Mechanosensitive Channel MscS

Japanese Lantern Protein MscS
Fig. 1. "Japanese Lantern" Protein MscS.

Mechanosensitive channels are membrane proteins that open and close in response to mechanical forces produced by sound, gravity or osmotic pressure, among other mechanical stimuli. In the open state, these proteins allow passage of ions across the membrane, thus generating an ionic current that eventually becomes an electrical signal (mechanotransduction) or that simply helps, for instance, in the regulation of cell volume. In bacteria, a protein that looks like a Japanese lantern (formed by a transmembrane pore and a cytoplasmic "balloon" with seven small openings around its equator) apparently prevents cell burst upon osmotic shocks by controlling passage of water and ions in response to membrane tension. Mechanical channel gating and transport of ions through this Japanese lantern protein, called MscS, have been studied by combining different experimental and computational methodologies: patch-clamp experiments and electron paramagnetic resonance (EPR) measurements (Perozo's Lab), all-atom molecular dynamics simulations (using NAMD), a coarse-grained model (using BioMOCA, Ravaioli's Lab), and restrained molecular dynamics (Roux's Lab). The experiments revealed that MscS can be found in inactive, closed, or open conformations, while the MscS simulations (all-atom involving 220,000-atom systems lasting tens of nanoseconds and coarse-grained lasting 100 nanoseconds or more) suggest that the structure depicted by X-ray crystallography is not that of a completely open, non-selective channel. Finally, restrained molecular dynamics incorporating data from EPR experiments provided a model for the closed conformation of MscS in its native environment.

Molecular Architecture of MscS

MscS, with a conductance of 1 nanoSiemens and a slight anionic selectivity, is formed by seven identical subunits, as revealed by its crystal structure. The transmembrane domain has three alpha-helices per subunit, and the third helix is divided by a pronounced kink into two subunits. The transmembrane pore appears to be in an open state with a diameter of 6.5 Angstroms in its narrowest section. On the other hand, the cytoplasmic domain of MscS is formed by a large chamber or "balloon" thought to act as a molecular filter (but otherwise of unknown function), with seven openings on the sides and one at the bottom.

The crystal structure, as described above, allows one to observe the MscS at atomic resolution and to identify domains and residues relevant for its function. However, despite the availability of the structure, key questions remain unanswered: Does the structure explain the physiological role of MscS, i.e., is MscS only a safety valve? What residues are relevant for gating? Does the crystal structure show the fully open form of the channel? What is the role of the large (balloon shaped) cytoplasmic domain?

Mechanosensitive Channel of Small Conductance MscS
Fig. 2. Mechanosensitive Channel of Small Conductance MscS. The left panel shows a top view of the transmembrane domain and pore of MscS (from the periplasm). Side view of transmembrane helices of MscS are shown in the center panel along with the proposed position of the membrane and the axis of the channel. A side view of the cytoplasmic "balloon" of MscS is shown in the right panel.

Turning MscS On and Off

Simulated conformations of MscS
Fig. 3. Simulated conformations of MscS.

In order to determine if MscS is in its open state and how it transitions to a closed or "off" state ("gating"), several all-atom molecular dynamics simulations were performed on a system that included the whole crystal structure of MscS embedded in a lipid bilayer with water and salt (220,000 atoms!). After a short equilibration with the protein backbone restrained to the crystal structure, a close packing of lipids against the protein was achieved (see panels A and B of Figure 3) favored by close interactions between lipid head groups and charged side chains of the protein. Despite restraining the backbone of the protein, the radius of the transmembrane pore in its narrowest part decreased, indicating a strong tendency of the channel to close in the membrane environment.

The channel at native physiological conditions should be closed, preventing leakage of solutes and dissipation of the cellular potential. Simulations in which restraints on the backbone of the protein were eliminated allowed us to investigate if MscS does relax to a closed state in a membrane without tension. Indeed, gradual elimination of backbone restraints resulted in the spontaneous, asymmetric, closure of the transmembrane pore (see panel C of Fig. 3). Whether this occluded state represents a closed ("off"), collapsed ("out of service"), or inactive ("on vacation") state remains uncertain, but clearly MscS relaxes to a state in which ions cannot be transported.

Key interactions mediating structural rearrangements of MscS
Fig. 4. Key interactions mediating structural rearrangements of MscS.

Mscs is controlled in vivo by tension in the cell membrane, and its activity is recorded in patch clamp experiments where curvature and tension in the membrane are induced by application of a small pressure. The resulting tension mimics the effect of an osmotic downshock, where cell volume increases due to the intracellular/extracellular difference in ion concentration (osmotic pressure). Similar external conditions were simulated and the ensuing dynamics of MscS explored. A top view of the final state shows how the collapse of the pore is prevented and an increase in its size is induced (see panel D of Fig. 3). Two additional open conformations through which ions and water can permeate were obtained by performing steered molecular dynamics (SMD) simulations. During the first simulation, peripheral residues of the transmembrane domain were subject to forces applied radially, pointing away from the center of the channel (see panel E of Fig. 3). During the second simulation, forces were applied radially pointing away from the center of the channel to the transmembrane helices lining the pore (the resulting conformation is shown in panel F of Fig. 3). In both cases, opening results from straightening of TM3 helices. The only way to determine which of these open conformations is really open is by computing ionic currents (see below). However, the simulations already hint at how membrane tension controls the functional state of MscS. In all simulations, two amino acids of opposite charge glue together forming a so-called salt-bridge (see Fig. 4). This interaction provides a link between external transmembrane domains of MscS moving due to interactions with lipids, and the pore lining helices controlling the passage of ions across the channel

  • Click here for a movie (mpeg, 3.9M) showing closure of MscS transmembrane pore.
  • Click here for a movie (mpeg, 3.5M) showing widening of MscS transmembrane pore.

Flooding MscS

Water Permeation and Ionic Distribution
Fig. 5. Left: Intermittent water permeation through the transmembrane pore of MscS. Right: Charged amino acids located at the cytoplasmic and periplasmic ends of MscS's transmembrane pore.

How do we know if MscS is in its fully-open conformation? For this particular protein, the open state is characterized by conduction of ions accompanied by water. Therefore, the degree of water permeation gives an idea of how open or closed the conformations obtained through simulations are. Surprisingly, two different states were observed when the protein was restrained to the crystal conformation: the pore transited from being completely filled with water, to being partially empty. This dewetting transitions may be relevant for transport of ions, although they were not observed in wider conformations of the channel that exhibited enhanced transport of water (see Fig. 5).

What about ions? Only spontaneous diffusion of charges should be observed when no electrostatic bias is applied (resulting in a zero net transport of charge across the channel). The simulations showed indeed spontaneous diffusion of ions across the cytoplasmic side openings of the "balloon", but not through the transmembrane domain. Moreover, a particular pattern for the distribution of ions was observed: chloride ions (negative) accumulated at the ends of the transmembrane pore, while potassium ions (positive) seemed to accumulate at the distal part (from the membrane) of the cytoplasmic "balloon". Since the time scale of these simulations was too short and no biasing voltage was applied, further simulations using a coarse-grained description were carried out to determine the degree of "openness" of different conformations.

  • Click here for a movie (mpeg, 8.6M) showing permeation of water molecules through MscS.

MscS as an Ion Channel

In order to explore transport of ions across MscS subject to a biasing electrostatic potential over long time scales (100 nanoseconds or more) we simplified the description of MscS and its environment and modeled the protein and lipids as fixed dielectric materials, while ions were explicitly simulated using an implicit solvent. The dynamics of the ions was described by the Boltzmann transport equation and determined by electrostatic fields computed using Poisson's equation. The ions were therefore slowly pushed across the channel and the degree of "openness" of different conformations of MscS were determined by counting how many ions crossed through the MscS. The simulations revealed that ions permeated MscS through side openings of the cytoplasmic domain ("balloon") rather than through the distal cytoplasmic opening of the balloon. In addition, time-averaged electrostatic potential maps and ion densities featured a strong localized separation of chlorides and potassium ions inside and around the cytoplasmic domain and the transmembrane pore (see Fig. 6). Ionic currents computed for MscS conformations similar to those of the crystal structure were found to be too small to represent an open state, while currents computed for wider conformations reached through steered molecular dynamics simulations reproduce the experimentally determined values of the fully open channel. However, ionic currents were found to be mainly driven by chloride ions, indicating a high selectivity for anions over cations, in relative disagreement with present experimental results that suggest slight anionic selectivity.

Electrostatic Potential and Ionic Distribution
Fig. 6. Electrostatic potential, chloride and potassium concentrations along the MscS channel averaged over 100 nanoseconds of BioMOCA simulation.

Ionic Currents through MscS with All-atom MD Simulations

The approximations used in the static model of MscS (fixed protein and lipids, implicit solvent) permit one to reach simulation time scales of hundreds of nanoseconds. However, many details are dropped and one may wonder how important those details are. Therefore, all-atom molecular dynamics simulations were performed to test the validity of the previous results. In order to obtain meaningful results using the more expensive all-atom model, large voltages had to be applied, and only short trajectories (tens of nanoseconds) could be monitored. Ions were therefore rushed through the transmembrane pore of MscS by applying large electrostatic potentials.

The electrostatic potential maps depicted in Fig. 7 confirm that we are applying the right potentials. The biased, all-atom simulations revealed that MscS in its crystal conformation poorly conducts negative ions, while a wider state obtained upon application of biases exhibits a conductance that matches the experimentally observed values. All the open states tested feature straightened TM3 helices.

PME Electrostatic Potential Map
Fig. 7. The electrostatic potential computed using the particle mesh Ewald method, averaged over 2 trajectories (10 nanoseconds each) at 0.6 V (middle) and 0 V (right).
  • Click here for a movie (mpeg, 13.5M) showing a two-dimensional slice through the simulation cell during 12 ns of restrained MscS dynamics with an applied bias of 1.2 V.
  • Click here for a movie (mpeg, 8.2M) showing ion translocation through the restrained MscS pore during 12 ns of dynamics with an applied bias of 1.2 V.
  • Click here for a movie (mpeg, 8.8M) showing ion translocation through the unrestrained MscS pore during 10 ns of dynamics with an applied bias of 1.2 V.

Using Experimental Restraints to Obtain a Refined Model of MscS in its Closed State

EPR based refinement of MscS
Fig. 8. EPR based refinement of MscS.

The simulations of MscS in its native-like environment revealed that the crystal conformation does not represent a stable open or closed state. Likely, the structure is distorted by crystallization conditions: much like a pudding baked without an appropriate container, MscS crystallized without the membrane is out of shape. In order to solve this problem, one can use electron paramagnetic resonance (EPR) measurements on MscS spin-labeled cysteine mutants.

The EPR data is obtained from closed channels that are embedded in a controlled membrane environment, thus decreasing artifacts arising from the absence of a lipid bilayer observed in other experimental setups. The EPR spectra is used to determine whether a particular residue is facing a membrane or aqueous environment, located at the interface between the membrane and the solvent, or buried inside the protein.

Molecular dynamics simulations are then used to incorporate the information provided by the EPR experiments into a symmetrized model of MscS. First, pseudo-atoms representing the spin labels utilized in EPR experiments are assigned to each amino-acid of the structure (see Fig. 8). Second, the interactions between these pseudo-atoms and additional particles representing the membrane or the solvent are assigned for each residue. The assignment is done by taking into account the EPR data: pseudo-atoms of amino-acids exposed to aqueous environments (according to experiments) are set to interact favorably (in the simulation) with particles representing the solvent, while pseudo-atoms of amino-acids exposed to a membrane environment are set to interact favorably with particles representing the membrane, and pseudo-atoms of buried amino-acids are set to repel solvent and membrane particles. Finally, a molecular dynamics simulation is performed in which the pseudo atoms guide a dynamic refinement of the structure.

The model of the MscS closed state is similar to the crystal structure but with significant differences. The overall architecture is more compact and TM3 helices pack more tightly against the rest of the protein. The model also includes the previously unresolved N-terminus.

  • Click here for coordinates (Calpha atoms) of the EPR based model of MscS in its closed conformation

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