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. 2022 Apr 7;30(4):608-622.e5.
doi: 10.1016/j.str.2021.12.004. Epub 2022 Jan 4.

Pocket delipidation induced by membrane tension or modification leads to a structurally analogous mechanosensitive channel state

Bolin Wang  1 , Benjamin J Lane  1 , Charalampos Kapsalis  2 , James R Ault  3 , Frank Sobott  3 , Hassane El Mkami  4 , Antonio N Calabrese  3 , Antreas C Kalli  5 , Christos Pliotas  6
Affiliations

Pocket delipidation induced by membrane tension or modification leads to a structurally analogous mechanosensitive channel state

Bolin Wang et al. Structure. .

Abstract

The mechanosensitive ion channel of large conductance MscL gates in response to membrane tension changes. Lipid removal from transmembrane pockets leads to a concerted structural and functional MscL response, but it remains unknown whether there is a correlation between the tension-mediated state and the state derived by pocket delipidation in the absence of tension. Here, we combined pulsed electron paramagnetic resonance spectroscopy and hydrogen-deuterium exchange mass spectrometry, coupled with molecular dynamics simulations under membrane tension, to investigate the structural changes associated with the distinctively derived states. Whether it is tension- or modification-mediated pocket delipidation, we find that MscL samples a similar expanded subconducting state. This is the final step of the delipidation pathway, but only an intermediate stop on the tension-mediated path, with additional tension triggering further channel opening. Our findings hint at synergistic modes of regulation by lipid molecules in membrane tension-activated mechanosensitive channels.

Keywords: EPR spectroscopy; ESSEM; HDX; MD; MscL; MscS; force-from-lipid; lipids; mass spectrometry; mechanosensitive channels.

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Conflict of interest statement

Declaration of interests The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Global changes on MscL induced by pocket delipidation observed by HDX-MS (A) Differences in the deuterium uptake of regions of TbMscL (PDB: 2OAR) when comparing the WT and the L89W (pocket lipid removal) modified protein. Regions highlighted in red are deprotected following the L89W modification. Regions of the protein in gray show no significant difference between the 2 conditions. L89W modification site is depicted as a cyan sphere. (B) Wood’s plots showing the summed differences in deuterium uptake in MscL over all 5 HDX time points, comparing WT with L89W MscL (Wood’s plots were generated using Deuteros (Lau et al., 2019). Peptides colored in red are deprotected from exchange in L89W MscL. No peptides were significantly protected from exchange in L89W MscL compared with wild-type MscL. Peptides with no significant difference between conditions, determined using a 99% confidence interval (dotted line), are shown in gray. Example deuterium uptake curves and a map of the peptides detected are shown in Figure S1. Note that only 5 residues from residues 1–120 were not covered by peptides.
Figure 2
Figure 2
Single-residue mapping of MscL by 3pESEEM (A) Solvent accessibility of spin-labeled residues are shown as red and pale yellow spheres for the most and the least solvent accessible, respectively. Spin-labeled residues are colored based on the quartile of their relative accessibility, which has been normalized on a scale of 0% and 100%, where the residue with the highest accessibility corresponds to 100%. (B) Background-corrected time-domain 3pESEEM experimental spectra with fitting for representative spin-labeled mutants. Residue F5R1 is found on the S1 amphipathic helix, I23R1 and L42R1 on TM1, and K100R1 and E102R1 are at the interface between TM2 and the CHB.
Figure 3
Figure 3
Effect of pocket delipidation on MscL structure investigated by 3pESEEM and HDX-MS (A) Background-corrected time-domain 3pESEEM experimental spectra (orange) of the single mutants N13R1, V21R1, L42R1, N70R1, L72R1, L73R1, and K100R1 in DDM overlaid with their associated L89W double-mutant spectra (purple). (B) Differences in solvent accessibility for TbMscL (PDB: 2OAR) following L89W modification. Spin-labeled mutation sites used for 3pESEEM accessibility measurements are represented by spheres, and peptides that demonstrate a change in accessibility in HDX-MS following the L89W modification are represented as highlighted helices. Red regions or spheres highlight areas that are deprotected, while blue spheres and regions show areas that are protected following the L89W modification. There was no significant difference in the solvent accessibility of N13R1, L42R1, and N70R1 compared to their 89W double-mutant counterpart. Solvent accessibility increased for V21R1 and L72R1 and decreased for L71R1 and K100R1 following the L89W modification. The associated column bar representation including errors are described in Figure S6.
Figure 4
Figure 4
Tension-mediated expanded MscL state (A) TM comparison between closed TbMscL (tan, PDB: 2OAR) and open state WT MscLs generated by MD simulations (cyan, under tension)—side, top, and single subunit views. The latter shows a substantial tilting of TM1 and TM2 toward the membrane plane. These conformational changes occurring under tension are more evident in Videos S1, S2, and S3. (B) The pocket’s surface dramatically decreases upon tension application, limiting lipid access. Uniform membrane thinning of 1.2 nm (3.8–2.6 nm) also occurs during channel expansion, due to tension application.
Figure 5
Figure 5
MscL pore hydration investigation under applied bilayer tension WT under no tension (left column), WT under bilayer tension (right column), and L89W under bilayer tension (right column). (A) Solvent density profile of the MD simulations using the CHAP (Klesse et al., 2019) software, with the vapor-lock position of MscL marked with blue arrows. (B) Membrane becomes thinner when tension is applied, but only the WT TbMscL pore is hydrated (blue spheres), in contrast to L89W channel. Lipid (olive sticks) availability is larger in the closed state, and lipids have easier access within the pockets to provide force on the back of the vapor lock, keeping the MscL pore closed. This is in contrast to WT MscL under tension (center column), where the pockets become smaller, while lipid availability decreases, and the pore becomes hydrated. However, when lipids are trapped in the pockets of the L89W modified channel, the pore does not become hydrated, despite structural rearrangements occurring (right column). (C) Surface visualization of the pore pathway using the program HOLE (Smart et al., 1993). Red color indicates a pore radius smaller than 1.15 Å (water molecules cannot go through such an opening), blue represents a radius larger than 2.3 Å, and green represents a radius between 1.15 and 2.3 Å.
Figure 6
Figure 6
The effect of membrane tension to MscL protein-lipid contact interactions (A and B) F79 and L89W form a molecular bridge in the L89W under tension state and consequently trap the lipids within the pockets (right column). F79 and L89 come into greater proximity under bilayer tension in the WT channel (expanded MscL, center column) compared to the closed state. However, they do not prevent the lipids from exchanging with the bulk bilayer during tension application. The L89W mutation locks lipids in the pockets, preventing the channel from transiting to a hydrated state. (C) Relative changes in the number of lipid contacts following stimulated tension application in the membrane during MD. The blue regions show a decrease in lipid contacts, while the red regions show an increase in lipid contacts. From left to right: surface view, single subunit illustration representation, and internal MscL surface view. Internal channel regions become more exposed to lipids, while initially, membrane-exposed regions (closed state) become less exposed to lipids, suggesting a MscL TM helical rotation upon tension application and channel expansion or opening.
Figure 7
Figure 7
Comparison between tension- and modification-induced pocket delipidation MscL states and proposed model (A and B) TM1 and TM2 tilt and rotate to initiate MscL opening. Comparison between expanded MaMscL (orchid, PDB: 4Y7J) and the open state WT TbMscL generated by MD (cyan, under tension) (A) and the open WT TbMscL state generated by MD (cyan, under tension) and L89W TbMscL (green, under tension) (B). (C) Proposed gating model for pocket lipid removal and tension-activated modes. Closed (vertical TM helices, large pockets, tightly associated annular lipids, and non-hydrated pore) and expanded (tilted TM helices, smaller pockets, loosely associated annular lipids, and hydrated pore). Extra membrane tension is required to fully open MscL.
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