Approaches for the modulation of mechanosensitive MscL channel pores

Abstract

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.

Keywords: MscL; agonists; antibiotics; mechanosensitive channel; membrane pores; modulators.

FIGURE 1

Key methods used to probe and investigate the modulation of MscL. Electrophysiology is a key functional methodology for understanding how modulation in the form of mutations, post-translational modification, agonists and indirect modulators alter the functional parameters of the protein. In early studies of MscL, this was often paired with cell viability and osmotic down-shock assays. X-ray crystallography allowed the visualisation of the structure of TbMscL. Pulsed EPR techniques, such as PELDOR and ESEEM, allow the structural dynamics of the protein to be followed through Å resolution distance measurements and by monitoring changes in solvent accessibility (Kapsalis et al., 2019; Wang et al., 2022). HDX-MS also informs on changes in solvent accessibility, albeit at lower resolution than ESSEEM spectroscopy (Lane et al., 2022; Wang et al., 2022). Native mass spectrometry was key to determining the effect of detergents and lipids on channel stoichiometry (Reading et al., 2015), while ion mobility mass spectrometry defined key subconducting states of MscL in response to cysteine-specific post-translation modification in the pore (Konijnenberg et al., 2020). MD simulations have been crucial in understanding modulation and mechanism of MscL (Bavi et al., 2016; Wray et al., 2016; Melo et al., 2017; Wang et al., 2022). Finally, fluorescence resonance energy transfer (FRET) was used in establishing a helix-tilt model for MscL following opening of the channel using LPC (Wang et al., 2014).

FIGURE 2

Binding sites for lipids and agonists of MscL. The TM pocket (red) of MscL is occupied by several lipid acyl chains that are proposed to determine the conformational state of the MS channel proteins, according to the lipid moves first model initially proposed for MscS and then extended to MscL (Pliotas et al., 2015; Pliotas and Naismith, 2017; Kapsalis et al., 2019). This was defined when either a bulky L89W (pink) mutation or a L89C sulfhydryl modification (MTSSL) succeeded in stabilising an expanded subconducting state of the TbMscL, consistent with the modifications at the entrance of these pockets restricting lipid acyl chain access (Kapsalis et al., 2019). This is proposed to have disrupted the link between the membrane and the channel, destabilising the closed state. Several agonists of MscL have been identified and they all bind at the interface between the S1 and TM1 region of one subunit with the TM2 of another at the membrane-cytoplasmic interface (Wray et al., 2016; Wray et al., 2020; Wray et al., 2021). These agonists all bind close to the TM pocket and so these molecules could be disrupting protein-lipid interactions that are key to determining the conformational state of the protein. Agonist studies were done on EcMscL but equivalent residues were highlighted on the structure of TbMscL (2OAR) in the absence of a structure for EcMscL.