Highlights d Pocket delipidation of TM pockets stabilizes an expanded MscL state d HDX and EPR probe conformational transitions of MscL d Bilayer stretching MD independently generates a tensionactivated state d Structural analogy between these states has implications in MS channel regulation Authors
MscL was the first mechanosensitive ion channel identified in bacteria. The channel opens its large pore when the turgor pressure of the cytoplasm increases close to the lytic limit of the cellular membrane. Despite their ubiquity across organisms, their importance in biological processes, and the likelihood that they are one of the oldest mechanisms of sensory activation in cells, the exact molecular mechanism by which these channels sense changes in lateral tension is not fully understood. Modulation of the channel has been key to understanding important aspects of the structure and function of MscL, but a lack of molecular triggers of these channels hindered early developments in the field. Initial attempts to activate mechanosensitive channels and stabilize functionally relevant expanded or open states relied on mutations and associated post-translational modifications that were often cysteine reactive. These sulfhydryl reagents positioned at key residues have allowed the engineering of MscL channels for biotechnological purposes. Other studies have modulated MscL by altering membrane properties, such as lipid composition and physical properties. More recently, a variety of structurally distinct agonists have been shown bind to MscL directly, close to a transmembrane pocket that has been shown to have an important role in channel mechanical gating. These agonists have the potential to be developed further into antimicrobial therapies that target MscL, by considering the structural landscape and properties of these pockets.
The mechanosensitive channel of large conductance MscL gates in response to tension changes in the membrane to allow the exchange of molecules through its pore. Native ligands that bind and modulate MscL are unknown and trapping an activated state has been challenging. Disruption of lipid access to tension-sensitive transmembrane pockets by modification leads to a concerted structural and functional MscL response. However, it is unknown whether there is structural correlation between tension mediated and molecular activation in mechanosensitive channels. Here, we combine HDX mass spectrometry and ESEEM solvent accessibility measurements on MscL, coupled with molecular dynamics under bilayer tension, to investigate the structural changes associated with the two distinctively derived states. Membrane thinning is not sufficient to hydrate the MscL pore, when lipids are trapped in the pockets, under tensions capable to gate the native channel. Tension and molecule stabilised states present analogous MscL structures, suggesting a link between these two distinct activation mechanisms. These findings could hint at synergistic modes of regulation in mechanosensitive ion channels with implications for their multimodality.
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