Sawada Yasuyuki, Nomura Takeshi, Martinac Boris, Sokabe Masahiro
Department of Physiology, Nagoya University Graduate School of Medicine, Nagoya, Japan.
Institute of Materials Innovation, Institutes of Innovation for Future Society, Nagoya University, Nagoya, Japan.
Front Chem. 2023 Jun 6;11:1175443. doi: 10.3389/fchem.2023.1175443. eCollection 2023.
The bacterial mechanosensitive channel of large conductance MscL is activated exclusively by increased tension in the membrane bilayer. Despite many proposed models for MscL opening, its precise mechano-gating mechanism, particularly how the received force at the tension sensor transmits to the gate remains incomplete. Previous studies have shown that along with amphipathic -terminus located near the cytoplasmic surface of the membrane, Phe78 residue near the outer surface also acts as a "tension sensor," while Gly22 is a central constituent of the "hydrophobic gate." Present study focused on elucidating the force transmission mechanism from the sensor Phe78 in the outer transmembrane helix (TM2) to the gate in the inner transmembrane helix (TM1) of MscL by applying the patch clamp and molecular dynamics (MD) simulations to the wild type MscL channel and its single mutants at the sensor (F78N), the gate (G22N) and their combination (G22N/F78N) double mutant. F78N MscL resulted in a severe loss-of-function, while G22N MscL caused a gain-of-function channel exhibiting spontaneous openings at the resting membrane tension. We initially speculated that the spontaneous opening in G22N mutant might occur without tension acting on Phe78 residue. To test this hypothesis, we examined the (G22N/F78N) double mutant, which unexpectedly exhibited neither spontaneous activity nor activity by a relatively high membrane tension. To understand the underlying mechanism, we conducted MD simulations and analyzed the force transduction pathway. Results showed that the mutation at the tension sensor (F78N) in TM2 caused decreased interaction of this residue not only with lipids, but also with a group of amino acids (Ile32-Leu36-Ile40) in the neighboring TM1 helix, which resulted in an inefficient force transmission to the gate-constituting amino acids on TM1. This change also induced a slight tilting of TM1 towards the membrane plane and decreased the size of the channel pore at the gate, which seems to be the major mechanism for the inhibition of spontaneous opening of the double mutant channel. More importantly, the newly identified interaction between the TM2 (Phe78) and adjacent TM1 (Ile32-Leu36-Ile40) helices seems to be an essential force transmitting mechanism for the stretch-dependent activation of MscL given that substitution of any one of these four amino acids with Asn resulted in severe loss-of-function MscL as reported in our previous work.
大电导机械敏感通道MscL仅通过膜双层张力增加而被激活。尽管针对MscL开放提出了许多模型，但其精确的机械门控机制，特别是张力传感器接收到的力如何传递到门控部位仍不完整。先前的研究表明，除了位于膜细胞质表面附近的两亲性C末端外，膜外表面附近的苯丙氨酸78残基也充当“张力传感器”，而甘氨酸22是“疏水门”的核心组成部分。本研究通过对野生型MscL通道及其在传感器位点（F78N）、门控位点（G22N）的单突变体以及它们的组合（G22N/F78N）双突变体应用膜片钳和分子动力学（MD）模拟，着重阐明从外跨膜螺旋（TM2）中的传感器苯丙氨酸78到MscL内跨膜螺旋（TM1）中的门控部位的力传递机制。F78N MscL导致严重的功能丧失，而G22N MscL导致功能增强型通道，在静息膜张力下呈现自发开放。我们最初推测G22N突变体中的自发开放可能在没有张力作用于苯丙氨酸78残基的情况下发生。为了验证这一假设，我们研究了（G22N/F78N）双突变体，其出乎意料地既没有自发活性，也没有在相对较高的膜张力下表现出活性。为了理解其潜在机制，我们进行了MD模拟并分析了力转导途径。结果表明，TM2中张力传感器（F78N）的突变不仅导致该残基与脂质的相互作用减少，而且与相邻TM1螺旋中的一组氨基酸（异亮氨酸32 - 亮氨酸36 - 异亮氨酸40）的相互作用减少，这导致向TM1上构成门控的氨基酸的力传递效率低下。这种变化还诱导TM1向膜平面轻微倾斜，并减小了门控处通道孔的大小，这似乎是双突变体通道自发开放受到抑制的主要机制。更重要的是，新发现的TM2（苯丙氨酸78）与相邻TM1（异亮氨酸32 - 亮氨酸36 - 异亮氨酸40）螺旋之间的相互作用似乎是MscL拉伸依赖性激活的一种基本力传递机制，因为如我们先前工作中所报道的，将这四个氨基酸中的任何一个替换为天冬酰胺都会导致严重功能丧失的MscL。
