Zhang Xuning, Zhang Yan, Tang Siyang, Ma Shaojie, Shen Yang, Chen Yanke, Tong Qiong, Li Yuezhou, Yang Jun
Key Laboratory of Molecular Biophysics of Ministry of Education, College of Life Science and Technology and the Collaborative Innovation Center for Brain Science, Huazhong University of Science and Technology, Wuhan 430074, China.
National Center for Magnetic Resonance in Wuhan, Key Laboratory of Magnetic Resonance in Biological Systems, State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Wuhan Institute of Physics and Mathematics, Innovation Academy for Precision Measurement Science and Technology, Chinese Academy of Sciences, Wuhan 430071, P. R. China.
J Phys Chem B. 2021 Mar 18;125(10):2477-2490. doi: 10.1021/acs.jpcb.0c07487. Epub 2021 Mar 8.
The bacterial mechanosensitive channel of large conductance (MscL) functions as a pressure-relief safety valve to prevent cells from lysing during sudden hypo-osmotic shock. The hydrophobic gate of MscL in the closed state forms a barrier to the permeation of ions and water molecules and can be switched to the open state for releasing solutions and ions. Currently, the gate-constituting residues and the functional role of these residues in the hydrophobic gate of MscL remain elusive and controversial. Here, we employ magic angle spinning solid-state nuclear magnetic resonance (ssNMR) techniques and functional assays to investigate the hydrophobic gate of MscL from (Ma-MscL) in lipid bilayers. We obtain chemical shift assignments of ∼70% residues of Ma-MscL and predict its 3D structure. Based on the structural characterization, we identify that the residues I21-T30 in the transmembrane helix 1 constitute the hydrophobic gate by detecting water distributions in the transmembrane pore using ssNMR H/D exchange and water-edited experiments. By using ssNMR structural characterization and functional assays, we reveal that the packing of aromatic rings of F23 in each subunit of Ma-MscL is critical to the hydrophobic gate, and hydrophilic substitutions of the other functionally important residues A22 and G26 modulate channel gating by attenuating hydrophobicity of constriction of F23.
大电导细菌机械敏感通道（MscL）作为一种压力释放安全阀，可防止细胞在突然的低渗休克中裂解。处于关闭状态的MscL的疏水门对离子和水分子的渗透形成屏障，并可切换到开放状态以释放溶液和离子。目前，构成MscL疏水门的残基及其在疏水门中的功能作用仍然难以捉摸且存在争议。在这里，我们采用魔角旋转固态核磁共振（ssNMR）技术和功能测定法来研究脂质双层中来自[具体来源未给出]（Ma-MscL）的MscL的疏水门。我们获得了Ma-MscL约70%残基的化学位移归属，并预测了其三维结构。基于结构表征，我们通过使用ssNMR H/D交换和水编辑实验检测跨膜孔中的水分布，确定跨膜螺旋1中的I21-T30残基构成疏水门。通过使用ssNMR结构表征和功能测定法，我们揭示Ma-MscL每个亚基中F23的芳香环堆积对疏水门至关重要，其他功能重要残基A22和G26的亲水性取代通过减弱F23收缩处的疏水性来调节通道门控。
