Three-dimensional architecture of membrane-embedded MscS in the closed conformation

Abstract

The mechanosensitive channel of small conductance (MscS) is part of a coordinated response to osmotic challenges in Escherichia coli. MscS opens as a result of membrane tension changes, thereby releasing small solutes and effectively acting as an osmotic safety valve. Both the functional state depicted by its crystal structure and its gating mechanism remain unclear. Here, we combine site-directed spin labeling, electron paramagnetic resonance spectroscopy, and molecular dynamics simulations with novel energy restraints based on experimental electron paramagnetic resonance data to investigate the native transmembrane (TM) and periplasmic molecular architecture of closed MscS in a lipid bilayer. In the closed conformation, MscS shows a more compact TM domain than in the crystal structure, characterized by a realignment of the TM segments towards the normal of the membrane. The previously unresolved NH(2)-terminus forms a short helical hairpin capping the extracellular ends of TM1 and TM2 and is in close interaction with the bilayer interface. The present three-dimensional model of membrane-embedded MscS in the closed state represents a key step in determining the molecular mechanism of MscS gating.

Figure 1

MscS architecture and cysteine mutants expression (A) Linear representation of MscS topology. The TM helices (as suggested from the crystal structure) of the MscS monomer (TM1, TM2, and TM3) are represented by rectangles. The scale corresponds to the amino acid residue numbering. The red line denotes the residues that were mutated to cysteine. (B) Single MscS subunit showing amino acid residues (black spheres) probed through site directed spin labeling of cysteines. A single MscS monomer is represented as part of the heptamer according to the crystal structure of the channel. The NH₂-terminus segment (26 residues) is represented by a schematic line. (C) Expression of cysteine mutants. The total amount of cell and protein obtained is shown for each residue after expressing single cysteine mutants in vector pET28 (containing MscS with His6 epitope at the NH₂-termini), in E. coli Rosetta^™ cells. Grayed areas represent the membrane embedded areas derived from the MscS crystal structure.

Figure 2

EPR spectroscopy of MscS (A) Representative X-band EPR spectra of consecutively spin-labeled mutants from TM1 segment of MscS reconstituted in DOPC:POPG liposomes. All spectra were obtained from samples with the same protein to lipid ratio, and using a loop-gap resonator with the microwave power set to 2 mW. (B) Residue-specific environmental parameter profiles obtained for the NH₂ terminal and TM segments: mobility parameter ΔH_o⁻¹ (top, black circles), O₂ accessibility parameter ΠO₂ (middle, red squares, the dotted box pinpoints the patch of higher O₂ accessibility in TM3), and NiEdda accessibility parameter ΠNiEdda (bottom, blue triangles, the broken line represents the NiEdda average for the TMs). Grayed areas represent the TM segment assignment derived from the MscS crystal structure. (C) Residue environmental parameter profiles mapped onto a molecular surface of two representative MscS monomers. Mobility parameter ΔH_o⁻¹ (top panel), O₂ accessibility parameter ΠO₂ (middle panel), and NiEdda accessibility parameter ΠNiEdda (bottom panel). Arrows on bottom panel pinpoint residues with high NiEdda accessibility in the middle of the bilayer.

Figure 3

Identification of MscS interfacial residues with DOGS-NTA[Ni(II)]lipids (A) Ribbon diagram of the TM segments of the MscS crystal structure (a single MscS monomer is highlighted) embedded in a schematic membrane consisting of DOPC:POPG and DOGS-NTA[Ni(II)]lipids (few lipid molecules are shown for clarity). Black spheres show the location of cysteine mutants that were used to obtain the data. (B) Residue environmental parameter profile derived from Y27C (black arrow), TM1–TM2 loop and TM3B. Experimental controls (I39C and G41C) are identified with red arrows. Gray box highlights the region that corresponds to background collisional level. (C) Results mapped onto a molecular surface of MscS′ TM segments.

Figure 4

Functional and residue-specific environmental parameter profiles of NH₂-terminal cysteine mutants (A) Functional assays of cells containing NH₂-terminal cysteine mutants over-expressed in pQE70 vector. MJF465 and WT MscS were used as a negative and positive control, respectively. Red dotted lines represent the mean value of WT MscS (right) and the mean value of MJF465 (left). (B) X-band EPR spectra of consecutively spin-labeled mutants from the NH₂-terminal segment of MscS reconstituted in DOPC:POPG liposomes. (C) Residue-specific environmental parameter profiles obtained for the NH₂-terminal segment: mobility parameter ΔH_o⁻¹ (top, black circles), O₂ accessibility parameter ΠO₂ (middle, red squares), NiEdda accessibility parameter ΠNiEdda (middle, blue triangles), and interfacial accessibility parameter ΠDOGS-NTA[Ni(II)]lipids (bottom, green rhombuses). Gray box highlights the region that corresponds to background collisional level.

Figure 5

Structural Analysis of the NH₂-terminal segment. (A) Helical wheel representation of the NH₂-termini. Environmental parameters have been superimposed in a polar coordinate representation. A resultant vector was calculated for the O₂ accessibility (left panel, red arrow) and NiEdda accessibility (right panel, blue arrow). (B) Box plot analysis of the NH₂-termini and TM segments. Squares represent the mean, boxes the data distribution, and bars the standard deviation. (C) Windowed periodicity analysis for the NiEdda parameter ΠNiEdda. The α-helical periodicity index was calculated as described earlier with an angular range from 80°–120° and a sliding window of seven residues. The horizontal line represents the threshold at which the periodicity of the windowed segment is significantly α-helical (10 to 14, and 20 to 27).

Figure 6

Topology and structural model of MscS′ NH₂-terminal domain, obtained by the pseudo-atom driven solvent accessibility refinement. (A) Secondary structure prediction, from the linear sequence (left panel) of residues 1 to 178, using the software Rosetta ; . The right panel shows a conformation of the NH₂-terminal domain obtained after energy minimization of the obtained model. The resulting structure was used in the EPR based refinement of the MscS closed conformation. (B) Left panel, cartoon representation of MscS model used as initial structure for refinement. The model includes residues 1 to 178. Pseudo-atoms representing the spin label attached to each residue are shown for one subunit and color coded as follows: red particles represent buried residues, blue particles represent aqueous residues, yellow particles represent residues facing the membrane, and green particles represent residues at the water-membrane interface. Red particles are shown for each C-α atom representing PROT particles attached to each residue. Inset shows protein residues in thin licorice representation and pseudo-spin probes in CPK representation. Unbounded particles in red and blue represent O₂ and NiEdda virtual environment particles, respectively. View of the NH₂-terminal model after pseudo-atom driven solvent accessibility refinement is shown on the right panel. Residues 1 to 28 are shown in gray, resultant vectors for NiEdda and O₂ accessibilities are shown in blue and red respectively.

Figure 7

MscS closed conformation obtained from EPR based refinement (A) Ribbon representation of the MscS closed state model (two subunits are shown for clarity). Individual TM segments are color-coded as follows: NH₂-terminus, green, TM1, yellow; TM2, blue; and TM3, red. (B–E) Mobility parameter ΔH_o⁻¹, O₂ accessibility parameter ΠO₂, NiEdda accessibility parameter ΠNiEdda, and interfacial accessibility parameter ΠDOGS-NTA[Ni(II)]lipids, respectively.

Figure 8

Comparison of MscS crystal structure and outcome of EPR based refinement. (A) Crystal structure model. Left panel, ribbon representation of the TM segments of the MscS (two subunits are shown for clarity). Individual TM segments are color-coded as follows: TM1, yellow; TM2, blue; and TM3, red. Right most panels show residue environmental parameter profiles mapped onto molecular surfaces of the x-ray model (B) Closed state model from EPR based refinement. Left panel, ribbon representation of the MscS closed state model. Individual TM segments are color-coded as above, except for the NH₂-terminus in green. Right most panel show residue environmental parameter profiles mapped onto molecular surfaces of the EPR refined MscS model. The arrows show the key rearrangement in the TM segments of MscS in a native closed state.