Structural basis of the gating mechanism of the large-conductance mechanosensitive channel from Escherichia coli

The mechanosensitive channel of large conductance (MscL) is a tension-gated, pore-forming protein that acts as a safety valve to protect bacteria from osmotic lysis. Escherichia coli MscL (EcMscL) was the first mechanosensitive channel discovered and subsequently served as a model system for understanding mechanical sensing, becoming one of the most decorated and well-studied systems. Despite extensive biophysical and functional characterisation spanning several decades, the precise mechanism of EcMscL gating has been poorly understood due to the lack of high-resolution structural information. Herein, we solved two EcMscL structures by cryoEM in the closed conformation in DMPC and DOPC lipid nanodiscs. Using PELDOR spectroscopy, we screened conditions and identified that in DSPC lipids, EcMscL open-like states are present within its conformational ensemble. We solved the structure in an expanded state by cryoEM, revealing an architecture with pore properties consistent with previous electrophysiology reports. By combining hydrogen-deuterium exchange mass spectrometry and molecular dynamics simulations, we investigated the dynamics of EcMscL gating in lipid bilayers, identifying sites involved in the closed-open transition. Combined, this has enabled us to inform on the elusive structural mechanism of EcMscL mechanosensitive channel function.