In-Lipid Structure of Pressure-Sensitive Domains Hints Mechanosensitive Channel Functional Diversity

Abstract

The mechanosensitive channel of large conductance (MscL) from Mycobacterium tuberculosis has been used as a structural model for rationalizing functional observations in multiple MscL orthologs. Although these orthologs adopt similar structural architectures, they reportedly present significant functional differences. Subtle structural discrepancies on mechanosensitive channel nanopockets are known to affect mechanical gating and may be linked to large variability in tension sensitivity among these membrane channels. Here, we modify the nanopocket regions of MscL from Escherichia coli and M. tuberculosis and employ PELDOR/DEER distance and 3pESEEM deuterium accessibility measurements to interrogate channel structure within lipids, in which both channels adopt a closed conformation. Significant in-lipid structural differences between the two constructs suggest a more compact E. coli MscL at the membrane inner-leaflet, as a consequence of a rotated TM2 helix. Observed differences within lipids could explain E. coli MscL’s higher tension sensitivity and should be taken into account in extrapolated models used for MscL gating rationalization.

Tension mediated mechanical activation and pocket delipidation lead to an analogous MscL state

“…Disruption of lipid access into the TbMscL's pockets promoted a mechanical response in the absence of applied tension (2), while subtle structural differences between these pockets in MscL orthologues dramatically altered their function (15). This pocket region in TbMscL, which is important for lipid binding (2,15), also binds, specific to EcMscL molecules, which could modulate its activity (12,13). TbMscL L89 is located at the entrance of the pockets and is structurally aligned to EcMscL M94 and in close proximity to I92, F93, I96 and K97, modifications of which, influence MscL's activation by ultrasound pulses (65), decrease its tension threshold for use in a multicompartmental lipid vesicle framework (66), and modulate its function (12) .…”

“…The protocols followed in this study were similar to the ones previously described (2,15,54). For details and also HDX-MS sample preparation see the Appendix.…”

“…The former involves the exchange of protons on a protein with deuterium to generate accessibility information, and has been used to provide key insights into the role(s) of lipids in modulating membrane protein conformation(46–49). The latter measures weak hyperfine coupling between unpaired electrons and nuclear spins to probe accessibility to solvent (D 2 O) and has been used to investigate the dynamics of membrane proteins(8, 19, 34, 35, 50–54). Here we endeavored to investigate whether there is a structural analogy between the physiologically relevant tension-activated state, and the one stabilized by modifications that result in pocket lipid removal.…”

“…Indeed, the function of MscL (11)(12)(13) and MscS (5,14) can be modulated by molecule occupancy changes within these pockets. Subtle structural differences within the same regions lead to substantial functional differences in orthologous MscL proteins (15). This suggests a common regulatory mechanism, tailored to the unique structural landscape of MS channels, which may guide ligand specificity (2,3).…”

“…The former involves the exchange of protons on a protein with deuterium generates accessibility information and has been used to provide key insights into the role of lipids in determining the conformational state, but also secondary structure changes in membrane transporters (40)(41)(42)(43)(44). The latter measures weak hyperfine coupling between unpaired electrons and nuclear spins to probe accessibility to solvent (D2O) and protons (residues, ions and lipids) and has been used to investigate the dynamics of membrane proteins (2,15,(45)(46)(47)(48)(49)(50)(51).…”