetric conduction. Here, we use extensive Multiscale Reactive Molecular Dynamics (MS-RMD) and quantum mechanics/molecular mechanics (QM/MM) molecular dynamics simulations with an explicit, reactive excess proton to calculate the free energy of proton transport in this M2 mutant and to study the dynamic molecular-level behavior of D44N M2. We find that this mutation significantly lowers the barrier of His37 deprotonation in the activated state and shifts the barrier for entry to the Val27 tetrad. These free energy changes are reflected in structural shifts. Additionally, we show that the increased hydration around the His37 tetrad diminishes the effect of the His37 charge on the channel's water structure, facilitating proton transport and enabling activation from the viral interior. Altogether, this work provides key insight into the fundamental characteristics of PT in WT M2 and how the D44N mutation alters this PT mechanism, and it expands understanding of the role of emergent mutations in viroporins.
甲型流感 M2 通道是病毒孔道蛋白的原型，是一种酸激活的病毒孔道蛋白，能够在病毒膜上传导质子，这是病毒生命周期中的关键步骤。四个中心的 His37 残基通过结合来自病毒外部的后续质子来控制通道的激活，从而打开 Trp41 门并允许质子流向内部。Asp44 对于在高 pH 值下保持 Trp41 门处于关闭状态至关重要，从而导致不对称传导。流行的 D44N 突变破坏了这个门，并打开了通道的 C 末端，导致传导增加和这种不对称传导的丧失。在这里，我们使用广泛的多尺度反应分子动力学（MS-RMD）和量子力学/分子力学（QM/MM）分子动力学模拟，使用显式、反应性的过剩质子来计算这个 M2 突变体中质子传输的自由能，并研究 D44N M2 的动态分子水平行为。我们发现，这种突变显著降低了激活状态下 His37 去质子化的势垒，并改变了进入 Val27 四联体的势垒。这些自由能的变化反映在结构的变化上。此外，我们表明，His37 四联体周围的水化程度增加，降低了 His37 电荷对通道水结构的影响，从而促进质子传输并使激活能够从病毒内部进行。总的来说，这项工作提供了关于 WT M2 中质子转移的基本特征以及 D44N 突变如何改变这种质子转移机制的关键见解，并扩展了对新兴突变在病毒孔道蛋白中的作用的理解。
