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

Abstract

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 ∼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.

Water Nanoconfined in a Hydrophobic Pore: Molecular Dynamics Simulations of Transmembrane Protein 175 and the Influence of Water Models

“…e ., channel) systems. However, recent experimental studies have attempted to estimate water profiles within the hydrophobic gate of an ion channel (but not yet for TMEM175). In the meantime, we can state that the five additive water models explored appear to be equally transferrable when moving from bulk water to water confined in a nanopore.…”

“…In the absence of direct experimental data, it is difficult to assess whether the water models used are transferrable from bulk to nanoconfined (i.e., channel) systems. However, recent experimental studies have attempted to estimate water profiles within the hydrophobic gate of an ion channel 58 (but not yet for TMEM175).…”

“…Combining recent computational (this study) and experimental ( e.g ., ref ) approaches would, therefore, allow us to exploit biological nanopores/channels to characterize the effects of confinement on water behavior on smaller scales than those currently addressed (down to ∼1.5 nm) by studies of nanofabricated devices . In the limiting case, this may enable us to use biological systems to examine the behavior of water under extreme nanoconfinement with just one or two water molecules present.…”

Water Nanoconfined in a Hydrophobic Pore: MD Simulations and Water Models

“…Combining recent computational (this study) and experimental (e.g. 51 ) approaches would therefore allow us to exploit biological nanopores/channels to characterise the effects of confinement on water behaviour on smaller scales than those currently addressed (down to ~1.5 nm) by studies of nanofabricated devices 39 . In the limiting case, this may enable us to use biological systems to examine the behaviour of water under extreme nanoconfinement with just one or two water molecules present.…”

“…channel) systems. However, recent experimental studies have attempted to estimate water profiles within the hydrophobic gate of an ion channel 51 (but not yet for TMEM175). In the meantime, we can state that the five additive water models explored appear to be equally transferrable when moving from bulk water to water confined in a nanopore.…”