Curcumin activation of a bacterial mechanosensitive channel underlies its membrane permeability and adjuvant properties.

Curcumin, a natural compound isolated from the rhizome of turmeric, has been shown to have antibacterial properties. It has several physiological effects on bacteria including an apoptosis-like response involving RecA, membrane permeabilization, inhibiting septation, and it can also work synergistically with other antibiotics. The mechanism by which curcumin permeabilizes the bacterial membrane has been unclear. Most bacterial species contain a Mechanosensitive channel of large conductance, MscL, which serves the function of a biological emergency release valve; these large-pore channels open in response to membrane tension from osmotic shifts and, to avoid cell lysis, allow the release of solutes from the cytoplasm. Here we show that the MscL channel underlies the membrane permeabilization by curcumin as well as its synergistic properties with other antibiotics, by allowing access of antibiotics to the cytoplasm; MscL also appears to have an inhibitory role in septation, which is enhanced when activated by curcumin.

An agonist of the MscL channel affects multiple bacterial species and increases membrane permeability and potency of common antibiotics.

The bacterial MscL channel normally functions as an emergency release valve discharging cytoplasmic solutes upon osmotic stress. The channel opens and passes molecules up to 30 Å and its pore is the largest of any gated channel. Opening the MscL pore inappropriately is detrimental to the bacterial cell, suggesting MscL as a potential novel drug target. A small-molecule compound, 011A, has been shown to increase sensitivity of the Escherichia coli MscL channel, slow growth, and even decrease viability of quiescent cultures. The mscL gene is highly conserved and found in the vast majority of bacterial species, including pathogens. Here, we test the hypothesis that 011A can influence the growth and viability of other bacterial species, specifically Staphylococcus aureus and Mycobacterium smegmatis, in a MscL-dependent manner. Furthermore, we demonstrate that the 011A compound can increase potency of other antibiotics, presumably by permeabilizing the membrane and allowing easier access of the antibiotic into the cytoplasm. Thus, MscL activators have potential as novel broad-spectrum antibiotics or adjuvants that work with antibiotics to selectively allow passage across bacterial membranes.

Novel compounds that specifically bind and modulate MscL: insights into channel gating mechanisms.

The bacterial mechanosensitive channel of large conductance (MscL) normally functions as an emergency release valve discharging cytoplasmic solutes upon osmotic stress. Opening the large pore of MscL inappropriately is detrimental to the cell, and thus it has been speculated to be a potential antibiotic target. Although MscL is one of the best studied mechanosensitive channels, no chemical that influenced bacterial growth by modulating MscL is known. We therefore used a high-throughput screen to identify compounds that slowed growth in an MscL-dependent manner. We characterized 2 novel sulfonamide compounds identified in the screen. We demonstrated that, although both increase MscL gating, one of these compounds does not work through the folate pathway, as other antimicrobial sulfonamides; indeed, the sulfonamide portion of the compound is not needed for activity. The only mode of action appears to be MscL activation. The binding pocket is where an α-helix runs along the cytoplasmic membrane and interacts with a neighboring subunit; analogous motifs have been observed in several prokaryotic and eukaryotic channels. The data not only demonstrate that MscL is a viable antibiotic target, but also give insight into the gating mechanisms of MscL, and they may have implications for developing agonists for other channels.-Wray, R., Iscla, I., Kovacs, Z., Wang, J., Blount, P. Novel compounds that specifically bind and modulate MscL: insights into channel gating mechanisms.

Dihydrostreptomycin Directly Binds to, Modulates, and Passes through the MscL Channel Pore.

The primary mechanism of action of the antibiotic dihydrostreptomycin is binding to and modifying the function of the bacterial ribosome, thus leading to decreased and aberrant translation of proteins; however, the routes by which it enters the bacterial cell are largely unknown. The mechanosensitive channel of large conductance, MscL, is found in the vast majority of bacterial species, where it serves as an emergency release valve rescuing the cell from sudden decreases in external osmolarity. While it is known that MscL expression increases the potency of dihydrostreptomycin, it has remained unclear if this effect is due to a direct interaction. Here, we use a combination of genetic screening, MD simulations, and biochemical and mutational approaches to determine if dihydrostreptomycin directly interacts with MscL. Our data strongly suggest that dihydrostreptomycin binds to a specific site on MscL and modifies its conformation, thus allowing the passage of K+ and glutamate out of, and dihydrostreptomycin into, the cell.

Structure and molecular mechanism of an anion-selective mechanosensitive channel of small conductance.

Mechanosensitive (MS) channels are universal cellular membrane pores. Bacterial MS channels, as typified by MS channel of small conductance (MscS) from Escherichia coli (EcMscS), release osmolytes under hypoosmotic conditions. MS channels are known to be ion selective to different extents, but the underlying mechanism remains poorly understood. Here we identify an anion-selective MscS channel from Thermoanaerobacter tengcongensis (TtMscS). The structure of TtMscS closely resembles that of EcMscS, but it lacks the large cytoplasmic equatorial portals found in EcMscS. In contrast, the cytoplasmic pore formed by the C-terminal ß-barrel of TtMscS is larger than that of EcMscS and has a strikingly different pattern of electrostatic surface potential. Swapping the ß-barrel region between TtMscS and EcMscS partially switches the ion selectivity. Our study defines the role of the ß-barrel in the ion selection of an anion-selective MscS channel and provides a structural basis for understanding the ion selectivity of MscS channels.

Sensing and responding to membrane tension: the bacterial MscL channel as a model system.

Mechanosensors are important for many life functions, including the senses of touch, balance, and proprioception; cardiovascular regulation; kidney function; and osmoregulation. Many channels from an assortment of families are now candidates for eukaryotic mechanosensors and proprioception, as well as cardiovascular regulation, kidney function, and osmoregulation. Bacteria also possess two families of mechanosensitive channels, termed MscL and MscS, that function as osmotic emergency release valves. Of the two channels, MscL is the most conserved, most streamlined in structure, and largest in conductance at 3.6 nS with a pore diameter in excess of 30 Å; hence, the structural changes required for gating are exaggerated and perhaps more easily defined. Because of these properties, as well as its tractable nature, MscL represents a excellent model for studying how a channel can sense and respond to biophysical changes of a lipid bilayer. Many of the properties of the MscL channel, such as the sensitivity to amphipaths, a helix that runs along the membrane surface and is connected to the pore via a glycine, a twisting and turning of the transmembrane domains upon gating, and the dynamic changes in membrane interactions, may be common to other candidate mechanosensors. Here we review many of these properties and discuss their structural and functional implications.

An in vivo screen reveals protein-lipid interactions crucial for gating a mechanosensitive channel.

The bacterial mechanosensitive channel MscL is the best-studied mechanosensor, thus serving as a paradigm of how a protein senses and responds to mechanical force. Models for the transition of Escherichia coli MscL from closed to open states propose a tilting of the transmembrane domains in the plane of the membrane, suggesting dynamic protein-lipid interactions. Here, we used a rapid in vivo assay to assess the function of channels that were post-translationally modified at several different sites in a region just distal to the cytoplasmic end of the second transmembrane helix. We utilized multiple probes with various affinities for the membrane environment. The in vivo functional data, combined with site-directed mutagenesis, single-channel analyses, and tryptophan fluorescence measurements, confirmed that lipid interactions within this region are critical for MscL gating. The data suggest a model in which this region acts as an anchor for the transmembrane domain tilting during gating. Furthermore, the conservation of analogous motifs among many other channels suggests a conserved protein-lipid dynamic mechanism.

In Silico Screen Identifies a New Family of Agonists for the Bacterial Mechanosensitive Channel MscL.

MscL is a highly conserved mechanosensitive channel found in the majority of bacterial species, including pathogens. It functions as a biological emergency release valve, jettisoning solutes from the cytoplasm upon acute hypoosmotic stress. It opens the largest known gated pore and has been heralded as an antibacterial target. Although there are no known endogenous ligands, small compounds have recently been shown to specifically bind to and open the channel, leading to decreased cell growth and viability. Their binding site is at the cytoplasmic/membrane and subunit interfaces of the protein, which has been recently been proposed to play an essential role in channel gating. Here, we have targeted this pocket using in silico screening, resulting in the discovery of a new family of compounds, distinct from other known MscL-specific agonists. Our findings extended the study of this functional region, the progression of MscL as a viable drug target, and demonstrated the power of in silico screening for identifying and improving the design of MscL agonists.

Activation of a Bacterial Mechanosensitive Channel, MscL, Underlies the Membrane Permeabilization of Dual-Targeting Antibacterial Compounds.

Resistance to antibiotics is a serious and worsening threat to human health worldwide, and there is an urgent need to develop new antibiotics that can avert it. One possible solution is the development of compounds that possess multiple modes of action, requiring at least two mutations to acquire resistance. Compound SCH-79797 both avoids resistance and has two mechanisms of action: one inhibiting the folate pathway, and a second described as "membrane permeabilization"; however, the mechanism by which membranes from bacterial cells, but not the host, are disrupted has remained mysterious. The opening of the bacterial mechanosensitive channel of large conductance, MscL, which ordinarily serves the physiological role of osmotic emergency release valves gated by hypoosmotic shock, has been previously demonstrated to affect bacterial membrane permeabilization. MscL allows the rapid permeabilization of both ions and solutes through the opening of the largest known gated pore, which has a diameter of 30 Å. We found that SCH-79797 and IRS-16, a more potent derivative, directly bind to the MscL channel and produce membrane permeabilization as a result of its activation. These findings suggest that possessing or adding an MscL-activating component to an antibiotic compound could help to lower toxicity and evade antibiotic resistance.

Pivotal role of the glycine-rich TM3 helix in gating the MscS mechanosensitive channel.

The crystal structure of an open form of the Escherichia coli MscS mechanosensitive channel was recently solved. However, the conformation of the closed state and the gating transition remain uncharacterized. The pore-lining transmembrane helix contains a conserved glycine- and alanine-rich motif that forms a helix-helix interface. We show that introducing 'knobs' on the smooth glycine face by replacing glycine with alanine, and substituting conserved alanines with larger residues, increases the pressure required for gating. Creation of a glycine-glycine interface lowers activation pressure. The importance of residues Gly104, Ala106 and Gly108, which flank the hydrophobic seal, is demonstrated. A new structural model is proposed for the closed-to-open transition that involves rotation and tilt of the pore-lining helices. Introduction of glycine at Ala106 validated this model by acting as a powerful suppressor of defects seen with mutations at Gly104 and Gly108.

Life with Bacterial Mechanosensitive Channels, from Discovery to Physiology to Pharmacological Target.

General principles in biology have often been elucidated from the study of bacteria. This is true for the bacterial mechanosensitive channel of large conductance, MscL, the channel highlighted in this review. This channel functions as a last-ditch emergency release valve discharging cytoplasmic solutes upon decreases in osmotic environment. Opening the largest gated pore, MscL passes molecules up to 30 Å in diameter; exaggerated conformational changes yield advantages for study, including in vivo assays. MscL contains structural/functional themes that recur in higher organisms and help elucidate how other, structurally more complex, channels function. These features of MscL include (i) the ability to directly sense, and respond to, biophysical changes in the membrane, (ii) an α helix ("slide helix") or series of charges ("knot in a rope") at the cytoplasmic membrane boundary to guide transmembrane movements, and (iii) important subunit interfaces that, when disrupted, appear to cause the channel to gate inappropriately. MscL may also have medical applications: the modality of the MscL channel can be changed, suggesting its use as a triggered nanovalve in nanodevices, including those for drug targeting. In addition, recent studies have shown that the antibiotic streptomycin opens MscL and uses it as one of the primary paths to the cytoplasm. Moreover, the recent identification and study of novel specific agonist compounds demonstrate that the channel is a valid drug target. Such compounds may serve as novel-acting antibiotics and adjuvants, a way of permeabilizing the bacterial cell membrane and, thus, increasing the potency of commonly used antibiotics.

Novel MscL agonists that allow multiple antibiotics cytoplasmic access activate the channel through a common binding site.

The antibiotic resistance crisis is becoming dire, yet in the past several years few potential antibiotics or adjuvants with novel modes of action have been identified. The bacterial mechanosensitive channel of large conductance, MscL, found in the majority of bacterial species, including pathogens, normally functions as an emergency release valve, sensing membrane tension upon low-osmotic stress and discharging cytoplasmic solutes before cell lysis. Opening the huge ~30Å diameter pore of MscL inappropriately is detrimental to the cell, allowing solutes from and even passage of drugs into to cytoplasm. Thus, MscL is a potential novel drug target. However, there are no known natural agonists, and small compounds that modulate MscL activity are just now being identified. Here we describe a small compound, K05, that specifically modulates MscL activity and we compare results with those obtained for the recently characterized MscL agonist 011A. While the structure of K05 only vaguely resembles 011A, many of the findings, including the binding pocket, are similar. On the other hand, both in vivo and molecular dynamic simulations indicate that the two compounds modulate MscL activity in significantly different ways.

On the structure of the N-terminal domain of the MscL channel: helical bundle or membrane interface.

The mechanosensitive channel of large conductance, MscL, serves as a biological emergency release valve protecting bacteria from acute osmotic downshock and is to date the best characterized mechanosensitive channel. A well-recognized and supported model for Escherichia coli MscL gating proposes that the N-terminal 11 amino acids of this protein form a bundle of amphipathic helices in the closed state that functionally serves as a cytoplasmic second gate. However, a recently reexamined crystal structure of a closed state of the Mycobacterium tuberculosis MscL shows these helices running along the cytoplasmic surface of the membrane. Thus, it is unclear if one structural model is correct or if they both reflect valid closed states. Here, we have systematically reevaluated this region utilizing cysteine-scanning, in vivo functional characterization, in vivo SCAM, electrophysiological studies, and disulfide-trapping experiments. The disulfide-trapping pattern and functional studies do not support the helical bundle and second-gate hypothesis but correlate well with the proposed structure for M. tuberculosis MscL. We propose a functional model that is consistent with the collective data.

Scanning MscL Channels with Targeted Post-Translational Modifications for Functional Alterations.

Mechanosensitive channels are present in all living organisms and are thought to underlie the senses of touch and hearing as well as various important physiological functions like osmoregulation and vasoregulation. The mechanosensitive channel of large conductance (MscL) from Escherichia coli was the first protein shown to encode mechanosensitive channel activity and serves as a paradigm for how a channel senses and responds to mechanical stimuli. MscL plays a role in osmoprotection in E. coli, acting as an emergency release valve that is activated by membrane tension due to cell swelling after an osmotic down-shock. Using an osmotically fragile strain in an osmotic down-shock assay, channel functionality can be directly determined in vivo. In addition, using thiol reagents and expressed MscL proteins with a single cysteine substitution, we have shown that targeted post-translational modifications can be performed, and that any alterations that lead to dysfunctional proteins can be identified by this in vivo assay. Here, we present the results of such a scan performed on 113 MscL cysteine mutants using five different sulfhydryl-reacting probes to confer different charges or hydrophobicity to each site. We assessed which of these targeted modifications affected channel function and the top candidates were further studied using patch clamp to directly determine how channel activity was affected. This comprehensive screen has identified many residues that are critical for channel function as well as highlighted MscL domains and residues that undergo the most drastic environmental changes upon gating.

A new antibiotic with potent activity targets MscL.

The growing problem of antibiotic-resistant bacteria is a major threat to human health. Paradoxically, new antibiotic discovery is declining, with most of the recently approved antibiotics corresponding to new uses for old antibiotics or structurally similar derivatives of known antibiotics. We used an in silico approach to design a new class of nontoxic antimicrobials for the bacteria-specific mechanosensitive ion channel of large conductance, MscL. One antimicrobial of this class, compound 10, is effective against methicillin-resistant Staphylococcus aureus with no cytotoxicity in human cell lines at the therapeutic concentrations. As predicted from in silico modeling, we show that the mechanism of action of compound 10 is at least partly dependent on interactions with MscL. Moreover we show that compound 10 cured a methicillin-resistant S. aureus infection in the model nematode Caenorhabditis elegans. Our work shows that compound 10, and other drugs that target MscL, are potentially important therapeutics against antibiotic-resistant bacterial infections.

Streptomycin potency is dependent on MscL channel expression.

The antibiotic streptomycin is widely used in the treatment of microbial infections. The primary mechanism of action is inhibition of translation by binding to the ribosome, but how it enters the bacterial cell is unclear. Early in the study of this antibiotic, a mysterious streptomycin-induced potassium efflux preceding any decrease in viability was observed; it was speculated that this changed the electrochemical gradient such that streptomycin better accessed the cytoplasm. Here we use a high-throughput screen to search for compounds targeting the mechanosensitive channel of large conductance (MscL) and find dihydrostreptomycin among the 'hits'. Furthermore, we find that MscL is not only necessary for the previously described streptomycin-induced potassium efflux, but also directly increases MscL activity in electrophysiological studies. The data suggest that gating MscL is a novel mode of action of dihydrostreptomycin, and that MscL's large pore may provide a mechanism for cell entry.

Improving the Design of a MscL-Based Triggered Nanovalve.

The mechanosensitive channel of large conductance, MscL, has been proposed as a triggered nanovalve to be used in drug release and other nanodevices. It is a small homopentameric bacterial protein that has the largest gated pore known: greater than 30 Å. Large molecules, even small proteins can be released through MscL. Although MscL normally gates in response to membrane tension, early studies found that hydrophilic or charged residue substitutions near the constriction of the channel leads to pore opening. Researchers have successfully changed the modality of MscL to open to stimuli such as light by chemically modifying a single residue, G22, within the MscL pore. Here, by utilizing in vivo, liposome efflux, and patch clamp assays we compared modification of G22 with that of another neighboring residue, G26, and demonstrate that modifying G26 may be a better choice for triggered nanovalves used for triggered vesicular release of compounds.

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 oligomeric state of the truncated mechanosensitive channel of large conductance shows no variance in vivo.

The mechanosensitive channel of large conductance (MscL) from E. coli serves as an emergency release valve allowing the cell to survive acute osmotic downshock. It is one of the best studied mechanosensitive channels and serves as a paradigm for how a protein can sense and respond to membrane tension. Two MscL crystal structures of the orthologs M. tuberculosis and S. aureus have been solved showing pentameric and tetrameric structures, respectively. Several studies followed to understand whether the discrepancy in their stoichiometry was a species difference or a consequence of the protein manipulation for crystallization. Two independent studies now agree that the full-length S. aureus MscL is actually a pentamer, not tetramer. While detergents appear to play a role in modifying the oligomeric state of the protein, a cytoplasmic helical bundle has also been implicated. Here, we evaluate the role of the C-terminal region of S. aureus MscL in the oligomerization of the channel in native membranes by using an in vivo disulfide-trapping technique. We find that the oligomeric state of S. aureus MscLs with different C-terminal truncations, including the one used to obtain the tetrameric S. aureus MscL crystal structure, are pentamers in vivo. Thus, the C-terminal domain of the S. aureus protein only plays a critical role in the oligomeric state of the SaMscL protein when it is solubilized in detergent.

Disulfide trapping the mechanosensitive channel MscL into a gating-transition state.

The mechanosensitive channel of large conductance, MscL, serves as a biological emergency release valve protecting bacteria from acute osmotic downshock, and is to date the best characterized mechanosensitive channel. The N-terminal region of the protein has been shown to be critical for function by random, site-directed, and deletion mutagenesis, yet is structurally poorly understood. One model proposes that the extreme N-termini form a cluster of amphipathic helices that serves as a cytoplasmic second gate, separated from the pore-forming transmembrane domain by a "linker". Here, we have utilized cysteine trapping of single-cysteine mutated channels to determine the proximity, within the homopentameric complex, of residues within and just peripheral to this proposed linker. Our results indicate that all residues in this region can form disulfide bridges, and that the percentage of dimers increases when the channel is gated in vivo. Functional studies suggest that oxidation traps one of these mutated channels, N15C, into a gating-transition state that retains the capacity to obtain both fully open and closed states. The data are not easily explained by current models for the smooth transition from closed-to-open states, but predict that an asymmetric movement of one or more of the subunits commonly occurs upon gating.