Water dynamics and dewetting transitions in the small mechanosensitive channel MscS

Abstract

The dynamics of confined water in capillaries and nanotubes suggests that gating of ion channels may involve not only changes of the pore geometry, but also transitions between water-filled and empty states in certain locations. The recently solved heptameric structure of the small mechanosensitive channel of Escherichia coli, MscS, has revealed a relatively wide (7-15 A) yet highly hydrophobic transmembrane pore. Continuum estimations based on the properties of pore surface suggest low conductance and a thermodynamic possibility of dewetting. To test the predictions we performed molecular dynamics simulations of MscS filled with flexible TIP3P water. Irrespective to the initial conditions, several independent 6-ns simulations converged to the same stable state with the pore water-filled in the wider part, but predominantly empty in the narrow hydrophobic part, displaying intermittent vapor-liquid transitions. The polar gain-of-function substitution L109S in the constriction resulted in a stable hydration of the entire pore. Steered passages of Cl(-) ions through the narrow part of the pore consistently produced partial ion dehydration and required a force of 200-400 pN to overcome an estimated barrier of 10-20 kcal/mole, implying negligibly low conductance. We conclude that the crystal structure of MscS does not represent an open state. We infer that MscS gate, which is similar to that of the nicotinic ACh receptor, involves a vapor-lock mechanism where limited changes of geometry or surface polarity can locally switch the regime between water-filled (conducting) and empty (nonconducting) states.

FIGURE 1

Properties of MscS pore: radius, resistance, surface polarity, hydration energy, and evolutionary conservancy. (a) Crystal structure of E. coli MscS (Bass et al., 2002) in a schematic representation. The pore region considered for continuum estimations and then included in MD simulations is highlighted yellow. (b) The cross section of the central pore region with its solvent-accessible surface (probe R = 1.4 Å) is colored according to the residue type: acidic (red), basic (blue), polar (green), and nonpolar (white). Positions of residues defining the polarity are indicated. (c) Effective pore radius R (black solid line) and specific resistance ρ (red solid line) as functions of z coordinate. A hypothetical widening of the constriction and the corresponding drop of resistance are shown by dotted lines. (d) The pore region is colored according to the relative conservation index for the YggB family (data from Pivetti et al., 2003). (e) The surface of the pore is colored according to the atomic solvation energies with hydrophilic atoms shown in blue. (f) Hydration energy (red) and free energies of dewetting calculated with the surface tension parameter σ = 70 (blue) and 20 mJ/m² (green). The initiation of the vapor phase formation was inferred in the narrowest part of the pore (dashed line).

FIGURE 2

Water dynamics in wild-type and gain-of-function MscS channels. (a) A cross section through the simulation box with the protein (yellow boundary), water (cyan sticks), octane (gray lines), sodium (blue VDW sphere), and chloride (red VDW sphere) ions. Basic (blue) and polar (green) residues facing the pore lumen are shown as sticks. Snapshots of the pore region in the vapor-plugged (b) and partially water-filled (c) states of WT MscS, and of the stably hydrated L109S gain-of-function mutant (d).

FIGURE 3

Water occupancy of MscS pore during 6-ns simulations as a function of time. (a) The number of water molecules found in the narrowest pore region of WT MscS simulated in the presence of salt. (b) The z positions of water molecules in the pore region. Each point represents the position of a water oxygen at a given time step. Horizontal lines in b delineate the central region (−26 Å > z > −18 Å) where the water molecules were scored. (c) The average z coordinates of the terminal carbons of all seven L109 side chains (solid line) and their average distance from the axis of the pore (dashed line). (d) The average number of chloride and sodium ions in the periplasmic ( and ) and in the cytoplasmic ( and ) halves of the pore. (e) The pore occupancy by water presented as in a for the simulation of WT MscS without ions.

FIGURE 4

Water densities in wild-type and L109S gain-of-function MscS pores. The probability of the occurrence of water oxygens was calculated using a three-dimensional volumetric grid and normalized to the density of bulk water outside the pore. The maps represent 2 Å slices along the pore axis, averaged for the last 5.5 ns of WT MscS simulation with (a) and without (b) salt, and for 3.5 ns of L109S (c) in the presence of salt. Inset in a shows the density in the slice across the wider nonpolar region where water is organized as two concentric cylindrical layers.

FIGURE 5

Densities of hydrogen bonding in WT and GOF MscS. Hydrogen bonds defined by the geometric criteria (see Methods) were counted and averaged for entire simulations as in Fig. 4. The maps represent 2 Å slices along the pore axis through the center of the pore. (a) WT MscS simulated with 200 mM NaCl and (b) without ions. (c) L109S gain-of-function mutant simulated in the presence of salt, showing bulk-like density of H-bonds throughout the pore.

FIGURE 6

Steered movement of Cl⁻ ion through WT and L109S MscS pores. (a) The initial points for the outward (from left to right) and inward movements through the constriction are shown by red spheres. The ion was harmonically restrained to a plane which was moved with a velocity of 38 Å/ns along the z axis. In the course of transition, the ion was free to move in x and y directions. Three trajectories, two outward and one inward, are shown. Two of the trajectories are color-coded according to the force applied to the ion (red being the highest). The key residues are denoted. (b) The steering force (smoothed with a 50-ps running frame) as a function of z coordinate for three independent transitions in each direction. The state of the pore (water-filled or empty) at the moment of ion's entry into the constriction for each trace is shown in the legend. (c) The potential energy of the ion inside the pore calculated as the difference of total work produced by the steering force and the friction losses (see Methods). The force (d) and the energy (e) profiles for the steered movement of Cl⁻ through the permanently hydrated L109S MscS pore.

FIGURE 7

Sequential snapshots of the steered ion movement through a vapor-plugged MscS pore. The ion experiences the highest resistance when it enters the constriction and at the same time loses a part of the hydration shell (a and b). The ion regains its complete hydration shell only after exiting the constriction (g and h), which is accompanied by a small acceleration (see Fig. 6 and text). The presence of ion in the middle of the pore (c–f) does not result in the complete hydration of the wall.