Hydrophobic Gate of Mechanosensitive Channel of Large Conductance in Lipid Bilayers Revealed by Solid-State NMR Spectroscopy

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 Methanosarcina acetivorans (Ma-MscL) in lipid bilayers. We obtain chemical shift assignments of similar to 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.

Automated summary

In this study, the hydrophobic gate of MscL was investigated using solid-state nuclear magnetic resonance (ssNMR) techniques and functional assays. It was found that residues I21-T30 in transmembrane helix 1 constitute the hydrophobic gate, and the packing of aromatic rings of F23 in each subunit is critical to gate function. Hydrophilic substitutions of residues A22 and G26 modulate channel gating by affecting the hydrophobicity of F23 constriction.