Polymer-solubilized structure of the mechanosensitive channel MscS suggests the role of protein-lipid interactions in the functional gating cycle

Membrane protein structure determination is not only technically challenging but is further complicated by the removal or displacement of lipids, which can result in non-native conformations or a strong preference for certain states at the exclusion of others. This is especially applicable to mechanosensitive channels (MSCs) that evolved to gate in response to subtle changes in membrane tension, such as MscS, a model bacterial system for MSC gating with homologs found across all phyla of walled organisms. MscS is highly adaptive, it readily opens under super-threshold tension but under lower tensions it inactivates and can only recover when tension is released. Functional data strongly suggests a restructuring of the protein-lipid boundary during the slow inactivation and recovery processes. Existing cryo-EM structures fall into two categories depending on the method of solubilization: (1) non-conductive (lipid reconstituted or mixed micelles) characterized by kinked pore-lining helices and splayed lipidfacing helices, or (2) semi-open (pure detergent or short-chain lipids). These structures do not explain the full functional gating cycle consisting of three well defined states: open, closed, and inactivated. Here, we present a 3 Angstrom MscS structure in native nanodiscs generated with Glyco DIBMA polymer solubilization which eliminates the lipid removal step common to all previous structures. Besides the protein in the splayed conformation, we observe well resolved densities that represent phospholipids intercalating between the lipid-facing and pore-lining helices in preferred orientations. The structure illustrates the lipid-based mechanism for the uncoupling of the tension sensing helical pairs from the gate and prompts critical questions on whether the two distinct tension driven opening-closing and inactivation-recovery pathways are separated by the kinetic principle and what types of forces drive the recovery back to a more compact closed state.