Figure 2. A new lipid-protein interface for membrane-embedded MscS.

(A) Close-up of MscS-ND EM density (in Chimera’s ‘solid’ representation). Left, Side view. The location of the bilayer in the nanodisc in indicated by dashed yellow likes (approximately 38 Å in diameter). Density for the putative heptameric histidine tag complex is shown by a dotted arrow. Right, Top view. The yellow circle represents the putative average size of the E3D1 nanodisc (~130 Å) in relation to the density, which points to a partial averaging of the density likely due to MscS lateral mobility. (B) Comparison between the location of the membrane interface in MscS-ND, the FC14 crystal structure (2OAU) and the ‘Cryst’ deletion construct. Black dashed lines depict the limits of the lipid bilayer based on the nanodisc EM density. Left, EM density for the protein (red) and the nanodisc (grey) for MscS-ND, the black ovals highlight the fact that the prominent cavities formed between the TM1-TM2 hairpin and TM3 are fully located outside the membrane. Center, relative positioning of 2OAU based on a rigid fit of the structure onto MscS-ND EM density. The gray rectangle in the background represents the previous consensus membrane location. Right, the low-resolution cryoEM structure of MscS ∆2–27 (‘Cryst’,~20 Å) shows an overall architecture for the nanodisc-embedded channel. In spite of the N-terminal deletion, the nanodisc is located at the same position as in MscS-ND. (C) Probing the energetics of the membrane interface. A Potential of mean force (PMF) calculation was carried out by relocating a lipid bilayer from a coordinate origin (0 Å) predicted by the CHARM-GUI server (Jo et al., 2008) and moved up to 16 Å (the thickness of a lipid monolayer) along the Z-axis coordinate (see Figure 2—figure supplement 2). Left, free energy as a function of Z-axis displacement. A global minima was found at ~25 Å (~8 Å above the prediction) and the free energy increases exponentially beyond this point. The energy minima coincides with the location if the interface as defined by the EM density of MscS-ND. Right, evolution of MD simulation starting at three membrane interface locations: predicted by CHARM-GUI (0 Å, red trace), at the cryo-EM density (+ 8 Å, black trace) and a further +16 Å (Higher placement, blue trace). After ~60 ns simulation all membrane interfaces converge to that defined by the cryo-EM density.

Figure 2—figure supplement 1. MscS bilayer footprint is compatible with bilayer predictions and surface charge distribution.

(A) A map of all surface-exposed charged residues (in VDW sphere representation) fully agrees with the location of membrane interface based on the MscS-ND EM density. With the exception of Arginine 88 (which coordinates the phosphate group of the hook lipid), there are no charge residues in the proposed TM region. (B) OPM predicted the location of the bilayer for the crystal structure 2OAU (Lomize et al., 2012). (C) MemProtMD predicted location of the bilayer for the crystal structure 2OAU (Newport et al., 2019).

Figure 2—figure supplement 2. Details of MD simulations and PMF calculated from umbrella sampling for determining the optimum position of MscS with respect to the bilayer.

(A) Example of MscS (MscS Cryo-EM) embedded in a membrane for MD simulation. (B) Root mean square deviation (RMSD) of different MD models used in this study. (C) The reaction coordinate for the PMF calculations has been defined as the distance between the Z coordinate of the center mass of phosphate molecules of the lipid bilayer and the Z coordinate of center mass of the pore-forming helices of MscS (i.e. residue 105 to 115). (D) Counts with respect to different sampling windows (reaction coordinates ranging from 16 Å to 31 Å); 10 ns Simulation for each histogram.

Figure 2—figure supplement 3. Geometrical properties of MscS embedded in a lipid bilayer for PMF calculations.

(A) Surface representation of MscS with residue-type color map. Red color shows negatively charged residues, blue shows positively charged residues, green hydrophilic and grey hydrophobic residues of MscS while embedded in the bilayer. A van der Waals illustration of residues W16 and I48 at the lipid-protein interface has been shown for determining the hydrophobic length of the protein, $d_p$. (B) An example membrane thickness ($d_l$) distribution (from the simulation with Z distance = 31 $\AA$) shown in color spectrum ranging from ~ 30 $\AA$ (around the protein) to ~ 47 $\AA$ (near the edges). (C) Snapshots of the membrane with different curvatures (C), which are calculated based on the length (l) and the height (h) of the bilayer. (D–F) The mean-field free energy change due to change in the bilayer curvature and lipid-protein hydrophobic mismatch as a result of different MscS position with respect to the membrane. Top panel indicates the free energy change assuming constant curvature across the bilayer area and the bottom panel assume variable curvature across the bilayer area. (D) Indicates the free energy contribution of curvature change at different umbrella sampling windows, (E) shows the contribution of the free energy change due to hydrophobic mismatch between MscS and the bilayer, and (F) shows the combination of the free energy from both curvature and hydrophobic mismatch. Hydrophobic mismatch is the main contributing factor in change in the free energy level across different reaction coordinates. The values are mean ± std.

Figure 2—figure supplement 4. Cartoon representation of concentric areas and associated curvatures around membrane-embedded MscS.

The membrane is discretized into concentric ribbons of areas $a_1$ to $a_n$ with curvatures $c_1$ to $c_n$.