The role of lipids in mechanosensation

Abstract

The ability of proteins to sense membrane tension is pervasive in biology. A higher-resolution structure of the Escherichia coli small-conductance mechanosensitive channel MscS identifies alkyl chains inside pockets formed by the transmembrane helices (TMs). Purified MscS contains E. coli lipids, and fluorescence quenching demonstrates that phospholipid acyl chains exchange between bilayer and TM pockets. Molecular dynamics and biophysical analyses show that the volume of the pockets and thus the number of lipid acyl chains within them decreases upon channel opening. Phospholipids with one acyl chain per head group (lysolipids) displace normal phospholipids (with two acyl chains) from MscS pockets and trigger channel opening. We propose that the extent of acyl-chain interdigitation in these pockets determines the conformation of MscS. When interdigitation is perturbed by increased membrane tension or by lysolipids, the closed state becomes unstable, and the channel gates.

Figure 1. Pockets that are formed between the TM helices that change upon gating

A: The 3.0 Å structure of the D67R1 MscS heptamer shown in cartoon representation on left with a space fill diagram showing the alkyl chains shown on right. B: The voids between the TM helices undergo large changes upon gating. The pockets (highlighted by black box) in the closed (left) structure are larger than when compared to the open structure (right). The higher resolution of the new structure has allowed to visualize for the first time bound molecules in the pockets (see also Supplementary Fig. 1C). C: Surface view of the closed (2OAU) and open (D67R1) MscS structures. The open and closed diameters of the pore, which arises from the displacement of the TM3a helices are shown by disks placed along the channel axis.

Figure 2. Lipids pack in the pockets created by the TM helices

A: Native mass spectroscopy of wild type MscS, the expected theoretical weight of the heptamer is 223,727 Da. If this value is subtracted from the resolved first peak value of 224,344 Da (brown dot), then it results in around 620Da, consistent with a small lipid (brown dot in Fig 2A). Subsequent differences between peaks reveal additional lipid adducts. B: ES-MS of phospholipid extracted from a sample of DDM-solubilized MscS. C: Thin layer chromatogram of extracted lipids from DDM-solubilized MscS. Lipid separation was carried out in the solvent system chloroform:methanol:1M KCl (10:10:3, v/v/v ). Spots on the TLC plate were visualized by staining first with a) 0.1% ninhydrin and b) 0.05% primuline, both in acetone:water (80:20, v/v). The samples loaded are Lane [1], MscS-DDM (238 μg)*, [2] MscS-DDM (280 μg), [3] MscS-Fos-14 (305 μg)*, [4] POPG (5 μg), [5] POPE (2.4 μg), [6] E. coli lipids (8 μg), [7] DDM (5 μg), [8] Fos-14 (10 μg). The result is typical of three separate experiments. Lanes 1 & 3 were freshly purified, lane 2 was several months old. *mass refers to quantity of MscS protein loaded; others refer to mass of lipids. Note that PG, known to be present from mass spectrometry in preparations 1 & 2, is not visible due to the overlap with the DDM, which is much more abundant in the detergent-solubilized material.

Figure 3. Lipids exchange between the pockets and the bilayer

A cut away slice showing snapshots (at 100 ns) of atomistic simulations of the closed (A) and open (B) conformation of MscS in POPE:POPG (4:1) phospholipid bilayers. Movies of the lipid bilayer (with protein removed) are available as Supplementary files (movie 1 and movie 2). C: Comparison of the number of lipids that remain within 6 Å of the TM (i.e. residues 27 to 128, light bars) and the lower pocket (TM3b region, residues 106 to 122 (darker shaded bars) of closed (blue bars) and open state (magenta bars) MscS throughout the latter 0.5 μs of the CG-MD simulations. Error bars indicate one standard deviation of number of lipid contacts. A larger number of lipids are in rapid exchange between the pockets and bilayer (Supplementary Fig. 3E). D: Single-tryptophan mutants of MscS probed with brominated phospholipids and the degree of quenching is shown by color shading. E: Typical raw data of quenching experiments (selected mutants). Emission spectra are shown from MscS in 100% DOPC (black) or 100% BrPC (red). F: Quantitative results of BrPC quenching in form of the fractional quenching by brominated lipid for TM3b mutants in DOPC (black bars; s.d. shown as error bar from n= 17 reconstitutions) and 80% DOPE:20% DOPG (white bars; s.d. shown as error bar from n = 4 reconstitutions)).

Figure 4. MscS conformational state can be altered by perturbing the interactions between the phospholipid and the protein

A: A119W (left) and M47W (right) were reconstituted into DOPC (top row) or BrPC (bottom row) shown in black. Brominated LPC (top) or non-brominated LPC (bottom) was added (green) causing quenching or dequenching, respectively. B: ES-MS of phospholipid extracted from DDM-solubilised MscS after treatment with LPC 14:0. Survey scan in positive ion mode (465-500 m/z) showing the 490 m/z of the LPC 14:0. Survey scan in negative ion mode (600-1000 m/z) of MscS after treatment with LPC 14:0 (inset). Only LPC 14:0 [M+Na⁺] 490.3 was observed. C: Typical current recordings of single MscS channels in planar lipid bilayers. The applied potential was at +20 mV. Right panels: corresponding all-points amplitude histograms. In this experiment 3 μM LPC 18:1, was added to the cis compartment. D: As 4C but with 3 μM LPC 18:1 added to the trans compartment. E: As 4C but with 10 μM LPC 14:0 added to the cis compartment. F: As 4C but with 10 μM LPC 14:0, added to the trans compartment. Inset: recording at expanded time scale.

Figure 5. A model for mechanosensation

A: MscS is depicted as a simplified a line diagram, PE and PG molecules are shown with black headgroups, those inside the pockets are highlighted (for easy visualization) with a green headgroup. The phospholipids partition in the pockets and the lipid bilayer. As pressure is applied, the lateral tension increases and as a result the phospholipids repartition (blue arrows) from the protein pockets to the bilayer destabilizing the closed structure. The protein responds by undergoing a conformational change (orange arrow) to the open form. B: LPC (shown as a single chain with a yellow headgroup) enters first the bilayer then the pockets from the cytoplasmic side and as result the lipid content (acyl chains) falls inside the protein pockets destabilizing the closed structure. The protein undergoes a conformational change to a sub conducting state. C: Single channel bilayer recordings show that MscS D67C exhibits similar conductivity as WT protein when opened by addition of LPC 14:0 to the cis side (Fig 4E).