Membrane tension, lipid adaptation, conformational changes, and energetics in MscL gating

This study aims to explore gating mechanisms of mechanosensitive channels in terms of membrane tension, membrane adaptation, protein conformation, and energetics. The large conductance mechanosensitive channel from Mycobacterium tuberculosis (Tb-MscL) is used as a model system; Tb-MscL acts as a safety valve by releasing small osmolytes through the channel opening under extreme hypoosmotic conditions. Based on the assumption that the channel gating involves tilting of the transmembrane (TM) helices, we have performed free energy simulations of Tb-MscL as a function of TM helix tilt angle in a dimyristoylphosphatidylcholine bilayer. Based on the change in system dimensions, TM helix tilting is shown to be essentially equivalent to applying an excess surface tension to the membrane, causing channel expansion, lipid adaptation, and membrane thinning. Such equivalence is further corroborated by the observation that the free energy cost of Tb-MscL channel expansion is comparable to the work done by the excess surface tension. Tb-MscL TM helix tilting results in an expanded water-conducting channel of an outer dimension similar to the proposed fully open MscL structure. The free energy decomposition indicates a possible expansion mechanism in which tilting and expanding of TM2 facilitates the iris-like motion of TM1, producing an expanded Tb-MscL.     

The dynamics of protein-protein interactions between domains of MscL at the cytoplasmic-lipid interface

The bacterial mechanosensitive channel of large conductance, MscL, is one of the best characterized mechanosensitive channels serving as a paradigm for how proteins can sense and transduce mechanical forces. The physiological role of MscL is that of an emergency release valve that opens a large pore upon a sudden drop in the osmolarity of the environment. A crystal structure of a closed state of MscL shows it as a homopentamer, with each subunit consisting of two transmembrane domains (TM). There is consensus that the TM helices move in an iris like manner tilting in the plane of the membrane while gating. An N-terminal amphipathic helix that lies along the cytoplasmic membrane (S1), and the portion of TM2 near the cytoplasmic interface (TM2_ci), are relatively close in the crystal structure, yet predicted to be dynamic upon gating. Here we determine how these two regions interact in the channel complex, and study how these interactions change as the channel opens. We have screened 143 double-cysteine mutants of E. coli MscL for their efficiency in disulfide bridging and generated a map of protein-protein interactions between these two regions. Interesting candidates have been further studied by patch clamp and show differences in channel activity under different redox potentials; the results suggest a model for the dynamics of these two domains during MscL gating.     

The dynamics of protein-protein interactions between domains of MscL at the cytoplasmic-lipid interface

The bacterial mechanosensitive channel of large conductance, MscL, is one of the best characterized mechanosensitive channels serving as a paradigm for how proteins can sense and transduce mechanical forces. The physiological role of MscL is that of an emergency release valve that opens a large pore upon a sudden drop in the osmolarity of the environment. A crystal structure of a closed state of MscL shows it as a homopentamer, with each subunit consisting of two transmembrane domains (TM). There is consensus that the TM helices move in an iris like manner tilting in the plane of the membrane while gating. An N-terminal amphipathic helix that lies along the cytoplasmic membrane (S1), and the portion of TM2 near the cytoplasmic interface (TM2_ci), are relatively close in the crystal structure, yet predicted to be dynamic upon gating. Here we determine how these two regions interact in the channel complex, and study how these interactions change as the channel opens. We have screened 143 double-cysteine mutants of E. coli MscL for their efficiency in disulfide bridging and generated a map of protein-protein interactions between these two regions. Interesting candidates have been further studied by patch clamp and show differences in channel activity under different redox potentials; the results suggest a model for the dynamics of these two domains during MscL gating.     

Contributions of the different extramembranous domains of the mechanosensitive ion channel MscL to its response to membrane tension

MscL is a mechanosensitive channel that is gated by tension in the membrane bilayer alone. It is a homo-oligomer of a protein comprising two transmembrane segments connected by an external loop, with the NH(2) and COOH termini located in the cytoplasm. The contributions of the extramembranous domains of the channel to its activity were investigated by specific proteolysis during patch-clamp experiments. Limited proteolysis of the COOH terminus or the NH(2) terminus increased the mechanosensitivity of the channel without changing its conductance. Strikingly, after cleavage of the external loop of each monomer, the channel was still functional, and its mechanosensitivity was increased dramatically, indicating that the loop acts as a spring that resists the opening of the channel and promotes its closure when it is open. These results indicate that the integrity of most of the extramembranous domains is not essential for mechanosensitivity. They suggest that these domains counteract the movement of the transmembrane helices to which they are connected, thus setting the level of sensitivity of the channel to tension.     

A large iris-like expansion of a mechanosensitive channel protein induced by membrane tension

MscL, a bacterial mechanosensitive channel of large conductance, is the first structurally characterized mechanosensor protein. Molecular models of its gating mechanisms are tested here. Disulfide crosslinking shows that M1 transmembrane alpha-helices in MscL of resting Escherichia coli are arranged similarly to those in the crystal structure of MscL from Mycobacterium tuberculosis. An expanded conformation was trapped in osmotically shocked cells by the specific bridging between Cys 20 and Cys 36 of adjacent M1 helices. These bridges stabilized the open channel. Disulfide bonds engineered between the M1 and M2 helices of adjacent subunits (Cys 32-Cys 81) do not prevent channel gating. These findings support gating models in which interactions between M1 and M2 of adjacent subunits remain unaltered while their tilts simultaneously increase. The MscL barrel, therefore, undergoes a large concerted iris-like expansion and flattening when perturbed by membrane tension.     

The gating mechanism of the bacterial mechanosensitive channel MscL revealed by molecular dynamics simulations

One of the ultimate goals of the study on mechanosensitive (MS) channels is to understand the biophysical mechanisms of how the MS channel protein senses forces and how the sensed force induces channel gating. The bacterial MS channel MscL is an ideal subject to reach this goal owing to its resolved 3D protein structure in the closed state on the atomic scale and large amounts of electrophysiological data on its gating kinetics. However, the structural basis of the dynamic process from the closed to open states in MscL is not fully understood. In this study, we performed molecular dynamics (MD) simulations on the initial process of MscL opening in response to a tension increase in the lipid bilayer. To identify the tension-sensing site(s) in the channel protein, we calculated interaction energy between membrane lipids and candidate amino acids (AAs) facing the lipids. We found that Phe78 has a conspicuous interaction with the lipids, suggesting that Phe78 is the primary tension sensor of MscL. Increased membrane tension by membrane stretch dragged radially the inner (TM1) and outer (TM2) helices of MscL at Phe78, and the force was transmitted to the pentagon-shaped gate that is formed by the crossing of the neighboring TM1 helices in the inner leaflet of the bilayer. The radial dragging force induced radial sliding of the crossing portions, leading to a gate expansion. Calculated energy for this expansion is comparable to an experimentally estimated energy difference between the closed and the first subconductance state, suggesting that our model simulates the initial step toward the full opening of MscL. The model also successfully mimicked the behaviors of a gain of function mutant (G22N) and a loss of function mutant (F78N), strongly supporting that our MD model did simulate some essential biophysical aspects of the mechano-gating in MscL.     

The gating mechanism of the bacterial mechanosensitive channel MscL revealed by molecular dynamics simulations: From tension sensing to channel opening

One of the ultimate goals of the study on mechanosensitive (MS) channels is to understand the biophysical mechanisms of how the MS channel protein senses forces and how the sensed force induces channel gating. The bacterial MS channel MscL is an ideal subject to reach this goal owing to its resolved 3D protein structure in the closed state on the atomic scale and large amounts of electrophysiological data on its gating kinetics. However, the structural basis of the dynamic process from the closed to open states in MscL is not fully understood. In this study, we performed molecular dynamics (MD) simulations on the initial process of MscL opening in response to a tension increase in the lipid bilayer. To identify the tension-sensing site(s) in the channel protein, we calculated interaction energy between membrane lipids and candidate amino acids (AAs) facing the lipids. We found that Phe78 has a conspicuous interaction with the lipids, suggesting that Phe78 is the primary tension sensor of MscL. Increased membrane tension by membrane stretch dragged radially the inner (TM1) and outer (TM2) helices of MscL at Phe78, and the force was transmitted to the pentagon-shaped gate that is formed by the crossing of the neighboring TM1 helices in the inner leaflet of the bilayer. The radial dragging force induced radial sliding of the crossing portions, leading to a gate expansion. Calculated energy for this expansion is comparable to an experimentally estimated energy difference between the closed and the first subconductance state, suggesting that our model simulates the initial step toward the full opening of MscL. The model also successfully mimicked the behaviors of a gain of function mutant (G22N) and a loss of function mutant (F78N), strongly supporting that our MD model did simulate some essential biophysical aspects of the mechano-gating in MscL.     

Loss-of-function mutations at the rim of the funnel of mechanosensitive channel MscL

MscL is a bacterial mechanosensitive channel that is activated directly by membrane stretch. Although the gene has been cloned and the crystal structure of the closed channel has been defined, how membrane tension causes conformational changes in MscL remains largely unknown. To identify the site where MscL senses membrane tension, we examined the function of the mutants generated by random and scanning mutagenesis. In vitro (patch-clamp) and in vivo (hypoosmotic-shock) experiments showed that when a hydrophilic amino acid replaces one of the hydrophobic residues that are thought to make contact with the membrane lipid near the periplasmic end of the M1 or M2 transmembrane domain, MscL loses the ability to open in response to membrane tension. Hydrophilic (asparagine) substitution of the other residues in the lipid-protein interface did not impair the channel's mechanosensitivity. These observations suggest that the disturbance of the hydrophobic interaction between the membrane lipid and the periplasmic rim of the channel's funnel impairs the function of MscL.     

Gating of the mechanosensitive channel protein MscL: the interplay of membrane and protein

The mechanosensitive channel of large conductance (MscL) belongs to a family of transmembrane channel proteins in bacteria and functions as a safety valve that relieves the turgor pressure produced by osmotic downshock. MscL gating can be triggered solely by stretching of the membrane. This work reports an effort to understand this mechanotransduction by means of molecular dynamics (MD) simulation on the MscL of mycobacterium tuberculosis embedded in a palmitoyloleoylphosphatidylethanolamine membrane. Equilibrium MD under zero membrane tension produced a more compact protein structure, as measured by its radii of gyration, compared to the crystal structure, in agreement with previous experimental findings. Even under a large applied tension up to 1000 dyn/cm, the MscL lateral dimension largely remained unchanged after up to 20 ns of simulation. A nonequilibrium MD simulation of 3% membrane expansion showed a significant increase in membrane rigidity upon MscL inclusion, which can contribute to efficient mechanotransduction. Direct observation of channel opening was possible only when an explicit lateral bias force was applied to each of the five subunits of MscL in the radially outward direction. Using this force, open structures with a large pore of radius 10 Å could be obtained. The channel opening takes place in a stepwise manner and concurrently with the water chain formation across the channel, which occurs without direct involvement of protein hydrophilic residues. The N-terminal S1 helices stabilize the open structure, and the membrane asymmetry (different lipid density on the two leaflets of membrane) promotes channel opening.     

Gating of MscL studied by steered molecular dynamics

Steered molecular dynamics simulations of the mechanosensitive channel of large conductance, MscL, were used to investigate how forces arising from membrane tension induce gating of the channel. A homology model of the closed form of MscL from Escherichia coli was subjected to external forces of 35-70 pN applied to residues near the membrane-water interface. The magnitude and location of these forces corresponded to those determined from the lateral pressure profile computed from a lipid bilayer simulation. A fully expanded state was obtained on the 10-ns timescale that revealed the mechanism for transducing membrane forces into channel opening. The expanded state agrees well with proposed models of MscL gating, in that it entails an irislike expansion of the pore accompanied by tilting of the transmembrane helices. The channel was most easily opened when force was applied predominantly on the cytoplasmic side of MscL. Comparison of simulations in which gating progressed to varying degrees identified residues that pose steric hindrance to channel opening.     

Dynamics of Protein-Protein Interactions at the MscL Periplasmic-Lipid Interface

MscL, the highly conserved bacterial mechanosensitive channel of large conductance, is one of the best studied mechanosensors. It is a homopentameric channel that serves as a biological emergency release valve that prevents cell lysis from acute osmotic stress. We previously showed that the periplasmic region of the protein, particularly a single residue located at the TM1/periplasmic loop interface, F47 of Staphylococcus aureus and I49 of Escherichia coli MscL, plays a major role in both the open dwell time and mechanosensitivity of the channel. Here, we introduced cysteine mutations at these sites and found they formed disulfide bridges that decreased the channel open dwell time. By scanning a likely interacting domain, we also found that these sites could be disulfide trapped by addition of cysteine mutations in other locations within the periplasmic loop of MscL, and this also led to rapid channel kinetics. Together, the data suggest structural rearrangements and protein-protein interactions that occur within this region upon normal gating, and further suggest that locking portions of the channel into a transition state decreases the stability of the open state.     

Dynamics of Protein-Protein Interactions at the MscL Periplasmic-Lipid Interface

MscL, the highly conserved bacterial mechanosensitive channel of large conductance, is one of the best studied mechanosensors. It is a homopentameric channel that serves as a biological emergency release valve that prevents cell lysis from acute osmotic stress. We previously showed that the periplasmic region of the protein, particularly a single residue located at the TM1/periplasmic loop interface, F47 of Staphylococcus aureus and I49 of Escherichia coli MscL, plays a major role in both the open dwell time and mechanosensitivity of the channel. Here, we introduced cysteine mutations at these sites and found they formed disulfide bridges that decreased the channel open dwell time. By scanning a likely interacting domain, we also found that these sites could be disulfide trapped by addition of cysteine mutations in other locations within the periplasmic loop of MscL, and this also led to rapid channel kinetics. Together, the data suggest structural rearrangements and protein-protein interactions that occur within this region upon normal gating, and further suggest that locking portions of the channel into a transition state decreases the stability of the open state.     

Membrane topology and multimeric structure of a mechanosensitive channel protein of Escherichia coli.

We have studied the membrane topology and multimeric structure of a mechanosensitive channel, MscL, which we previously isolated and cloned from Escherichia coli. We have localized this 15-kDa protein to the inner membrane and, by PhoA fusion, have shown that it contains two transmembrane domains with both the amino and carboxyl termini on the cytoplasmic side. Mutation of the glutamate at position 56 to histidine led to changes in channel kinetics which were dependent upon the pH on the periplasmic, but not cytoplasmic side of the membrane, providing additional evidence for the periplasmic positioning of this part of the molecule. Tandems of two MscL subunits expressed as a single polypeptide formed functional channels, suggesting an even number of transmembrane domains per subunit (amino and carboxyl termini on the same side of the membrane), and an even number of subunits per functional complex. Finally, cross-linking studies suggest that the functional MscL complex is a homohexamer. In summary, these data are all consistent with a protein domain assignment and topological model which we propose and discuss.     

Nanomechanical properties of MscL α helices: A steered molecular dynamics study

Gating of mechanosensitive (MS) channels is driven by a hierarchical cascade of movements and deformations of transmembrane helices in response to bilayer tension. Determining the intrinsic mechanical properties of the individual transmembrane helices is therefore central to understanding the intricacies of the gating mechanism of MS channels. We used a constant-force steered molecular dynamics (SMD) approach to perform unidirectional pulling tests on all the helices of MscL in M. tuberculosis and E. coli homologs. Using this method, we could overcome the issues encountered with the commonly used constant-velocity SMD simulations, such as low mechanical stability of the helix during stretching and high dependency of the elastic properties on the pulling rate. We estimated Young's moduli of the α-helices of MscL to vary between 0.2 and 12.5 GPa with TM2 helix being the stiffest. We also studied the effect of water on the properties of the pore-lining TM1 helix. In the absence of water, this helix exhibited a much stiffer response. By monitoring the number of hydrogen bonds, it appears that water acts like a ‘lubricant’ (softener) during TM1 helix elongation. These data shed light on another physical aspect underlying hydrophobic gating of MS channels, in particular MscL.     

Mechanosensitive channels of bacteria and archaea share a common ancestral origin

The ubiquity of mechanosensitive (MS) ion channels set off a search for their functional homologues in archaea, the third domain of life. A new MS channel was identified in the archaeon Methanococcus jannaschii by using the TM1 transmembrane domain of the bacterial MS channel of large conductance, MscL, as a genetic probe to search the archaeal genomic database for MS channel homologues. The hypothetical protein MJ0170 (MscMJ) was found to harbor two MscL-like TM1 structural motifs and showed a high degree of se quence and secondary structure conservation with MscS (YggB) homologues. The alignment of sequences of MscL, MscS and MscMJ homologues further revealed that bacterial and archaeal channels form a phylogenetic tree composed of three main branches and share a common ancestral origin. This suggests the evolution of prokaryotic MS channels via gene duplication of a MscL-like progenitor gene followed by divergence, fur ther indicating that the common ancestor of the prokaryotic MS channels most likely resembled MscL. When expressed in E. coli and functionally examined by the patch clamp, the MscMJ protein behaved as a MS channel with a conductance of 270 pS in 200 mM KCl and a cation selectivity (PK/PC]) of approximately 6. The structural and functional homologue of MscMJ, MscMJLR, was identified as a second type of MS channel in M. jannaschii. The channel has a conductance of approximately 2 nS, rectifies with voltage and shares cation selectivity with MscMJ. The stoichiometry of both types of MS channels revealed that the free energy of activation, deltaG0 approximately 7kT, obtained for MscMJ matches the one calculated for MscS, deltaG0 approximately 5kT, whereas the free energy of activation approximately deltaG0 approximately 18kT of MscMJLR resembles more the deltaG0 = 14-19kT reported for MscL. The presence of two types of MS channels discovered in the cell envelope of M. jannaschii indicates that multiplicity of MS channels in prokaryotes is a necessary element for their survival in the habitats frequently challenged by sudden changes in osmolarity. Further functional and phylogenetic study of MS channels from all three domains of the universal phylogenetic tree may help to understand the evolution and common biophysical principles that govern mechanosensory transduction.     

Novel splice variants of amphiphysin I are expressed in retina

The MscL channel is a mechanosensitive channel which is gated by membrane stress or tension. Here, we describe a series of simulations which apply simulated mechanical stress to a molecular model of the MscL channel using two methods - direct force application to the transmembrane segments, and anisotropic pressure coupling. In the latter simulations, pressures less than that equivalent to a bilayer tension of 12 dyn/cm did not cause the channel to open, while pressures in excess of this value resulted in the channel opening. These results are in approximate agreement with experimental findings.     

Structural determinants of MscL gating studied by molecular dynamics simulations

The mechanosensitive channel of large conductance (MscL) in prokaryotes plays a crucial role in exocytosis as well as in the response to osmotic downshock. The channel can be gated by tension in the membrane bilayer. The determination of functionally important residues in MscL, patch-clamp studies of pressure-conductance relationships, and the recently elucidated crystal structure of MscL from Mycobacterium tuberculosis have guided the search for the mechanism of MscL gating. Here, we present a molecular dynamics study of the MscL protein embedded in a fully hydrated POPC bilayer. Simulations totaling 3 ns in length were carried out under conditions of constant temperature and pressure using periodic boundary conditions and full electrostatics. The protein remained in the closed state corresponding to the crystal structure, as evidenced by its impermeability to water. Analysis of equilibrium fluctuations showed that the protein was least mobile in the narrowest part of the channel. The gating process was investigated through simulations of the bare protein under conditions of constant surface tension. Under a range of conditions, the transmembrane helices flattened as the pore widened. Implications for the gating mechanism in light of these and experimental results are discussed.     

Structural models of the MscL gating mechanism.

Three-dimensional structural models of the mechanosensitive channel of large conductance, MscL, from the bacteria Mycobacterium tuberculosis and Escherichia coli were developed for closed, intermediate, and open conformations. The modeling began with the crystal structure of M. tuberculosis MscL, a homopentamer with two transmembrane alpha-helices, M1 and M2, per subunit. The first 12 N-terminal residues, not resolved in the crystal structure, were modeled as an amphipathic alpha-helix, called S1. A bundle of five parallel S1 helices are postulated to form a cytoplasmic gate. As membrane tension induces expansion, the tilts of M1 and M2 are postulated to increase as they move away from the axis of the pore. Substantial expansion is postulated to occur before the increased stress in the S1 to M1 linkers pulls the S1 bundle apart. During the opening transition, the S1 helices and C-terminus amphipathic alpha-helices, S3, are postulated to dock parallel to the membrane surface on the perimeter of the complex. The proposed gating mechanism reveals critical spatial relationships between the expandable transmembrane barrel formed by M1 and M2, the gate formed by S1 helices, and "strings" that link S1s to M1s. These models are consistent with numerous experimental results and modeling criteria.     

Structural Investigation of MscL Gating Using Experimental Data and Coarse Grained MD Simulations

The mechanosensitive channel of large conductance (MscL) has become a model system in which to understand mechanosensation, a process involved in osmoregulation and many other physiological functions. While a high resolution closed state structure is available, details of the open structure and the gating mechanism remain unknown. In this study we combine coarse grained simulations with restraints from EPR and FRET experiments to study the structural changes involved in gating with much greater level of conformational sampling than has previously been possible. We generated a set of plausible open pore structures that agree well with existing open pore structures and gating models. Most interestingly, we found that membrane thinning induces a kink in the upper part of TM1 that causes an outward motion of the periplasmic loop away from the pore centre. This previously unobserved structural change might present a new mechanism of tension sensing and might be related to a functional role in osmoregulation.     

Capping transmembrane helices of MscL with aromatic residues changes channel response to membrane stretch

Tyrosines and tryptophans that anchor both ends of the helices to membrane interfaces in many transmembrane proteins are not common in MscL and homologous mechanosensitive channels. This characteristic absence of two aromatic "belts" may be critical for MscL function as the opening transition is predicted to be associated with a strong helical reorientation. A single tyrosine (Y75) on the extracellular side of the M2 helix of pentameric EcoMscL is absent in TbMscL, which instead has a single tyrosine (Y87) on the cytoplasmic side of M2. Moving the tyrosine of EcoMscL to the intracellular side (Y75F/F93Y) or capping the TM2 helix on both sides (F93Y/W) slows the kinetics of gating and increases the threshold for activation, leading to a partial loss-of-function in osmotic shock survival assays. Increasing the distance between the caps (L98W, L102Y/W) partially restores channel function presumably by loosening restraints for tilting. Capping the TM2 helix with a charged residue (Y75E) causes a right shift of the activation curve ("stiff" phenotype) and abolishes function. Introducing a "cap" into the TM1 helix (I41W) decreases the activation threshold and shortens the mean open time but unexpectedly leads to a complete loss-of-function in vivo. The data are consistent with the view that restraining helical positions in MscL by introducing specific protein-lipid interactions at membrane interfaces compromises MscL function. Subtle differences in osmotic shock survival are more evident at low levels of mutant protein expression. We observed a correlation between the right shift of tension activation threshold and the loss-of-function channel phenotype, with a few exceptions that point to other parameters of gating that may define the osmotic rescuing ability in vivo.