Mechanosensitive Channel MscS

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## Mechanosensitive Channel 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, 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 two different methodologies: all-atom molecular dynamics simulations (using NAMD) and a coarse-grained model (using BioMOCA). Embedding the large MscS protein into a lipid bilayer and water led to all-atom simulations encompassing 220,000 atoms lasting tens of nanoseconds. On the other hand, the coarse-grained description permitted simulations lasting 100 nanoseconds or more (with a cumulative total of ~5 microseconds). These long simulations are required to compute ionic currents through different conformations of the channel and revealed a peculiar distribution of anions and cations inside and around the protein "balloon".

## 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?

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

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 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 lipids 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.

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). 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

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 the MscS is in its 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.

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

## All-atom Simulation of the MscS Ion Channel

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 performed. Ions were therefore rushed through the transmembrane pore of MscS by applying large electrostatic potentials. The results of these simulations will be presented in our next paper!

Fig. 7. The electrostatic potential computed using the particle mesh Ewald method, averaged over 3 trajectories (10 nanoseconds each) at 0.6 V (left), 1.2 V (middle) and 0 V (right).

## Publications

Electrostatic properties of the mechanosensitive channel of small conductance MscS. Marcos Sotomayor, Trudy A. van der Straaten, Umberto Ravaioli, and Klaus Schulten. Biophysical Journal, 90:3496-3510, 2006.

Towards understanding membrane channels. Emad Tajkhorshid, Jordi Cohen, Aleksij Aksimentiev, Marcos Sotomayor, and Klaus Schulten. In Boris Martinac and Andrzej Kubalski, editors, Bacterial ion channels and their eukaryotic homologues, pp. 153-190. ASM Press, Washington, DC, 2005.

Molecular dynamics study of gating in the mechanosensitive channel of small conductance MscS. Marcos Sotomayor and Klaus Schulten. Biophysical Journal, 87:3050-3065, 2004.

## Investigators

- Marcos Sotomayor
- Trudy van der Straaten
- Umberto Ravaioli
- Klaus Schulten

## Related TCB Group Projects

- Mechanosensitive Channel MscL
- Mechanical Proteins
- Molecular Basis of Hearing

Page created and maintained by Marcos Sotomayor.

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