Probing the Structure of the Mechanosensitive Channel of Small Conductance in Lipid Bilayers with Pulsed Electron-Electron Double Resonance

Mechanosensitive channel proteins are important safety valves against osmotic shock in bacteria, and are involved in sensing touch and sound waves in higher organisms. The mechanosensitive channel of small conductance (MscS) has been extensively studied. Pulsed electron-electron double resonance (PELDOR or DEER) of detergent-solubilized protein confirms that as seen in the crystal structure, the outer ring of transmembrane helices do not pack against the pore-forming helices, creating an apparent void. The relevance of this void to the functional form of MscS in the bilayer is the subject of debate. Here, we report PELDOR measurements of MscS reconstituted into two lipid bilayer systems: nanodiscs and bicelles. The distance measurements from multiple mutants derived from the PELDOR data are consistent with the detergent-solution arrangement of the protein. We conclude, therefore, that the relative positioning of the transmembrane helices is preserved in mimics of the cell bilayer, and that the apparent voids are not an artifact of detergent solution but a property of the protein that will have to be accounted for in any molecular mechanism of gating.     

Defining the Role of the Tension Sensor in the Mechanosensitive Channel of Small Conductance

Mutations that alter the phenotypic behavior of the Escherichia coli mechanosensitive channel of small conductance (MscS) have been identified; however, most of these residues play critical roles in the transition between the closed and open states of the channel and are not directly involved in lipid interactions that transduce the tension response. In this study, we use molecular dynamic simulations to predict critical lipid interacting residues in the closed state of MscS. The physiological role of these residues was then investigated by performing osmotic downshock assays on MscS mutants where the lipid interacting residues were mutated to alanine. These experiments identified seven residues in the first and second transmembrane helices as lipid-sensing residues. The majority of these residues are hydrophobic amino acids located near the extracellular interface of the membrane. All of these residues interact strongly with the lipid bilayer in the closed state of MscS, but do not face the bilayer directly in structures associated with the open and desensitized states of the channel. Thus, the position of these residues relative to the lipid membrane appears related to the ability of the channel to sense tension in its different physiological states.     

Structure, Dynamics, and Ion Conductance of the Phospholamban Pentamer

A 52-residue membrane protein, phospholamban (PLN) is an inhibitor of an adenosine-5′-triphosphate-driven calcium pump, the Ca²⁺-ATPase. Although the inhibition of Ca²⁺-ATPase involves PLN monomers, in a lipid bilayer membrane, PLN monomers form stable pentamers of unknown biological function. The recent NMR structure of a PLN pentamer depicts cytoplasmic helices extending normal to the bilayer in what is known as the bellflower conformation. The structure shows transmembrane helices forming a hydrophobic pore 4 Å in diameter, which is reminiscent of earlier reports of possible ion conductance through PLN pentamers. However, recent FRET measurements suggested an alternative structure for the PLN pentamer, known as the pinwheel model, which features a narrower transmembrane pore and cytoplasmic helices that lie against the bilayer. Here, we report on structural dynamics and conductance properties of the PLN pentamers from all-atom (AA) and coarse-grained (CG) molecular dynamics simulations. Our AA simulations of the bellflower model demonstrate that in a lipid bilayer membrane or a detergent micelle, the cytoplasmic helices undergo large structural fluctuations, whereas the transmembrane pore shrinks and becomes asymmetric. Similar asymmetry of the transmembrane region was observed in the AA simulations of the pinwheel model; the cytoplasmic helices remained in contact with the bilayer. Using the CG approach, structural dynamics of both models were investigated on a microsecond timescale. The cytoplasmic helices of the CG bellflower model were observed to fall against the bilayer, whereas in the CG pinwheel model the conformation of the cytoplasmic helices remained stable. Using steered molecular dynamics simulations, we investigated the feasibility of ion conductance through the pore of the bellflower model. The resulting approximate potentials of mean force indicate that the PLN pentamer is unlikely to function as an ion channel.     

Interaction between the cytoplasmic and transmembrane domains of the mechanosensitive channel MscS

The bacterial mechanosensitive channel MscS protects the bacteria from rupture on hypoosmotic shock. MscS is composed of a transmembrane domain with an ion permeation pore and a large cytoplasmic vestibule that undergoes significant conformational changes on gating. In this study, we investigated whether specific residues in the transmembrane and cytoplasmic domains of MscS influence each other during gating. When Asp-62, a negatively charged residue located in the loop that connects the first and second transmembrane helices, was replaced with either a neutral (Cys or Asn) or basic (Arg) amino acid, increases in both the gating threshold and inactivation rate were observed. Similar effects were observed after neutralization or reversal of the charge of either Arg-128 or Arg-131, which are both located near Asp-62 on the upper surface of the cytoplasmic domain. Interestingly, the effects of replacing Asp-62 with arginine were complemented by reversing the charge of Arg-131. Complementation was not observed after simultaneous neutralization of the charge of these residues. These findings suggest that the cytoplasmic domain of MscS affects both the mechanosensitive gating and the channel inactivation rate through the electrostatic interaction between Asp-62 and Arg-131.     

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

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.     

Lipid-protein interaction of the MscS mechanosensitive channel examined by scanning mutagenesis

The mechanosensitive channel of small conductance (MscS) is a bacterial mechanosensitive channel that opens in response to rapid hypoosmotic stress. Since MscS can be opened solely by membrane stretch without help from any accessory protein, the lipid-protein interface must play a crucial role in sensing membrane tension. In this study, the hydrophobic residues in the lipid-protein interface were substituted one by one with a hydrophilic amino acid, asparagine, to modify the interaction between the protein and the lipid. Function of the mutant MscSs was examined by patch-clamp and hypoosmotic shock experiments. An increase in the gating threshold and a decrease in the viability on hypoosmotic shock were observed when the hydrophobic residues near either end of the first or the second transmembrane helix (TM1 or TM2) were replaced with asparagine. This observation indicates that the lipid-protein interaction at the ends of both helices (TM1 and TM2) is essential to MscS function.     

Interaction of the Mechanosensitive Channel,MscS, with the Membrane Bilayer through Lipid Intercalation into Grooves and Pockets

All membrane proteins have dynamic and intimate relationships with the lipids of the bilayer that may determine their activity. Mechanosensitive channels sense tension through their interaction with the lipids of the membrane. We have proposed a mechanism for the bacterial channel of small conductance, MscS, that envisages variable occupancy of pockets in the channel by lipid chains. Here, we analyze protein–lipid interactions for MscS by quenching of tryptophan fluorescence with brominated lipids. By this strategy, we define the limits of the bilayer for TM1, which is the most lipid exposed helix of this protein. In addition, we show that residues deep in the pockets, created by the oligomeric assembly, interact with lipid chains. On the cytoplasmic side, lipids penetrate as far as the pore-lining helices and lipid molecules can align along TM3b perpendicular to lipids in the bilayer. Cardiolipin, free fatty acids, and branched lipids can access the pockets where the latter have a distinct effect on function. Cholesterol is excluded from the pockets. We demonstrate that introduction of hydrophilic residues into TM3b severely impairs channel function and that even “conservative” hydrophobic substitutions can modulate the stability of the open pore. The data provide important insights into the interactions between phospholipids and MscS and are discussed in the light of recent developments in the study of Piezo1 and TrpV4.     

Ion conduction through MscS as determined by electrophysiology and simulation

The mechanosensitive channel of small conductance (MscS) is a membrane protein thought to act as a safety valve in bacteria, regulating the release of ions and small solutes when gated by membrane tension under challenging osmotic conditions. The influence of voltage on channel activation and the functional state depicted by the available crystal structure of MscS remain debated. Therefore, in an effort to relate electrophysiological measurements on MscS and properties of the MscS crystal conformation, we report here MscS's response to voltage and pressure as determined by patch-clamp experiments, as well as MscS electrostatics and transport properties as determined through all-atom molecular dynamics simulations of the protein embedded in a lipid bilayer, a 224,000-atom system. The experiments reveal that MscS is a slightly anion-selective channel with a conductance of approximately 1 ns, activated by pressure and inactivated in a voltage-dependent manner. On the other hand, the simulations, covering over 200 ns and including biasing electrostatic potentials, show that MscS restrained to the crystal conformation exhibits low conductance; unrestrained it increases the channel radius upon application of a large electrostatic bias and exhibits then ion conduction that matches experimentally determined conductances. The simulated conductance stems mainly from Cl- ions.     

Negative and positive temperature dependence of potassium leak in MscS mutants: Implications for understanding thermosensitive channels

Bacterial mechanosensitive channel of small conductance (MscS) is a protein, whose activity is modulated by membrane tension, voltage and cytoplasmic crowding. MscS is a homoheptamer and each monomer consists of three transmembrane helices (TM1-3). Hydrophobic pore of the channel is made of TM3s surrounded by peripheral TM1/2s. MscS gating is a complex process, which involves opening and inactivation in response to the increase of membrane tension. A number of MscS mutants were isolated. Among them mutants affecting gating have been found including gain-of-function (GOF) and loss-of-function (LOF) that open at lower or at higher thresholds, respectively. Previously, using an in vivo screen we isolated multiple MscS mutants that leak potassium and some of them were GOF or LOF. Here we show that for a subset of these mutants K⁺ leak is negatively (NTD) or positively (PTD) temperature dependent. We show that temperature reliance of these mutants does not depend on how MS gating is affected by a particular mutation. Instead, we argue that NTD or PTD leak is due to the opposite allosteric coupling of the structures that determine the temperature dependence to the channel gate. In PTD mutants an increased hydration of the pore vestibule is directly coupled to the increase in the channel conductance. In NTD mutants, at higher temperatures an increased hydration of peripheral structures leads to complete separation of TM3 and a pore collapse.     

Two Families of Mechanosensitive Channel Proteins

Mechanosensitive (MS) channels that provide protection against hypoosmotic shock are found in the membranes of organisms from the three domains of life: bacteria, archaea, and eucarya. Two families of ubiquitous MS channels are recognized, and these have been designated the MscL and MscS families. A high-resolution X-ray crystallographic structure is available for a member of the MscL family, and extensive molecular genetic, biophysical, and biochemical studies conducted in many laboratories have allowed postulation of a gating mechanism allowing the interconversion of a tightly closed state and an open state that controls transmembrane ion and metabolite fluxes. In contrast to the MscL channel proteins, which are of uniform topology, the much larger MscS family includes protein members with topologies that are predicted to vary from 3 to 11 α-helical transmembrane segments (TMSs) per polypeptide chain. Sequence analyses reveal that the three C-terminal TMSs of MscS channel proteins are conserved among family members and that the third of these three TMSs exhibits a 20-residue motif that is shared by the channel-forming TMS (TMS 1) of the MscL proteins. We propose that this C-terminal TMS in MscS family homologues serves as the channel-forming helix in a homooligomeric structure. The presence of a conserved residue pattern for the putative channel-forming TMSs in the MscL and MscS family proteins suggests a common structural organization, gating mechanism, and evolutionary origin.     

Two families of mechanosensitive channel proteins.

Mechanosensitive (MS) channels that provide protection against hypoosmotic shock are found in the membranes of organisms from the three domains of life: bacteria, archaea, and eucarya. Two families of ubiquitous MS channels are recognized, and these have been designated the MscL and MscS families. A high-resolution X-ray crystallographic structure is available for a member of the MscL family, and extensive molecular genetic, biophysical, and biochemical studies conducted in many laboratories have allowed postulation of a gating mechanism allowing the interconversion of a tightly closed state and an open state that controls transmembrane ion and metabolite fluxes. In contrast to the MscL channel proteins, which are of uniform topology, the much larger MscS family includes protein members with topologies that are predicted to vary from 3 to 11 alpha-helical transmembrane segments (TMSs) per polypeptide chain. Sequence analyses reveal that the three C-terminal TMSs of MscS channel proteins are conserved among family members and that the third of these three TMSs exhibits a 20-residue motif that is shared by the channel-forming TMS (TMS 1) of the MscL proteins. We propose that this C-terminal TMS in MscS family homologues serves as the channel-forming helix in a homooligomeric structure. The presence of a conserved residue pattern for the putative channel-forming TMSs in the MscL and MscS family proteins suggests a common structural organization, gating mechanism, and evolutionary origin.     

Three-dimensional architecture of membrane-embedded MscS in the closed conformation

The mechanosensitive channel of small conductance (MscS) is part of a coordinated response to osmotic challenges in Escherichia coli. MscS opens as a result of membrane tension changes, thereby releasing small solutes and effectively acting as an osmotic safety valve. Both the functional state depicted by its crystal structure and its gating mechanism remain unclear. Here, we combine site-directed spin labeling, electron paramagnetic resonance spectroscopy, and molecular dynamics simulations with novel energy restraints based on experimental electron paramagnetic resonance data to investigate the native transmembrane (TM) and periplasmic molecular architecture of closed MscS in a lipid bilayer. In the closed conformation, MscS shows a more compact TM domain than in the crystal structure, characterized by a realignment of the TM segments towards the normal of the membrane. The previously unresolved NH₂-terminus forms a short helical hairpin capping the extracellular ends of TM1 and TM2 and is in close interaction with the bilayer interface. The present three-dimensional model of membrane-embedded MscS in the closed state represents a key step in determining the molecular mechanism of MscS gating.     

Functional analysis of conserved motifs in the mechanosensitive channel homolog MscS-Like2 from Arabidopsis thaliana

The Mechanosensitive channel of Small conductance (MscS) of Escherichia coli has become an excellent model system for the structural, biophysical, and functional study of mechanosensitive ion channels. MscS, a complex channel with multiple states, contributes to protection against lysis upon osmotic downshock. MscS homologs are widely and abundantly dispersed among the bacterial and plant lineages, but are not found in animals. Investigation into the eukaryotic branch of the MscS family is in the beginning stages, and it remains unclear how much MscS homologs from eukaryotes resemble E. coli MscS with respect to structure, function, and regulation. Here we test the effect of mutating three conserved motifs on the function of MscS-Like (MSL)2, a MscS homolog localized to the plastids of Arabidopsis thaliana. We show that 1) a motif at the top of the cytoplasmic domain, referred to here as the PN(X)(9)N motif, is essential for MSL2 function and for its proper intraplastidic localization; 2) substituting polar residues for two large hydrophobic residues located in the predicted pore-lining transmembrane helix of MSL2 produces a likely gain-of-function allele, as previously shown for MscS; and 3) mis-expression of this allele causes severe defects in leaf growth, loss of chloroplast integrity, and abnormal starch accumulation. Thus, two of the three conserved motifs we analyzed are critical for MSL2 function, consistent with the conservation of structure and function between MscS family members in bacteria and plants. These results underscore the importance of plastidic mechanosensitive channels in the maintenance of normal plastid and leaf morphology.     

Mechanosensitive channel MscS in the open state: modeling of the transition, explicit simulations, and experimental measurements of conductance

Mechanosensitive channels of small conductance (MscS) are ubiquitous turgor pressure regulators found in many walled cells and some intracellular organelles. Escherichia coli MscS acting as a tension-activated osmolyte release valve shows a nonsaturable conductance (1.2 nS in a 39 mS/cm electrolyte) and weak preference for anions. Pursuing the transition pathways in this channel, we applied the extrapolated motion protocol (cycles of displacements, minimizations, and short simulations) to the previously generated compact resting conformation of MscS. We observed tilting and straightening of the kinked pore-forming TM3 helices during the barrel expansion. Extended all-atom simulations confirmed the stability of the open conformation in the bilayer. A 53 degrees spontaneous axial rotation of TM3s observed after equilibration increased the width and polarity of the pore allowing for stable voltage-independent hydration and presence of both cations and anions throughout the pore. The resultant open state, characterized by a pore 1.6 nm wide, satisfied the experimental conductance and in-plane expansion. Applied transmembrane electric field (+/-100 to +/-200 mV) in simulations produced a flow of both K(+) and Cl(-), with Cl(-) current dominating at higher voltages. Electroosmotic water flux strongly correlated with the chloride current (approximately 8 waters per Cl(-)). The selectivity and rectification were in agreement with the experimental measurements performed in the same range of voltages. Among the charged residues surrounding the pore, only K169 was found to contribute noticeably in the rectification. We conclude that (a) the barrel expansion involving tilting, straightening, and rotation of TM3s provides the geometry and electrostatics that accounts for the conductive properties of the open pore; (b) the observed regimen of ion passage through the pore is similar to electrodiffusion, thus macroscopic estimations closely approximate the experimental and molecular dynamics-simulated conductances; (c) increased interaction of the opposing ionic fluxes at higher voltages may result in selectivities stronger than measured near the reversal potential.     

Transmembrane domains of viral ion channel proteins: a molecular dynamics simulation study

Nanosecond molecular dynamics simulations in a fully solvated phospholipid bilayer have been performed on single transmembrane alpha-helices from three putative ion channel proteins encoded by viruses: NB (from influenza B), CM2 (from influenza C), and Vpu (from HIV-1). alpha-Helix stability is maintained within a core region of ca. 28 residues for each protein. Helix perturbations are due either to unfavorable interactions of hydrophobic residues with the lipid headgroups or to the need of the termini of short helices to extend into the surrounding interfacial environment in order to form H-bonds. The requirement of both ends of a helix to form favorable interactions with lipid headgroups and/or water may also lead to tilting and/or kinking of a transmembrane alpha-helix. Residues that are generally viewed as poor helix formers in aqueous solution (e.g., Gly, Ile, Val) do not destabilize helices, if located within a helix that spans a lipid bilayer. However, helix/bilayer mismatch such that a helix ends abruptly within the bilayer core destabilizes the end of the helix, especially in the presence of Gly and Ala residues. Hydrogen bonding of polar side-chains with the peptide backbone and with one another occurs when such residues are present within the bilayer core, thus minimizing the energetic cost of burying such side-chains.     

CGDB: A database of membrane protein/lipid interactions by coarse-grained molecular dynamics simulations

Membrane protein function and stability has been shown to be dependent on the lipid environment. Recently, we developed a high-throughput computational approach for the prediction of membrane protein/lipid interactions. In the current study, we enhanced this approach with the addition of a new measure of the distortion caused by membrane proteins on a lipid bilayer. This is illustrated by considering the effect of lipid tail length and headgroup charge on the distortion caused by the integral membrane proteins MscS and FLAP, and by the voltage sensing domain from the channel KvAP. Changing the chain length of lipids alters the extent but not the pattern of distortion caused by MscS and FLAP; lipid headgroups distort in order to interact with very similar but not identical regions in these proteins for all bilayer widths investigated. Introducing anionic lipids into a DPPC bilayer containing the KvAP voltage sensor does not affect the extent of bilayer distortion.     

Genetic screen for potassium leaky small mechanosensitive channels (MscS) in Escherichia coli: recognition of cytoplasmic β domain as a new gating element

Mechanosensitive membrane channels in bacteria respond to the mechanical stretching of the membrane. They will open when bacteria are subjected to rapid osmotic down shock. MscS is a bacterial mechanosensitive channel of small conductance. It is a heptameric membrane protein whose transmembrane part, including the gate and its kinetics, has been well characterized. MscS has a large cytoplasmic domain of a cage-like shape that changes its conformation upon gating, but its involvement in gating is not understood. We screened MscS for mutations that cause potassium leak in Escherichia coli strains deficient in potassium transport systems. We did a phenotypic analysis of single and multiple mutants and recorded the single channel activities of some of them. After these analyses, we attributed the effects of a number of mutations to particular functional states of the channel. Our screen revealed that MscS leaks potassium in a desensitized and in an inactivated state. It also appeared that the lower part of TM3 (transmembrane, pore-forming helix) and the cytoplasmic β domain are tightly packed in the inactivated state but are dissociated in the open state. We attribute the TM3-β interaction to stabilization of the inactivated state in MscS and to the control of tight closure of its membrane pore.     

Transmembrane helices of membrane proteins may flex to satisfy hydrophobic mismatch

A novel mechanism for membrane modulation of transmembrane protein structure, and consequently function, is suggested in which mismatch between the hydrophobic surface of the protein and the hydrophobic interior of the lipid bilayer induces a flexing or bending of a transmembrane segment of the protein. Studies on model hydrophobic transmembrane peptides predict that helices tilt to submerge the hydrophobic surface within the lipid bilayer to satisfy the hydrophobic effect if the helix length exceeds the bilayer width. The hydrophobic surface of transmembrane helix 1 (TM1) of lactose permease, LacY, is accessible to the bilayer, and too long to be accommodated in the hydrophobic portion of a typical lipid bilayer if oriented perpendicular to the membrane surface. Hence, nuclear magnetic resonance (NMR) data and molecular dynamics simulations show that TM1 from LacY may flex as well as tilt to satisfy the hydrophobic mismatch with the bilayer. In an analogous study of the hydrophobic mismatch of TM7 of bovine rhodopsin, similar flexing of the transmembrane segment near the conserved NPxxY sequence is observed. As a control, NMR data on TM5 of lacY, which is much shorter than TM1, show that TM5 is likely to tilt, but not flex, consistent with the close match between the extent of hydrophobic surface of the peptide and the hydrophobic thickness of the bilayer. These data suggest mechanisms by which the lipid bilayer in which the protein is embedded modulates conformation, and thus function, of integral membrane proteins through interactions with the hydrophobic transmembrane helices.