On the osmotic signal and osmosensing mechanism of an ABC transport system for glycine betaine.

The osmosensing mechanism of the ATP-binding cassette (ABC) transporter OpuA of Lactococcus lactis has been elucidated for the protein reconstituted in liposomes. Activation of OpuA by osmotic upshift was instantaneous and reversible and followed changes in volume and membrane structure of the proteoliposomes. Osmotic activation of OpuA was dependent on the fraction of anionic lipids present in the lipid bilayer. Also, cationic and anionic lipophilic amphiphiles shifted the activation profile in a manner indicative of an osmosensing mechanism, in which electrostatic interactions between lipid headgroups and the OpuA protein play a major role. Further support for this notion came from experiments in which ATP-driven uptake and substrate-dependent ATP hydrolysis were measured with varying concentrations of osmolytes at the cytoplasmic face of the protein. Under iso-osmotic conditions, the transporter could be activated by high concentrations of ionic osmolytes, whereas neutral ones had no effect, demonstrating that intracellular ionic strength, rather than a specific signaling molecule or water activity, signals osmotic stress to the transporter. The data indicate that OpuA is under the control of a mechanism in which the membrane and ionic strength act in concert to signal osmotic changes.     

BetP of Corynebacterium glutamicum, a transporter with three different functions: betaine transport, osmosensing, and osmoregulation

In order to circumvent deleterious effects of hypo- and hyperosmotic conditions in its environment, Corynebacterium glutamicum has developed a number of mechanisms to counteract osmotic stress. The first response to an osmotic upshift is the activation of uptake mechanisms for the compatible solutes betaine, proline, or ectoine, namely BetP, EctP, ProP, LcoP and PutP. BetP, the most important uptake system responds to osmotic stress by regulation at the level of both protein activity and gene expression. BetP was shown to harbor three different properties, i.e. catalytic activity (betaine transport), sensing of appropriate stimuli (osmosensing) and signal transduction to the catalytic part of the carrier protein which adapts its activity to the extent of osmotic stress (osmoregulation). BetP is comprised of 12 transmembrane segments and carries N- and C-terminal domains, which are involved in osmosensing and/or osmoregulation. Recent results on molecular properties of these domains indicate the significance of particular amino acids within the terminal 25 amino acids of the C-terminal domain of BetP for the process of osmosensing and osmoregulation.     

Creation of a fully functional cysteine-less variant of osmosensor and proton-osmoprotectant symporter ProP from Escherichia coli and its application to assess the transporter's membrane orientation

Transporter ProP of Escherichia coli is an osmosensor and an osmoprotectant transporter. Previous results suggest that medium osmolality determines the proportions of ProP in active and inactive conformations. A cysteine-less (Cys-less) variant was created and characterized as a basis for structural and functional analyses based on site-directed Cys substitution and chemical labeling of ProP. Parameters describing the osmosensory and osmoprotectant transport activities of Cys-less ProP-(His)(6) variants were examined, including the threshold for osmotic activation and the absolute transporter activity at high osmolality (in both cells and proteoliposomes), the dependence of K(M) and V(max) for proline uptake on osmolality, and the rate constant for transporter activation in response to an osmotic upshift (in cells only). Variant ProP-(His)(6)-C112A-C133A-C264V-C367A (designated ProP) retained similar activities to ProP-(His)(6) in both cells and proteoliposomes. The bulky Val residue was favored over Ala or Ser at position 264, whereas Val strongly impaired function when placed at position 367, highlighting the importance of residues at those positions for osmosensing. In the ProP* background, variants with a single Cys residue at positions 112, 133, 241, 264, 293, or 367 retained full function. The native Cys at positions 112, 133, 264, and 367, predicted to be within transmembrane segments of ProP, were poorly reactive with membrane-impermeant thiol reagents. The reactivities of Cys at positions 241 and 293 were consistent with exposure of those residues on the cytoplasmic and periplasmic surfaces of the cytoplasmic membrane, respectively. These observations are consistent with the topology and orientation of ProP predicted by hydropathy analysis.     

A structural model for the osmosensor, transporter, and osmoregulator ProP of Escherichia coli

Transporter ProP of Escherichia coli, a member of the major facilitator superfamily (MFS), acts as an osmosensor and an osmoregulator in cells and after purification and reconstitution in proteoliposomes. H(+)-osmoprotectant symport via ProP is activated when medium osmolality is elevated with membrane impermeant osmolytes. The three-dimensional structure of ProP was modeled with the crystal structure of MFS member GlpT as a template. This GlpT structure represents the inward (or cytoplasm)-facing conformation predicted by the alternating access model for transport. LacZ-PhoA fusion analysis and site-directed fluorescence labeling substantiated the membrane topology and orientation predicted by this model and most hydropathy analyses. The model predicts the presence of a proton pathway within the N-terminal six-helix bundle of ProP (as opposed to the corresponding pathway found within the C-terminal helix bundle of its paralogue, LacY). Replacement of residues within the N-terminal helix bundle impaired the osmotic activation of ProP, providing the first indication that residues outside the C-terminal domain are involved in osmosensing. Some residues that were accessible from the periplasmic side, as predicted by the structural model, were more susceptible to covalent labeling in permeabilized membrane fractions than in intact bacteria. These residues may be accessible from the cytoplasmic side in structures not represented by our current model, or their limited exposure in vivo may reflect constraints on transporter structure that are related to its osmosensory mechanism.     

Requirements for osmosensing and osmotic activation of transporter ProP from Escherichia coli

Transporter ProP of Escherichia coli, a solute-H+ symporter, can sense and respond to osmotic upshifts imposed on cells, on membrane vesicles, or on proteoliposomes that incorporate purified ProP-(His)6. In this study, proline uptake catalyzed by ProP was used as a measure of its osmotic activation, and the requirements for osmosensing were defined using the proteoliposome system. The initial rate of proline uptake increased with decreasing external pH and increasing DeltaPsi, lumen negative. Osmotic upshifts increased DeltaPsi by concentrating lumenal K+, but osmotic activation of ProP could be distinguished from this effect. Osmotic activation of ProP resulted from changes in Vmax, though osmotic shifts also increased the KM for proline. Osmotic activation could be described as a reversible, osmotic upshift-dependent transition linking (at least) two transporter protein conformations. No correlation was observed between ProP activation and the position of the anions of activating sodium salts within the Hofmeister series of solutes. Both the magnitude of the osmotic upshift required to activate ProP and the ProP activity attained were similar for membrane-impermeant osmolytes, including NaCl, glucose, and PEG 600. The membrane-permeant osmolytes glycerol, urea, PEG 62, and PEG 106 failed to activate ProP. Two poly(ethylene glycol)s, PEG 150 and PEG 200, were membrane-permeant and did not cause liposome shrinkage, but they did partially activate ProP-(His)6.     

Regulatory properties and interaction of the C- and N-terminal domains of BetP, an osmoregulated betaine transporter from Corynebacterium glutamicum

The glycine betaine carrier BetP from Corynebacterium glutamicum responds to changes in external osmolality by regulation of its transport activity, and the C-terminal domain was previously identified to be involved in this process. Here we investigate the structural requirements of the C-terminal domain for osmoregulation as well as interacting domains that are relevant for intramolecular signal transduction in response to osmotic stress. For this purpose, we applied a proline scanning approach and amino acid replacements other than proline in selected positions. To analyze the impact of the surrounding membrane, BetP mutants were studied in both C. glutamicum and Escherichia coli, which strongly differ in their phospholipid composition. A region of approximately 25 amino acid residues within the C-terminal domain with a high propensity for alpha-helical structure was found to be essential in terms of its conformational properties for osmodependent regulation. The size of this region was larger in E. coli membranes than in the highly negatively charged C. glutamicum membranes. As a novel aspect of BetP regulation, interaction of the C-terminal domain with one of the cytoplasmic loops as well as with the N-terminal domain was shown to be involved in osmosensing and/or osmoregulation. These results support a functional model of BetP activation that involves the C-terminal domain shifting from interaction with the membrane to interaction with intramolecular domains in response to osmotic stress.     

Osmosensing and osmoregulatory compatible solute accumulation by bacteria

Bacteria inhabit natural and artificial environments with diverse and fluctuating osmolalities, salinities and temperatures. Many maintain cytoplasmic hydration, growth and survival most effectively by accumulating kosmotropic organic solutes (compatible solutes) when medium osmolality is high or temperature is low (above freezing). They release these solutes into their environment when the medium osmolality drops. Solutes accumulate either by synthesis or by transport from the extracellular medium. Responses to growth in high osmolality medium, including biosynthetic accumulation of trehalose, also protect Salmonella typhimurium from heat shock. Osmotically regulated transporters and mechanosensitive channels modulate cytoplasmic solute levels in Bacillus subtilis, Corynebacterium glutamicum, Escherichia coli, Lactobacillus plantarum, Lactococcus lactis, Listeria monocytogenes and Salmonella typhimurium. Each organism harbours multiple osmoregulatory transporters with overlapping substrate specificities. Membrane proteins that can act as both osmosensors and osmoregulatory transporters have been identified (secondary transporters ProP of E. coli and BetP of C. glutamicum as well as ABC transporter OpuA of L. lactis). The molecular bases for the modulation of gene expression and transport activity by temperature and medium osmolality are under intensive investigation with emphasis on the role of the membrane as an antenna for osmo- and/or thermosensors.     

Cardiolipin controls the osmotic stress response and the subcellular location of transporter ProP in Escherichia coli

The phospholipid composition of the membrane and transporter structure control the subcellular location and function of osmosensory transporter ProP in Escherichia coli. Growth in media of increasing osmolality increases, and entry to stationary phase decreases, the proportion of phosphatidate in anionic lipids (phosphatidylglycerol (PG) plus cardiolipin (CL)). Both treatments increase the CL:PG ratio. Transporters ProP and LacY are concentrated with CL (and not PG) near cell poles and septa. The polar concentration of ProP is CL-dependent. Here we show that the polar concentration of LacY is CL-independent. The osmotic activation threshold of ProP was directly proportional to the CL content of wild type bacteria, the PG content of CL-deficient bacteria, and the anionic lipid content of cells and proteoliposomes. CL was effective at a lower concentration in cells than in proteoliposomes, and at a much lower concentration than PG in either system. Thus, in wild type bacteria, osmotic induction of CL synthesis and concentration of ProP with CL at the cell poles adjust the osmotic activation threshold of ProP to match ambient conditions. ProP proteins linked by homodimeric, C-terminal coiled-coils are known to activate at lower osmolalities than those without such structures and coiled-coil disrupting mutations raise the osmotic activation threshold. Here we show that these mutations also prevent polar concentration of ProP. Stabilization of the C-terminal coiled-coil by covalent cross-linking of introduced Cys reverses the impact of increasing CL on the osmotic activation of ProP. Association of ProP C termini with the CL-rich membrane at cell poles may raise the osmotic activation threshold by blocking coiled-coil formation. Mutations that block coiled-coil formation may also block association of the C termini with the CL-rich membrane.     

Structure and function of transmembrane segment XII in osmosensor and osmoprotectant transporter ProP of Escherichia coli

Escherichia coli transporter ProP acts as both an osmosensor and an osmoregulator. As medium osmolality rises, ProP is activated and mediates H+-coupled uptake of osmolytes like proline. A homology model of ProP with 12-transmembrane (TM) helices and cytoplasmic termini was created, and the protein's topology was substantiated experimentally. Residues 468-497, at the end of the C-terminal domain and linked to TM XII, form an intermolecular, homodimeric alpha-helical coiled-coil that tunes the transporter's response to osmolality. We aim to further define the structure and function of ProP residues Q415-E440, predicted to include TM XII. Each residue was replaced with cysteine (Cys) in a histidine-tagged, Cys-less ProP variant (ProP*). Cys at positions 415-418 and 438-440 were most reactive with Oregon Green Maleimide (OGM), suggesting that residues 419 through 437 are in the membrane. Except for V429-I433, reactivity of those Cys varied with helical periodicity. Cys predicted to face the interior of ProP were more reactive than Cys predicted to face the lipid. The former may be exposed to hydrated polar residues in the protein interior, particularly on the periplasmic side. Intermolecular cross-links formed when ProP* variants with Cys at positions 419, 420, 422, and 439 were treated with DTME. Thus TM XII can participate, along its entire length, in the dimer interface of ProP. Cys substitution E440C rendered ProP* inactive. All other variants retained more than 30% of the proline uptake activity of ProP* at high osmolality. Most variants with Cys substitutions in the periplasmic half of TM XII activated at lower osmolalities than ProP*. Variants with Cys substitutions on one face of the cytoplasmic half of TM XII required a higher osmolality to activate. They included elements of a GXXXG motif that are predicted to form the interface of TM XII with TM VII. These studies define the position of ProP TM XII within the membrane, further support the predicted structure of ProP, reveal the dimerization interface, and show that the structure of TM XII influences the osmolality at which ProP activates.     

The osmotic activation of transporter ProP is tuned by both its C-terminal coiled-coil and osmotically induced changes in phospholipid composition

Transporter ProP of Escherichia coli (ProPEc) senses extracellular osmolality and mediates osmoprotectant uptake when it is rising or high. A replica of the ProPEc C terminus (Asp468-Arg497) forms an intermolecular alpha-helical coiled-coil. This structure is implicated in the osmoregulation of intact ProPEc, in vivo. Like that from Corynebacterium glutamicum (ProPCg), the ProP orthologue from Agrobacterium tumefaciens (ProPAt) sensed and responded to extracellular osmolality after expression in E. coli. The osmotic activation profiles of all three orthologues depended on the osmolality of the bacterial growth medium, the osmolality required for activation rising as the growth osmolality approached 0.7 mol/kg. Thus, each could undergo osmotic adaptation. The proportion of cardiolipin in a polar lipid extract from E. coli increased with extracellular osmolality so that the osmolality activating ProPEc was a direct function of membrane cardiolipin content. Group A ProP orthologues (ProPEc, ProPAt) share the C-terminal coiled-coil domain and were activated at low osmolalities. Like variant ProPEc-R488I, in which the C-terminal coiled-coil is disrupted, ProPEc derivatives that lack the coiled-coil and Group B orthologue ProPCg required a higher osmolality to activate. The amplitude of ProPEc activation was reduced 10-fold in its deletion derivatives. The coiled-coil structure is not essential for osmotic activation of ProP per se. However, it tunes Group A orthologues to osmoregulate over a low osmolality range. Coiled-coil lesions may impair both coiled-coil formation and interaction of ProPEc with amplifier protein ProQ. Cardiolipin may contribute to ProP adaptation by altering bulk membrane properties or by acting as a ProP ligand.     

Impacts of the Osmolality and the Lumenal Ionic Strength on Osmosensory Transporter ProP in Proteoliposomes

H(+) symporter ProP serves as a paradigm for the study of osmosensing. ProP attains the same activity at the same osmolality when the medium outside cells or proteoliposomes is supplemented with diverse, membrane-impermeant solutes. The osmosensory mechanism of ProP has been probed by varying the solvent within membrane vesicles and proteoliposomes. ProP activation was not ion specific, did not require K(+), and could be elicited by large, uncharged solutes polyethylene glycols (PEGS). We hypothesized that ProP is an ionic strength sensor and lumenal macromolecules activate ProP by altering ion activities. The attainable range of lumenal ionic strength was expanded by lowering the phosphate concentration within proteoliposomes. ProP activity at high osmolality, but not the osmolality, yielding half-maximal activity (Π(½)/RT), decreased with the lumenal phosphate concentration. This was attributed to acidification of the proteoliposome lumen due to H(+)-proline symport. The ionic strength yielding half-maximal ProP activity was more anion-dependent than Π(½)/RT for proteoliposomes loaded with citrate, sulfate, phosphate, chloride, or iodide. The anion effects followed the Hofmeister series. Lumenal bovine serum albumin (BSA) lowered the lumenal ionic strength at which ProP became active. Osmolality measurements documented the non-idealities of solutions including potassium phosphate and other solutes. The impacts of PEGS and BSA on ion activities did not account for their impacts on ProP activity. The effects of the tested solutes on ProP appear to be non-coulombic in nature. They may arise from effects of preferential interactions and macromolecular crowding on the membrane or on ProP.     

Osmosensor ProP of Escherichia coli responds to the concentration,chemistry, and molecular size of osmolytes in the proteoliposome lumen

Transporter ProP of Escherichia coli mediates the cellular accumulation of organic zwitterions in response to increased extracellular osmolality. We compared and characterized the osmoregulation of ProP activity in cells and proteoliposomes to define the osmotic shift-induced cellular change(s) to which ProP responds. ProP-(His)(6) activity in cells and proteoliposomes was correlated with medium osmolality, not osmotic shift, turgor pressure, or membrane strain. Both K(M) and V(max) for proline uptake via ProP-(His)(6) increased with increasing medium osmolality, as would be expected if osmolality controls the proportions of transporter with inactive and active conformations. The osmolality yielding half-maximal ProP-(His)(6) activity was higher in proteoliposomes than in cells. The osmolality response of ProP is also attenuated in bacteria lacking soluble protein ProQ. Indeed, the catalytic constant (k(cat)) for ProP-(His)(6) in proteoliposomes approximated that of ProP in intact bacteria lacking ProQ. Thus, the proteoliposome system may replicate a primary osmosensory response that can be further amplified by ProQ. ProP-(His)(6) is designated as an osmosensor because its activity is dependent on the osmolality, but not the composition, of the assay medium to which the cell surface is exposed. In contrast, ProP-(His)(6) activity was dependent on both the osmolality and the composition of the lumen in osmolyte-loaded proteoliposomes. For proteoliposomes containing inorganic salts, glucose, or poly(ethylene glycol) 503, transporter activity correlated with total lumenal cation concentration. In contrast, for proteoliposomes loaded with larger poly(ethylene glycol)s, the osmolality, the lumenal cation concentration, and the lumenal ionic strength at half-maximal transporter activity decreased systematically with poly(ethylene glycol) radius of gyration (range 0.8-1.8 nm). These data suggest that ProP-(His)(6) responds to osmotically induced changes in both cytoplasmic K(+) levels and the concentration of cytoplasmic macromolecules.     

Formation of an antiparallel, intermolecular coiled coil is associated with in vivo dimerization of osmosensor and osmoprotectant transporter ProP in Escherichia coli

Membrane transporter ProP from Escherichia coli senses extracellular osmolality and responds by mediating the uptake of osmoprotectants such as glycine betaine when osmolality is high. Earlier EPR and NMR studies showed that a peptide replica of the cytoplasmic ProP carboxyl terminus (residues D468-R497) forms a homodimeric, antiparallel, alpha-helical coiled coil in vitro stabilized by electrostatic interactions involving R488. Amino acid replacement R488I disrupted coiled-coil formation by the ProP peptide, elevated the osmolality at which ProP became active, and rendered the osmolality response of ProP transient. In the present study, either E480 or K473 was replaced with cysteine (Cys) in ProP, a Cys-less, fully functional, histidine-tagged ProP variant, to use Cys-specific cross-linking approaches to determine if antiparallel coiled-coil formation and dimerization of the intact protein occur in vivo. The Cys at positions 480 would be closer in an antiparallel dimer than those at positions 473. These replacements did not disrupt coiled-coil formation by the ProP peptide. Partial homodimerization of variant ProP-E480C could be demonstrated in vivo and in membrane preparations via Cys-specific cross-linking with dithiobis(maleimidoethane) or by Cys oxidation to cystine by copper phenanthroline. In contrast, these reagents did not cross-link ProP with Cys at position 133 or 241. Cross-linking of ProP with Cys at position 473 was limited and occurred only if ProP was overexpressed, consistent with an antiparallel orientation of the coiled coil in the intact protein in vivo. Although replacement E480C did not alter transporter activity, replacement K473C reduced the extent and elevated the threshold for osmotic activation. K473 may play a role in ProP structure and function that is not reflected in altered coiled-coil formation by the corresponding peptide. Substitution R488I affected the activities of ProP-(His)(6), ProP-E480C, and ProP-K473C as it affected the activity of ProP. Surprisingly, it did not eliminate cross-linking of Cys at position 480, and it elevated cross-linking at position 473, even when ProP was expressed at physiological levels. This suggested that the R488I substitution may have changed the relative orientation of the C-termini within the dimeric protein from antiparallel to parallel, resulting in only transient osmotic activation. These results suggest that ProP is in monomer-dimer equilibrium in vivo. Dimerization may be mediated by C-terminal coiled-coil formation and/or by interactions between other structural domains, which in turn facilitate C-terminal coiled-coil formation. Antiparallel coiled-coil formation is required for activation of ProP at low osmolality.     

Solution structure of the C-terminal antiparallel coiled-coil domain from Escherichia coli osmosensor ProP

Bacteria respond to increasing medium osmolality by accumulating organic solutes that are compatible with cellular functions. Transporter ProP of Escherichia coli, a proton symporter and a member of the major facilitator superfamily, senses osmotic shifts and responds by importing osmolytes such as glycine betaine. ProP contains a cytoplasmic, C-terminal extension that is essential for its activity. A peptide corresponding to the C-terminal extension of ProP forms a homodimeric alpha-helical coiled-coil even though some of its heptad a positions are not occupied by hydrophobic amino acid residues. Unexpectedly, amino acid replacement R488I, occurring at a heptad a position, destabilized the coiled-coil formed by the ProP peptide and attenuated the response of the intact transporter to osmotic upshifts in vivo. Thus, ProP was proposed to dimerize via an antiparallel coiled-coil. We used nuclear magnetic resonance (NMR) spectroscopy to determine the structure of the synthetic peptide corresponding to residues 468-497 of ProP. This region did form an antiparallel coil-coil in which critical residue R488 specifies the antiparallel coiled-coil orientation by forming stabilizing salt-bridges. Charged residues (both acidic and basic) are clustered on the c/g surface of the coiled-coil whereas polar residues are distributed on the b/e surface. This causes the structure to be bent, in contrast to other known antiparallel coiled-coils (those from the hepatitis delta antigen (PDB ID code 1A92) and the bovine F(1) ATPase inhibitor protein (PDB ID code 1HF9)). The coiled-coil and its possible importance for osmosensing are discussed.     

Osmosensor and osmoregulator properties of the betaine carrier BetP from Corynebacterium glutamicum in proteoliposomes

The secondary glycine betaine uptake system BetP of Corynebacterium glutamicum was purified from Escherichia coli membranes in strep-tagged form after heterologous expression of the betP gene and was reconstituted in E. coli lipids. BetP retained its kinetic properties (V(max) and K(m) for betaine and Na(+)) as compared with intact cells. The influence of driving forces (Na(+) gradient and/or electrical potential) on betaine uptake was quantified in proteoliposomes. BetP was effectively regulated by the external osmolality and was stimulated by the local anesthetic tetracaine. A shift of the optimum of osmotic stimulation to higher osmolalities was linearly correlated with an increasing share of phosphatidyl glycerol, the major lipid of the C. glutamicum plasma membrane in the E. coli lipid proteoliposomes. This finding correlates with results demonstrating an identical shift when betP was expressed in E. coli instead of C. glutamicum. These data indicate that (i) BetP comprises all elements of osmosensing and osmoregulatory mechanisms of betaine uptake, (ii) osmoregulation of BetP is directly related to protein/membrane interactions, (iii) the turgor pressure presumably plays no major role in osmoregulation of BetP, and (iv) the regulatory properties of BetP may be related to the physical state of the surrounding membrane.     

How do membrane proteins sense water stress?

Maintenance of cell turgor is a prerequisite for almost any form of life as it provides a mechanical force for the expansion of the cell envelope. As changes in extracellular osmolality will have similar physicochemical effects on cells from all biological kingdoms, the responses to osmotic stress may be alike in all organisms. The primary response of bacteria to osmotic upshifts involves the activation of transporters, to effect the rapid accumulation of osmoprotectants, and sensor kinases, to increase the transport and/or biosynthetic capacity for these solutes. Upon osmotic downshift, the excess of cytoplasmic solutes is released via mechanosensitive channel proteins. A number of breakthroughs in the last one or two years have led to tremendous advances in our understanding of the molecular mechanisms of osmosensing in bacteria. The possible mechanisms of osmosensing, and the actual evidence for a particular mechanism, are presented for well studied, osmoregulated transport systems, sensor kinases and mechanosensitive channel proteins. The emerging picture is that intracellular ionic solutes (or ionic strength) serve as a signal for the activation of the upshift-activated transporters and sensor kinases. For at least one system, there is strong evidence that the signal is transduced to the protein complex via alterations in the protein-lipid interactions rather than direct sensing of ion concentration or ionic strength by the proteins. The osmotic downshift-activated mechanosensitive channels, on the other hand, sense tension in the membrane but other factors such as hydration state of the protein may affect the equilibrium between open and closed states of the proteins.     

Engineering of Ion Sensing by the Cystathionine ��-Synthase Module of the ABC Transporter OpuA

We have previously shown that the C-terminal cystathionine β-synthase (CBS) domains of the nucleotide-binding domains of the ABC transporter OpuA, in conjunction with an anionic membrane surface function, act as sensor of internal ionic strength (I_in). Here, we show that a surface-exposed cationic region in the CBS module domain is critical for ion sensing. The consecutive substitution of up to five cationic residues led to a gradual decrease of the ionic strength dependence of transport. In fact, a 5-fold mutant was essentially independent of salt in the range from 0 to 250 mm KCl (or NaCl), supplemented to medium of 30 mm potassium phosphate. Importantly, the threshold temperature for transport was lowered by 5–7 °C and the temperature coefficient Q₁₀ was lowered from 8 to ∼1.5 in the 5-fold mutant, indicating that large conformational changes are accompanying the CBS-mediated regulation of transport. Furthermore, by replacing the anionic C-terminal tail residues that extend the CBS module with histidines, the transport of OpuA became pH-dependent, presumably by additional charge interactions of the histidine residues with the membrane. The pH dependence was not observed at high ionic strength. Altogether the analyses of the CBS mutants support the notion that the osmotic regulation of OpuA involves a simple biophysical switching mechanism, in which nonspecific electrostatic interactions of a protein module with the membrane are sufficient to lock the transporter in the inactive state.In their natural habitats microorganisms are often exposed to changes in the concentration of solutes in the environment (1). A sudden increase in the medium osmolality results in loss of water from the cell, loss of turgor, a decrease in cell volume, and an increase in intracellular osmolyte concentration. Osmoregulatory transporters such as OpuA in Lactococcus lactis, ProP in Escherichia coli, and BetP in Corynebacterium glutamicum diminish the consequences of the osmotic stress by mediating the uptake of compatible solutes upon an increase in extracellular osmolality (2–4). For the ATP-binding cassette (ABC)⁵ transporter OpuA, it has been shown that the system, reconstituted in proteoliposomes, is activated by increased concentrations of lumenal ions (increased internal ionic strength) (2, 5, 6). This activation is instantaneous both in vivo and in vitro and only requires threshold levels of ionic osmolytes. Moreover, the ionic threshold for activation is highly dependent of the ionic lipid content (charge density) of the membrane and requires the presence of so-called cystathionine β-synthase (CBS) domains, suggesting that the ionic signal is transduced to the transporter via critical interactions of the protein with membrane lipids.The ABC transporter OpuA consists of two identical nucleotide-binding domains (NBD) fused to CBS domains and two identical substrate-binding domains fused to transmembrane domains. The NBD-CBS and substrate-binding domain-transmembrane domain subunits are named OpuAA and OpuABC, respectively. Two tandem CBS domains are linked to the C-terminal end of the NBD; each domain (CBS1 and CBS2) has a β-α-β-β-α secondary structure (5) (Fig. 1A). The CBS domains are widely distributed in most if not all species of life but their function is largely unknown. Most of the CBS domains are found as tandem repeats but data base searches have also revealed tetra-repeat units (5). The crystal structures of several tandem CBS domains have been elucidated (7–9, 32), and in a number of cases it has been shown that two tandem CBS domains form dimeric structures with a total of four CBS domains per structural module (hereafter referred to as CBS module). The crystal structures of the full-length MgtE Mg²⁺ transporter confirm the dimeric configuration and show that the CBS domains undergo large conformational changes upon Mg²⁺ binding or release (10, 11). In general, ABC transporters are functional as dimers, which implies that two tandem CBS domains are present in the OpuA complex. Preliminary experiments with disulfides engineered at the interface of two tandem CBS domains in OpuA suggest that large structural rearrangements (association-dissociation of the interfaces) play a determining role in the ionic strength-regulated transport. Finally, a subset of CBS-containing proteins has a C-terminal extension, which in OpuA is highly anionic (sequence: ADIPDEDEVEEIEKEEENK) and modulates the ion sensing activity (6).Open in a separate windowFIGURE 1.Domain structure of CBS module of OpuA. A, sequence of tandem CBS domains. The predicted secondary structure is indicated above the sequence. The residues modified in this study are underlined. The amino acid sequence end-points of OpuAΔ61 and OpuAΔ119 are indicated by vertical arrows. B, homology model of tandem CBS domain of OpuA. The CBS domains were individually modeled on the crystal structure of the tandem CBS protein Ta0289 from T. acidophilum (PDB entry 1PVM), using Phyre. Ta0289 was used for the initial modeling, because its primary sequence was more similar to the CBS domains of OpuA than those of the other crystallized CBS proteins. The individual domain models were then assembled with reference to the atomic coordinates of the tandem CBS domains of IMPDH from Streptococcus pyogenes (PDB entry 1ZFJ) to form the tandem CBS pair, using PyMOL (DeLano). The positions of the (substituted) cationic residues are indicated.In this study, we have engineered the surface-exposed cationic residues of the CBS module and the C-terminal anionic tail of OpuA (Fig. 1B). The ionic strength and lipid dependence of the OpuA mutants were determined in vivo and in vitro. We show that substitution of five cationic residues for neutral amino acids is sufficient to inactivate the ionic strength sensor and convert OpuA into a constitutively active transporter. Moreover, by substituting six anionic plus four neutral residues of the C-terminal anionic tail for histidines, the transport reaction becomes strongly pH-dependent.     

Ion specificity and ionic strength dependence of the osmoregulatory ABC transporter OpuA

The ATPase subunit of the osmoregulatory ATP-binding cassette transporter OpuA from Lactococcus lactis has a C-terminal extension, the tandem cystathionine beta-synthase (CBS) domain, which constitutes the sensor that allows the transporter to sense and respond to osmotic stress (Biemans-Oldehinkel, E., Mahmood, N. A. B. N., and Poolman, B. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 10624-10629). C-terminal of the tandem CBS domain is an 18-residue anionic tail (DIPDEDEVEEIEKEEENK). To investigate the ion specificity of the full transporter, we probed the activity of inside-out reconstituted wild-type OpuA and the anionic tail deletion mutant OpuADelta12; these molecules have the tandem CBS domains facing the external medium. At a mole fraction of 40% of anionic lipids in the membrane, the threshold ionic strength for activation of OpuA was approximately 0.15, irrespective of the electrolyte composition of the medium. At equivalent concentrations, bivalent cations (Mg(2+) and Ba(2+)) were more effective in activating OpuA than NH(4)(+), K(+), Na(+), or Li(+), consistent with an ionic strength-based sensing mechanism. Surprisingly, Rb(+) and Cs(+) were potent inhibitors of wild-type OpuA, and 0.1 mM RbCl was sufficient to completely inhibit the transporter even in the presence of 0.2 M KCl. Rb(+) and Cs(+) were no longer inhibitory in OpuADelta12, indicating that the anionic C-terminal tail participates in the formation of a binding site for large alkali metal ions. Compared with OpuADelta12, wild-type OpuA required substantially less potassium ions (the dominant ion under physiological conditions) for activation. Our data lend new support for the contention that the CBS module in OpuA constitutes the ionic strength sensor whose activity is modulated by the C-terminal anionic tail.