Molecular dynamics study of gating in the mechanosensitive channel of small conductance MscS

Abstract

Mechanosensitive channels are a class of ubiquitous membrane proteins gated by mechanical strain in the cellular membrane. MscS, the mechanosensitive channel of small conductance, is found in the inner membrane of Escherichia coli and its crystallographic structure in an open form has been recently solved. By means of molecular dynamics simulations we studied the stability of the channel conformation suggested by crystallography in a fully solvated lipid (POPC) bilayer, the combined system encompassing 224,340 atoms. When restraining the backbone of the protein, the channel remained in the open form and the simulation revealed intermittent permeation of water molecules through the channel. Abolishing the restraints under constant pressure conditions led to spontaneous closure of the transmembrane channel, whereas abolishing the restraints when surface tension (20 dyn/cm) was applied led to channel widening. The large balloon-shaped cytoplasmic domain of MscS exhibited spontaneous diffusion of ions through its side openings. Interaction between the transmembrane domain and the cytoplasmic domain of MscS was observed and involved formation of salt bridges between residues Asp62 and Arg128; this interaction may be essential for the gating of MscS. K+ and Cl- ions showed distinctively different distributions in and around the channel.

FIGURE 1

Homoheptameric architecture of MscS. (A) Cartoon representation of the MscS crystal structure. Each subunit is presented in a different color. The suggested membrane position (Bass et al., 2002) is shown as a shaded bar. (B) Subunit B of the heptameric structure. This subunit is shown in cartoon and ribbons representation. The transmembrane domain is formed by three helices (TM1, TM2, and TM3A–TM3B) and a larger cytoplasmic domain. The TM3A–TM3B helix has a pronounced kink at residue Gly¹¹³, close to the narrowest part of the pore at the cytoplasmic side of the membrane. (C) Transmembrane helices of two monomers shown in surface and tube representations. Charged residues are shown in space-filling representation and colored blue (+) and red (−). Position of residues Leu¹⁰⁵ and Leu¹⁰⁹ (drawn in space-filling representation) is indicated by the arrow. (D) Top view of the transmembrane domain (residues 27–128). Pore residues (96–113) are shown in surface representation. Each subunit is labeled by a color and a letter. Note how the TM3B helix of one subunit is located just underneath the loop between TM1 and TM2 of the next subunit. (E) Surface representation of MscS. There are seven cytoplasmic openings located on the side and one at the bottom.

FIGURE 2

MscS in a membrane bilayer. (A) Stereo side view of MscS in a fully solvated membrane environment, 224,340 atoms. (B) Stereo view of MscS from its cytoplasmic side.

FIGURE 3

Summary of MscS simulations. Simulation sim0 consisted of 3 ns of equilibration with multiple substeps as described in the text. Permeation properties of MscS restrained to the crystal structure were studied in simulation sim1 (1.45 ns, protein backbone harmonically restrained). In simulation sim2a (4 ns), restraints were gradually suppressed leading to a nonsymmetrical closed state. The effect of surface tension on the open state was studied in simulation sim2b (3.7 ns). In simulation sim3a (1.2 ns), surface tension was applied to the closed state obtained in simulation sim2a; this resulted in a partially open state of MscS.

FIGURE 4

Simulated conformations of MscS. (A) Side view of the final state (4.45 ns) of simulation sim1. The protein is shown in surface representation and the lipid bilayer in licorice representation. Transmembrane domains (residues 27–128) of subunits A and B (see Fig. 1) along with part of the membrane have been eliminated to reveal the pore's interior. Water molecules inside the pore are shown in space-filling representation. The water box is drawn in transparent blue. (B–E) Top views of final conformations of simulations sim1 (4.45 ns), sim2a (8.45 ns), sim2b (9.05 ns), and sim3a (9.65 ns), respectively. The transmembrane domain is shown in surface representation and the lipid bilayer in space-filling representation. The color code for each protein subunit is the same as in Fig. 1 D.

FIGURE 5

Pore radius. (A, top) Pore of MscS in the crystal structure (excluding subunits C and D). The transmembrane domain is shown in transparent surface representation and colored by residue type (white, nonpolar residues; blue, basic residues; red, acidic residues; green, polar residues). Yellow spheres are drawn using the center and radii generated by HOLE. (A, bottom) Pore radius profile along the z axis for MscS with its backbone atoms restrained to the crystal structure (sim1) at t = 0 (black) and t = 4.45 ns (red). The shaded region marks the membrane boundaries. (B, top) Pore of MscS at the end of simulation sim2a (t = 8.45 ns) as in A. (B, bottom) Pore radius profile along the z axis of MscS at the end of simulations sim2a (t = 8.45 ns, green), sim2b (t = 9.05 ns, blue), and sim3a (t = 9.65 ns, yellow).

FIGURE 6

(A) Detail of TM3A deformation upon closing of the pore. The backbone of subunit-C helix (residues 96–113) is shown in tube representation. Residues Gly¹⁰¹, Ala¹⁰², Leu¹⁰⁵, and Ala¹⁰⁶ are shown in licorice representation. Hydrogen bonds are shown in blue. (Left) Snapshot at the end of simulation sim1 (4.45 ns). (Right) Snapshot at t = 6.04 ns of simulation sim2a. (B) View of pore residues (96–113, TM3A) and surrounding lipids (shown in van der Waals representation with hydrogen atoms in white, carbon atoms in cyan, and oxygen atoms in red). Subunits are colored as in Fig. 1 D. Residues Leu¹⁰⁵ of each subunit are shown in space-filling representation. Subunits A (light blue), C (red), and E (orange) show pronounced kinks and their respective Leu¹⁰⁵ side chains block the pore. Subunit A is in close contact with a lipid, whereas the other subunits are not.

FIGURE 7

RMSD with respect to the crystal structure. (A) RMSD of the whole protein backbone for simulations sim2a (red), sim2b (blue), and sim3a (green). The clearly discernible steps correspond to changes in harmonic restraints (see text). (B–D) RMSD per zone for simulations sim2a, sim2b, and sim3a, respectively. MscS zones are defined as follows: TM1–TM2 helices, residues 27–91 (black); TM3A–TM3B helix, residues 91–127 (red); pore (TM3A), residues 96–113 (green); and cytoplasmic domain, residues 128–280 (blue).

FIGURE 8

Water permeation. Occupancy of the hydrophobic (transmembrane) pore of MscS by water molecules is shown as a function of time for simulations sim1 (A), sim2a (B), sim2b (C), and sim3a (D). The number of water molecules inside the pore was monitored every picosecond assuming the positions of C_α atoms of residues 96 and 113 as pore ends. Snapshots of the pore (shown in surface representation and colored by residue type as described in Fig. 5) with water molecules are shown for representative states.

FIGURE 9

Ion diffusion across the cytoplasmic domain of MscS. (A) Stereo view of the diffusion of a Cl⁻ ion from the bulk to the interior of the MscS cytoplasmic domain in simulation sim2a. (B) Stereo view of the diffusion of a K⁺ ion from inside of the MscS cytoplasmic domain toward the bulk in simulation sim2a. In both cases, the cytoplasmic domain of MscS (residues 128–280) is drawn in tube representation and colored as in Fig. 1 D. The ion pathway is formed by the superposition of sampled positions of the respective ion along simulation sim2a, which is shown in space-filling representation (sphere). The color of the ion indicates time, with red corresponding to initial stages of simulation sim2a and blue to final stages of simulation sim2a.

FIGURE 10

Distribution of ions during simulation sim2a. (A) The average density of Cl⁻ ions as defined by the scale bar is shown at a transversal slice crossing the center of the channel in the z direction. The approximate shapes of MscS and the lipid bilayer are depicted by a solid line. (B) The crystal structure of MscS is shown in cartoon representation. The approximate volumetric shape of MscS and the position of the lipid bilayer are depicted by a solid line. (C) The average density of K⁺ ions is shown as in A.

FIGURE 11

Movement of transmembrane helices. Transmembrane domains are shown in ribbon representation for subunit E (A) and subunit A (B). Red shows the state of helices in simulation sim2a at t = 8.45 ns and green shows the state of helices in simulation sim2b at t = 9.05 ns. The movement observed in subunit E is in agreement with the gating mechanism proposed in Bass et al. (2002). Subunits D and F showed similar behavior. The movement observed in subunit A (along with subunits B and G) is in the opposite direction.

FIGURE 12

Contacts between the TM1–TM2 loop and the TM3B helix. (Top) Segments of helices TM1, TM2, and TM3 of subunit B are shown in blue along with helix TM3 of subunit A shown in light blue, all in tube representation. Residue Asp⁶² of subunit B and residue Arg¹²⁸ of subunit A are shown in space-filling representation. The interaction of both residues is clearly discernible. (Bottom) Segments of helices TM1 and TM2 of subunit F (yellow), and helix TM3 along with part of the cytoplasmic domain of subunit E (orange) are shown in transparent surface and tube representation. The illustration shows how the TM1–TM2 loop closely interacts with the TM3B helix.

FIGURE 13

Salt-bridge formation. Separation between the C_ζ atom of residue Arg¹²⁸ of one subunit and the C_γ atom of residue Asp⁶² of the subsequent subunit are shown for simulations sim1 (A) and sim2a (B–C). (The colors denote the subunits where the residue Arg¹²⁸ is located: subunit A, black; subunit B, red; subunit C, green; subunit D, blue; subunit E, orange; subunit F, brown; and subunit G, gray.)

FIGURE 14

Stereo side views of part of the transmembrane domain. Phosphorus atoms of lipid bilayer headgroups are shown as tan spheres. The MscS transmembrane domain is shown in surface representation and colored by residue type (nonpolar residues, white; basic residues, blue; acidic residues, red; and polar residues, green). (Top) Initial conformation. (Bottom) Final conformation (both of simulation sim1). The deformation of the lipid bilayer close to the membrane-protein interface can be observed. One can discern a clear rearrangement of lipid headgroups around reoriented charged and polar residues (blue, red, and green) on the cytoplasmic side (bottom) of MscS.