The Multidrug Transporter LmrP Protein Mediates Selective Calcium Efflux

Background: The multidrug transporter LmrP confers drug resistance on cells by mediating efflux of structurally dissimilar cytotoxic substrates.

Results: Surprisingly, LmrP catalyzes the selective, high affinity binding and extrusion of Ca²⁺, which inhibits multidrug transport by LmrP.

Conclusion: LmrP can act as a calcium/proton antiporter.

Significance: Multidrug transporters might fulfill additional physiological roles, which could promote their persistence in the absence of antibiotics.

Abstract

LmrP is a major facilitator superfamily multidrug transporter from Lactococcus lactis that mediates the efflux of cationic amphiphilic substrates from the cell in a proton-motive force-dependent fashion. Interestingly, motif searches and docking studies suggested the presence of a putative Ca²⁺-binding site close to the interface between the two halves of inward facing LmrP. Binding experiments with radioactive ⁴⁵Ca²⁺ demonstrated the presence of a high affinity Ca²⁺-binding site in purified LmrP, with an apparent K_d of 7.2 μm, which is selective for Ca²⁺ and Ba²⁺ but not for Mn²⁺, Mg²⁺, or Co²⁺. Consistent with our structure model and analogous to crystal structures of EF hand Ca²⁺-binding proteins, two carboxylates (Asp-235 and Glu-327) were found to be critical for ⁴⁵Ca²⁺ binding. Using ⁴⁵Ca²⁺ and a fluorescent Ca²⁺-selective probe, calcium transport measurements in intact cells, inside-out membrane vesicles, and proteoliposomes containing functionally reconstituted purified protein provided strong evidence for active efflux of Ca²⁺ by LmrP with an apparent K_t of 8.6 μm via electrogenic exchange with three or more protons. These observations demonstrate for the first time that LmrP mediates selective calcium/proton antiport and raise interesting questions about the functional and physiological links between this reaction and that of multidrug transport.

Introduction

Multidrug transporters are fascinating proteins that mediate the extrusion of structurally dissimilar chemotherapeutic agents away from their targets in the cell. The expression of multidrug exporters can significantly contribute to the development of drug resistance among (pathogenic) microorganisms. LmrP is a well studied member of the major facilitator superfamily that can transport a wide range of amphiphilic cationic drugs from Lactoccocus lactis (1). Previous work indicates that LmrP exports monovalent cationic ethidium by electrogenic exchange with protons (2) in a reaction that is dependent on the transmembrane H⁺ gradient (ΔpH) and membrane potential (Δψ) components of the proton-motive force (Δp).

To further investigate this exchange reaction, an inward facing three-dimensional homology model of LmrP was constructed that was based on the crystal structure of the glycerol-3P/P_i antiporter GlpT from Escherichia coli (3). In this model, LmrP is predicted to contain an internal cavity formed at the interface between the two halves of the transporter. On the surface of this cavity lie two clusters of polar, aromatic, and carboxyl residues with potentially important functions in proton shuttling and substrate interactions (3). Cluster 1 in the C-terminal half contains Asp-235 and Glu-327 in immediate proximity (<3.5 Å) of each other and is located near the apex of the cavity, whereas Cluster 2 in the N-terminal half contains Asp-142. Mutational analyses of these carboxylates suggested that both clusters act as separate proton conduction points (3) by a mechanism in which the carboxylates are protonated in the outward facing conformation and deprotonated in the inward facing conformation.

Recent studies on the energetics of ethidium⁺ and propidium²⁺ transport by LmrP point to a variable proton-substrate stoichiometry, which is thought to be related to substrate-dependent changes in the geometry and distance between Asp-235 and Glu-327 in the inward facing substrate-binding chamber (4). During transport of ethidium, binding of this substrate from the inside surface would decrease the proximity between the side chains of Glu-327 and Asp-235, thus allowing the formation of a carboxyl-carboxylate pair containing Asp-235 as a single proton release site that is stabilized through hydrogen bonding with undissociated Glu-327. In contrast, during the binding of propidium, the side chains of Glu-327 and Asp-235 would not directly interact with each other, and both carboxylates would function as independent proton release sites (4). The observation that in many other secondary active multidrug transporters, catalytic carboxylates are located too far away from each other to directly interact raised the question of why Asp-235 and Glu-327 are localized in close proximity in LmrP. Here, we describe (i) in silico analyses suggesting that Asp-235 and Glu-327 are part of a metal ion binding site with selectivity for Ca²⁺ and (ii) our experimental analyses demonstrating that LmrP mediates the selective binding and proton-coupled efflux of Ca²⁺.

EXPERIMENTAL PROCEDURES

Bacterial Strains, Plasmids, and Growth Conditions

L. lactis strain NZ9000 ΔlmrA ΔlmrCD (5), harboring empty expression vector pNZ8048 (6) or derivatives encoding C-terminally His₆-tagged WT LmrP (pHLP5) (2) or His₆-tagged double D235N/E327Q (DE) mutant LmrP (3, 4) downstream of a nisin A inducible promoter, was grown at 30 °C in M17 Broth (Oxoid) supplemented with 0.5% glucose and 5 μg/ml chloramphenicol. Medium was inoculated with a 1:50 dilution of an overnight culture, and cells were grown to an A₆₆₀ of 0.5–0.6. Expression of LmrP proteins was then induced by previously described methods (3, 4) for 1 h at 30 °C in the presence of 0.001% (v/v) of nisin A-containing supernatant of the nisin-producing strain L. lactis NZ9700 (6).

Preparation of Inside-out Membrane Vesicles

The cells were harvested by centrifugation at 13,000 × g for 10 min at 4 °C. The pellet was washed with 50 ml of ice-cold 100 mm K-HEPES (pH 7.0). The cells were resuspended in 25 ml of 100 mm K-HEPES (pH 7.0) containing 2 mg/ml of lysozyme and one tablet of Complete protease inhibitor (Roche Applied Science) and incubated at 30 °C for 30 min. The cells were disrupted by passing them twice through a Basic Z 0.75-kilowatt Benchtop Cell Disruptor (Constant Systems, Northants, UK) at 20,000 p.s.i. Disrupted cells were incubated for 30 min in the presence of 10 μg/ml DNase, 2 μg/ml RNase, and 10 mm MgSO₄. K-EDTA was added to a final concentration of 15 mm. Cell debris and undisrupted cells were removed by centrifugation at 13,000 × g for 15 min at 4 °C. Inside-out membrane vesicles were harvested by centrifugation of the supernatant at 125,000 × g for 30 min, resuspended in 100 mm K-HEPES (pH 7.0) containing 10% glycerol, and stored in liquid nitrogen. Protein concentration was determined using the Bio-Rad DC assay kit, and expression of the His₆-tagged proteins was confirmed on Western blot probed with anti-His₅ tag antibody (Sigma-Aldrich).

Protein Purification

Inside-out membrane vesicles (40–60 mg of total protein) were solubilized in 7.5 ml of solubilization buffer (50 mm K-HEPES buffer, pH 8.0, containing 100 mm NaCl, 10% (v/v) glycerol, and 1.5% β-d-dodecyl maltoside (DDM)³ (Melford)). The solubilization mix was incubated on a rotating wheel for 4 h at 4 °C. Unsolubilized particles were removed by centrifugation at 125,000 × g for 30 min at 4 °C, and the supernatant was immediately used in subsequent steps. For purification of the protein by nickel-affinity chromatography 400 μl of nickel-nitriloacetic acid (Ni-NTA)-agarose resin suspension (50% (w/w) in 30% (v/v) ethanol) (Sigma-Aldrich) was used. The resin was first equilibrated by washing three times with 5 ml of Milli-Q water and twice with 5 ml of Buffer A (50 mm K-HEPES, pH 8.0, containing 100 mm NaCl, 10% (v/v) glycerol, 0.05% (w/v) DDM, and 20 mm imidazole). The solubilized protein was added to the resin, after which the suspension was incubated on a rotating wheel at 4 °C overnight. The resin was washed five times with 5 ml of Buffer A and then six times with 5 ml of Buffer B (50 mm K-HEPES, pH 7.0, containing 100 mm NaCl, 10% (v/v) glycerol, 0.05% (w/v) DDM, and 20 mm imidazole). The buffer was discarded, and the resin was transferred to a Bio-spin column (Bio-Rad) and eluted with 500 μl elution buffer (Buffer B supplemented with 200 mm imidazole and 5% glycerol). As a control protein in drug binding studies, the His₆-tagged galactose-H⁺ symporter GalP from E. coli (7) was solubilized using the solubilization buffer as described above containing 300 mm NaCl instead of 100 mm (4, 8). The protein was purified on Ni-NTA resin by the method as described for LmrP using salt-free elution buffer. Protein concentrations were determined using the Micro-BCA assay kit (Pierce). The purity of protein samples was checked on Coomassie-stained SDS-PAGE and quantified by densitometric analysis of individual lanes using ImageJ software, version 1.43 (National Institutes of Health).

Substrate Binding to Purified Protein

WT LmrP, DE mutant LmrP, and GalP were purified as described under “Protein Purification” and diluted to a final concentration of 400 μg/ml. For ⁴⁵Ca²⁺ binding measurements, samples contained 10 μg of purified protein each and were prepared in 50 mm Tris-Cl at the pH indicated in the legend to Fig. 3, in a total volume of 500 μl. SDS-PAGE was run to confirm that the amount of protein in each samples was identical. ⁴⁵CaCl₂ (1.66 or 1.04 GBq/mg; PerkinElmer Life Sciences) was added with a specific activity of 0.57 TBq/mol for the single concentrations as indicated in legend to Fig. 3 and 0.12–0.57 TBq/mol for the concentration range in the kinetic experiment in Fig. 3B, after which the samples were incubated at room temperature for 30 min under mild shaking. The samples were subsequently filtered over nitrocellulose filters (Whatman; 0.2-μm diameter) using a vacuum pump to enable rapid filtration, during which the protein strongly binds to the filter and is separated from buffer containing free ⁴⁵Ca²⁺. Filters were immediately washed twice with 3 ml of ice-cold 50 mm Tris-Cl of the same pH as the assay buffer used for the experiment. The filters were transferred to tubes containing scintillation Ultima Gold XR (PerkinElmer Life Sciences) and subjected to liquid scintillation counting. Binding data were corrected for binding of ⁴⁵Ca²⁺ to the filters.

FIGURE 3.

⁴⁵Ca²⁺ binding by LmrP. A, ⁴⁵Ca²⁺ binding to affinity-purified LmrP (black bar), DE mutant LmrP (light gray bar), and GalP (control, dark gray bar) (10 μg each) in detergent solution was measured using a rapid filtration technique. The purified proteins were incubated for 30 min in the presence of 10.3 μm ⁴⁵Ca²⁺ and were subsequently filtered over 0.2-μm nitrocellulose filters. The filters were washed, after which the radioactivity retained on the filters was determined. B, purified WT LmrP (●) and GalP (○) were incubated for 30 min with ⁴⁵Ca²⁺ in a concentration range between 0 and 20.3 μm, after which ⁴⁵Ca²⁺ binding was measured as described under A. Specific binding to LmrP (corrected for binding to GalP) (▴) was fitted to a hyperbola to determine the apparent K_d and B_max of binding. C, pH dependence of ⁴⁵Ca²⁺ binding to purified WT LmrP (●) and DE mutant LmrP (○). ⁴⁵Ca²⁺ binding (10.3 μm) by both proteins was corrected for nonspecific binding to the GalP control. D, competition between ⁴⁵Ca²⁺ and nonradioactive divalent cations for binding to LmrP. Binding of 1.1 μm ⁴⁵Ca²⁺ to WT LmrP (black bar), DE mutant LmrP (gray bar), and GalP control was measured in the absence or presence of 100 μm of nonradioactive Ca²⁺, Ba²⁺, Mn²⁺, Mg²⁺, or Co²⁺. The observations for GalP were identical to those for the DE mutant and are not shown for clarity of presentation. The data represent the means ± S.E. of at least four separate determinations with independent protein preparations.

In Hoechst 33342 binding assays, 25 μg of purified protein was added to 2 ml of 100 mm K-HEPES (pH 7.0) containing 5 mm MgSO₄ and CaCl₂ at concentrations as indicated in Fig. 7, in a cuvette under mild stirring in a LS 55B fluorescence spectrometer (PerkinElmer). Hoechst 33342 was added in a stepwise manner (2 × 0.125 μm followed by 1 × 0.25 μm and 15 × 0.5 μm) every 20 s. Fluorescence was monitored over time until a plateau phase was reached. Hoechst 33342 fluorescence was monitored at excitation and emission wavelengths of 355 and 457 nm, respectively, with a slit width of 10 nm for emission and 5 nm for excitation. Elution buffer was used as a control for background binding to components of the buffer, and purified GalP served as a control for nonspecific binding.

FIGURE 7.

Inhibition by Ca²⁺ of Hoechst 33342-LmrP interactions. A and B, Δp-dependent Hoechst 33342 transport in inside-out membrane vesicles containing WT LmrP (A) or control membrane vesicles without LmrP was measured as described in the legend to Fig. 5D, using K-HEPES rather than KP_i, in the absence or presence of 1 mm Ca²⁺ or Mn². The buffer contained a standard amount of 5 mm Mg²⁺ that did not significantly inhibit transport. C and D, the binding of Hoechst 33342 to 0.28 μm purified WT LmrP (C) or DE mutant LmrP (D) was measured over a range of Hoechst 33342 concentrations (0.5–6.0 μm) in the absence (○) or presence of a fixed concentration of 1 mm Ca²⁺ (▾) or 2 mm Ca²⁺ (●). Kinetic parameters in the main text represent the means ± S.E. of three independent experiments.

Ca²⁺ Transport in Intact Cells

L. lactis cells were grown to an A₆₆₀ of 0.4–0.6 at 30 °C, after which LmrP proteins were expressed as described under “Bacterial Strains, Plasmids, and Growth Conditions.” The cells were subsequently harvested by centrifugation at 6,500 × g for 8 min at 4 °C, washed three times with ice-cold 50 mm K-HEPES (pH 7.0), and then resuspended in the same buffer to an A₆₆₀ of 5.0. The cells were preloaded with 0.5 μm Fura-2-AM (Invitrogen) and 250 μm CaCl₂ (when indicated) for 1.5 h at 30 °C under shaking. The cells were subsequently washed three times with ice-cold 50 mm K-HEPES (pH 7.0) in the same volume that was used for resuspension to remove any remaining extracellular Fura-2-AM. The cells were diluted 40-fold into a cuvette containing 2 ml of 50 mm K-HEPES (pH 7.0) containing 1.8 mm probenicid to inhibit endogenous anion efflux activity (9–12) and block background export of Fura-2 from L. lactis cells. Upon the addition of 25 mm glucose, Fura-2 fluorescence was monitored at an excitation wavelength of 340 nm and emission wavelength of 510 nm with slit widths of 5 or 10 nm, respectively.

Substrate Transport in Inside-out Membrane Vesicles

Inside-out membrane vesicles containing WT LmrP, DE mutant LmrP, or no LmrP protein (control) were prepared as described under “Preparation of Inside-out Membrane Vesicles.” The samples contained 1 mg/ml membrane vesicles, 2.5 mm ATP, 0.1 mg/ml creatine kinase, 5 mm phosphocreatine, and, where indicated, ionophores nigericin and/or valinomycin to a final concentration of 0.5 μm each in a buffer composed of 10 mm Tris-Cl, 10 mm KP_i, and 150 mm KCl (pH 7.5) in a total volume of 500 μl. The samples were prepared in sulfuric acid-washed glass tubes and incubated for 3 min before start of the measurements. ⁴⁵CaCl₂ (0.57 TBq/mol) was then added at concentrations as given in Fig. 5 and its legend. The samples were filtered over nitrocellulose filters (Whatman; 0.45 μm) to allow binding of the membrane vesicles to the filters, after which the filters were washed with 3 ml of ice-cold 100 mm LiCl to eliminate buffer containing free ⁴⁵Ca²⁺. Radioactivity retained on the filters was determined by liquid scintillation counting.

FIGURE 5.

Substrate transport in inside-out membrane vesicles. A and B, for measurements of transport of ⁴⁵Ca²⁺ in inside-out membrane vesicles containing WT LmrP (A) or without LmrP protein (B), membrane vesicles were diluted to a final concentration of 1 mg/ml into buffer (pH 7.0) containing 10 mm KP_i, 10 mm Tris-Cl, and 150 mm KCl supplemented with an ATP regenerating system and 2.5 mm MgATP. The latter is used by the F₁F₀ H⁺-ATPase to generate a Δp (interior positive and acid). At t = 0 s, 10.3 μm ⁴⁵Ca²⁺ was added in the absence of nigericin (Δp, ▾) or presence of nigericin (Δψ only, ○) or valinomycin (ΔpH only, ●) or both ionophores (no Δp, △). Ionophores were added to a final concentration of 0.5 μm each, followed by the addition of the ATP and a preincubation for 3 min prior to the addition of ⁴⁵Ca²⁺. ⁴⁵Ca²⁺ accumulation in the membrane vesicles was assessed by rapid filtration. C, initial velocities of Δp-dependent ⁴⁵Ca²⁺ transport in inside-out membrane vesicles containing WT LmrP (●), DE mutant LmrP (○), or without LmrP proteins (□) were determined from uptake over a 20–90-s time span, at ⁴⁵Ca²⁺ concentrations between 0.1 and 100.3 μm. D, Hoechst 33342 transport was measured in inside-out membrane vesicles (0.2 mg of protein/ml) containing WT LmrP or DE mutant LmrP or without LmrP proteins (Control) in 100 mm KP_i (pH 7.8) supplemented with 5 mm MgSO₄ and an ATP-regenerating system. After 30 s, Hoechst 33342 was added to a final concentration of 0.25 μm, followed by the addition of 2 mm ATP at 150 s. Hoechst transport was measured by fluorimetry. The traces represent observations in three independent experiments. The data in A–C represent the means ± S.E. of three separate experiments.

Transport of Hoechst 33342 in inside-out membrane vesicles was measured as described previously (8). Membrane vesicles (400 μg of total protein) were diluted into 2 ml of 100 mm KP_i buffer (pH 7.8) containing 5 mm MgSO₄, 0.1 mg/ml creatine kinase, and 5 mm phosphocreatine. For measurements in the presence of Ca²⁺, the KP_i was replaced by K-HEPES. After 30 s, Hoechst 33342 (0.25 μm) was added, and the fluorescence was monitored until a steady state was reached. Na-ATP was added to a final concentration of 2 mm to provide metabolic energy for the generation of a Δp via the F₁F₀ H⁺ ATPase. Hoechst 33342 fluorescence was followed further over time and was measured in a LS 55B fluorescence spectrometer (PerkinElmer) at excitation and emission wavelengths of 355 and 457 nm, respectively, and slit widths of 10 and 5 nm, respectively.

Purification of E. coli Lipids for Reconstitution

Lipids from E. coli total lipid extract (Avanti Polar lipids) containing approximately 67% phosphatidylethanolamine, 23% phosphatidylglycerol, and 10% cardiolipin were purified before use. 200 mg of the lipid was weighed out and dissolved in 3 ml of chloroform. The lipid solution was added dropwise into ice-cold acetone perfused with liquid N₂ and containing 2 μl/ml β-mercaptoethanol. The mixture was stirred gently at 4 °C overnight, during which contaminants dissolved in the acetone phase. The insoluble lipids were collected by centrifugation (3,000 × g rpm, 20 min, 4 °C). The pellet was solubilized with 40 ml of diethylether containing 2 μl/ml β-mercaptoethanol and perfused with N₂. The tube was vortexed until the pellet was dissolved. Subsequently, the soluble lipid fraction was separated from insoluble contaminants by centrifugation at 3,000 × g for 10 min. The supernatant was completely evaporated in a rotation evaporator at 25 °C. The lipids were then dissolved in chloroform and transferred into a preweight glass tube to allow accurate dilution of the lipid in chloroform to a concentration of 100 mg/ml. The lipid solution was kept at −20 °C in the dark under a N₂ atmosphere.

Transport Measurements in Proteoliposomes

WT LmrP and DE mutant LmrP were purified as described under “Protein Purification.” Reconstitution was performed using a combination of previously published methods (13, 14). Phospholipids (1.5 mg) were dried by evaporation under a stream of N₂ gas. Liposomes were prepared by sonication for 8 min in 2.7 ml of 10 mm Tris-Cl (pH 7.4), 0.5 mm K-EDTA, 1 mm β-mercaptoethanol, and 75 mm NaCl. Detergent DDM was added to the lipid suspension in a 1:2 w/w lipid/detergent ratio. Purified LmrP proteins in elution buffer, or equal volume of elution buffer as a control, were added in a 1:10 protein/lipid ratio (w/w). The samples were incubated at 30 min at room temperature under gentle agitation. Subsequently, the detergent DDM was removed by two incubations with 80 mg of biobeads for 2 h at room temperature under mild stirring. The third incubation with 80 mg of biobeads was performed overnight at 4 °C. Proteoliposomes were collected by centrifugation at 18,000 × g for 30 min and resuspended in 3 ml of buffer I (10 mm Tris-Cl, 10 mm KP_i, 150 mm KCl and 150 mm NH₄Cl, pH 6.8). The samples were frozen in liquid nitrogen and thawed at room temperature. Subsequently, the proteoliposomes were collected by centrifugation at 18,000 × g for 30 min, resuspended to a final concentration of 2 mg/ml in buffer I, and immediately used in an experiment.

For measurement of Hoechst 33342 transport in (proteo)liposomes in the presence of an imposed ΔpH (inside acidic) (Fig. 6A), proteoliposomes or empty liposomes (control) were diluted 200-fold in Buffer II (10 mm Tris-Cl, 10 mm KP_i, 150 mm KCl, and 150 mm NaCl, pH 7.5) in a total reaction volume of 2 ml. After 40 s, Hoechst 33342 was added to a final concentration of 0.25 μm, and changes in fluorescence were recorded in a PerkinElmer LS 55B fluorimeter for 800 s at excitation and emission wavelengths of 355 and 457 nm, respectively, and slit widths of 10 nm each. For Hoechst 33342 transport measurements in the absence of the ΔpH (Fig. 6B), proteoliposomes were diluted 200-fold in buffer I, in which they were prepared.

FIGURE 6.

Substrate transport in proteoliposomes. A and B, Hoechst 33342 transport in proteoliposomes containing purified and functionally reconstituted WT LmrP, DE mutant LmrP, or empty liposomes (Control) in the presence (A) or absence (B) of an imposed ΔpH. (Proteo)liposomes were prepared in Buffer I (10 mm Tris-Cl, 10 mm KP_i, 150 mm KCl, and 150 mm NH₄Cl, pH 6.8) and diluted 200-fold at t = 0 s into Buffer II (10 mm Tris-Cl, 10 mm KP_i, 150 mm KCl and 150 mm NaCl, pH 7.5) (A) to establish a ΔpH (interior acidic) or buffer I to prevent formation of ion gradients (B). At t = 40 s, 0.25 μm Hoechst 33342 was added. Hoechst 33342 transport was followed by fluorimetry. Shown are representative traces from three independent experiments using different batches of proteoliposomes. C and D, ⁴⁵Ca²⁺ transport in proteoliposomes containing purified and functionally reconstituted WT LmrP (●), DE mutant LmrP (○), or empty liposomes (Control) (▾) in the presence (C) or absence (D) of an imposed ΔpH. (Proteo)liposomes were prepared and diluted 50-fold as described under A and B in dilution buffers containing ⁴⁵Ca²⁺ at a final concentration of 1.1 μm. ⁴⁵Ca²⁺ transport into the (proteo)liposomes was estimated by rapid filtration. The values represent the means ± S.E. from four separate experiments.

⁴⁵Ca²⁺ transport measurements in (proteo)liposomes or empty liposomes in the presence (Fig. 6C) and absence (Fig. 6D) of an imposed ΔpH (interior acidic) started with the 50-fold dilution of the (proteo)liposomes in Buffers II and I, respectively, as described for Hoechst 33342 transport in a total reaction volume of 500 μl. Valinomycin (0.5 μm) was added to dissipate the formation of a reversed Δψ. The dilution buffer contained ⁴⁵CaCl₂ (0.57 TBq/mol) at a concentration as specified in the legend to Fig. 6 (C and D). The samples were rapidly filtered over nitrocellulose filters (Whatman; 0.2-μm diameter) as described under “Substrate Transport in Inside-out Membrane Vesicles” and washed two times with 3 ml of ice-cold 10 mm KP_i, 10 mm Tris-Cl, 150 mm KCl (pH 7.5). Filters were transferred into scintillation vials, and the radioactivity was determined.

RESULTS

In Silico Prediction of a Ca²⁺-binding Site in LmrP

LmrP contains two acidic residues Asp-235 and Glu-327 that are predicted to be located in close proximity of each other in our inward facing structure model (3, 4). A motif search was performed in the data base SPASM (Spatial Arrangements of Side chains and Main chains; Uppsala Software Factory) to test the occurrence of this arrangement of carboxyl residues in other proteins. Surprisingly, when only the coordinates of Asp-235 and Glu-327 were used as input, the search revealed that the Asp-235/Glu-327 arrangement is very similar to that of two key carboxyl residues in certain EF hand Ca²⁺-binding proteins such as calmodulin, troponin C, and calsequestrin (Fig. 1). To confirm and extend these predictions, we used the maximum entropy-based docking web server MEDock for efficient prediction of whether and how Ca²⁺ would bind in our LmrP model. MEDock predicted the binding of Ca²⁺, but not of Mg²⁺ in a fashion that involves coordination by the carboxyl oxygens of Asp-235 (transmembrane helix 7) and Glu-327 (transmembrane helix 9) in addition to the main chain carbonyl oxygen from Ile-264 (transmembrane helix 8) and a cation-pi electron interaction on the aromatic ring of Tyr-265 (transmembrane helix 8), which are all located in close proximity in the C-terminal half of LmrP (Fig. 1).

FIGURE 1.

Stereo views of molecular models of Ca²⁺ binding. A, model of Ca²⁺ coordination in LmrP was obtained in docking simulations by MEDock using our homology model of LmrP (3). Residue Glu-327 and Asp-235 have a predicted distance and geometry that is analogous to that of two key carboxylates in the Ca²⁺-binding site of many EF hand Ca²⁺-binding proteins. B, example of co-crystallized Ca²⁺-troponin C complex (Protein Data Bank 1A2X) at 2.3 Å resolution (45), in which, for clarity of presentation, only the positions of the two carboxyl residues (Asp-143 and Glu-150) are shown. The images were generated in MacPyMOL v1.3.

High Affinity Binding of Ca²⁺ to Purified LmrP

Following the in silico predictions of Ca²⁺ binding by LmrP, binding experiments with radioactive ⁴⁵Ca²⁺ were performed using purified LmrP in detergent solution. In these measurements, the major facilitator superfamily galactose/H⁺ symporter GalP from E. coli (7), with a similar overall architecture as LmrP and a dedicated role in the transport of monosaccharides, was used as a control for nonspecific substrate binding (4, 8). Using the attached His tag, WT LmrP, DE mutant LmrP, and GalP were purified from detergent-solubilized total membrane proteins in inside-out membrane vesicles by Ni-NTA affinity chromatography to purities above 95% (Fig. 2). The purified proteins were incubated for 30 min in the presence of ⁴⁵Ca²⁺ at a concentration of 10.3 μm and subsequently filtered over 0.2-μm nitrocellulose filters. The radioactivity retained on the filters was determined. Fig. 3A shows that ⁴⁵Ca²⁺ binding to WT LmrP was substantially elevated compared with the GalP control. Post-hoc analysis using Tukey's test suggested a significant difference between the means for LmrP and GalP (p < 0.03). Analysis by analysis of variance confirmed that the difference between the data sets was significant (p < 0.01). These Ca²⁺ binding experiments with WT LmrP and GalP were repeated using a range of Ca²⁺ concentrations. Subtraction of the nonspecific binding obtained for GalP from the total binding obtained for LmrP allowed determination of specific binding to LmrP as a function of the Ca²⁺ concentration (Fig. 3B). Fitting of the specific binding data to a hyperbola showed Ca²⁺ binding to LmrP with an apparent dissociation constant (K_d) of 7.2 ± 4.3 μm Ca²⁺ and a maximum binding (B_max) of 3.7 ± 0.9 nmol/mg protein.

FIGURE 2.

Protein preparations used in this study. Lanes 1 and 2, total membrane protein from lactococcal inside-out membrane vesicles containing WT LmrP (8 μg/lane) (lane 1) or DE mutant LmrP (11 μg/lane) (lane 2). Lane 3, total membrane protein (8 μg/lane) from E. coli inside-out membrane vesicles containing GalP. Lane 4–6, Ni-NTA affinity-purified WT LmrP (4 μg/lane) (lane 4), DE mutant LmrP (4.8 μg/lane) (lane 5), and GalP (6 μg/lane) (lane 6). Lane 7 and 8, proteoliposomes containing purified WT LmrP (1.2 μg/lane) (lane 7) or DE mutant LmrP (1.8 μg/lane) (lane 8). Lane 9, empty liposomes (equivalent to the amount of proteoliposomes containing DE mutant LmrP in lane 8).

Importance of Asp-235 and Glu-327 in Ca²⁺ Binding

To test the predicted role of Asp-235 and Glu-327 in Ca²⁺ coordination, ⁴⁵Ca²⁺ binding to purified double mutant D235N/E327Q (DE) LmrP was determined. ⁴⁵Ca²⁺ binding to the DE mutant was strongly reduced compared with WT LmrP and was close to the GalP control (Fig. 3A). Analysis of the data both by Tukey's test and by analysis of variance indicated a significant difference between the binding to WT LmrP and DE LmrP (p < 0.02 and 0.01, respectively) but not between DE LmrP and GalP. As carboxyl moieties coordinate Ca²⁺ in their dissociated form, it was interesting to compare the pH dependence of ⁴⁵Ca²⁺ binding to WT LmrP and DE LmrP. Whereas ⁴⁵Ca²⁺ binding to WT LmrP increased with increasing pH until the optimum pH for binding was reached around pH 8, ⁴⁵Ca²⁺ binding to the double mutant was low and independent of the pH between 6 and 9 (Fig. 3C). Hence, Asp-235 and Glu-327 play an important role in Ca²⁺ binding by LmrP.

Selectivity for Ca²⁺

The ability of LmrP to interact with divalent metal ions other than Ca²⁺ was tested in competition experiments in which the binding of 1.1 μm ⁴⁵Ca²⁺ by purified WT LmrP, DE LmrP, and the GalP control was measured in the absence and presence of a 91-fold excess of nonradioactive Ca²⁺, Ba²⁺, Mn²⁺, Mg²⁺, or Co²⁺. As shown in Fig. 3D, only nonradioactive Ca²⁺ and Ba²⁺ competed with ⁴⁵Ca²⁺ for binding to WT LmrP, suggesting that the cation-binding site in LmrP is selective for Ca²⁺ and Ba²⁺. None of the nonradioactive divalent cations competed with ⁴⁵Ca²⁺ for binding to DE mutant LmrP and GalP, which supports the notion that the DE mutation impairs specific binding of ⁴⁵Ca²⁺ to LmrP and that the residual binding to this mutant protein is nonspecific and, hence, comparable with ⁴⁵Ca²⁺ binding to the GalP control (Fig. 3A).

LmrP-mediated Ca²⁺ Extrusion in Intact Cells

To investigate whether LmrP can mediate Ca²⁺ efflux, a fluorescence-based assay was established for intact lactococcal cells based on the Ca²⁺-sensitive fluorescent probe Fura-2. When added to a suspension of ATP-depleted cells, the hydrophobic acetoxymethyl ester derivative of Fura-2 (Fura-2-AM) diffuses across the membrane into the cell, after which it is cleaved by nonspecific esterases and trapped intracellularly in its hydrophilic form. When cells were preloaded with 0.5 μm Fura-2-AM in presence of 250 μm Ca²⁺, the binding of Ca²⁺ to intracellular Fura-2 was associated with a strong fluorescence intensity enhancement of the probe. After a short lag phase during which metabolic energy was generated, the addition of 25 mm glucose initiated a large decrease in Fura-2 fluorescence in LmrP-expressing cells compared with the nonexpressing control and a smaller decrease in cells expressing DE mutant LmrP (Fig. 4A). In control experiments, no significant changes in the Fura-2 fluorescence were observed in control cells or cells expressing WT LmrP or DE mutant LmrP in the absence of the preloading with Ca²⁺ (Fig. 4B). Furthermore, Fura-2 remained undetectable in the external buffer during these experiments, indicating that LmrP did not mediate Fura-2 efflux at a significant level (data not shown). Because WT LmrP and the DE mutant are equally well expressed in the lactococcal cells under the experimental conditions (4), our findings demonstrate the Ca²⁺ extrusion activity of LmrP in intact cells and the importance of Asp-235 and Glu-327 for this activity.

FIGURE 4.

Ca²⁺ transport in intact cells. Lactococcal cells expressing WT LmrP, DE mutant LmrP, or no LmrP protein (control) were preloaded with 0.5 μm Fura-2 in the presence of 250 μm CaCl₂ (A) or in the absence of the CaCl₂ (B). The cells were washed and diluted 40-fold into 100 mm K-HEPES (pH 7.0). Ca²⁺ efflux was initiated by the addition of 25 mm glucose as a source of metabolic energy and was followed over time by fluorimetry. Traces are typical for data obtained in three independent experiments using different batches of cells.

Transport Studies in Inside-out Membrane Vesicles

To further test the ability of LmrP to mediate Ca²⁺ transport, ⁴⁵Ca²⁺ uptake was measured in inside-out membrane vesicles. For this purpose, inside-out membrane vesicles were diluted to a final protein concentration of 2 mg/ml into 10 mm Tris-Cl, 10 mm KP_i, 150 mm KCl (pH 7.0) containing an ATP-regenerating system and 2.5 mm ATP, which is the source of metabolic energy for the formation of the Δp ( = Δψ − ZΔpH in which Z = 58.1 mV at 20 °C, interior positive and acidic) through proton pumping by the F₁F₀ H⁺-ATPase. The transport reaction was started with the addition of ⁴⁵Ca²⁺ to a final concentration of 10.3 μm, after which samples were taken over time, and the amount of ⁴⁵Ca²⁺ accumulated in the inside-out membrane vesicles was determined by rapid filtration. In these experiments, the magnitude and composition of the Δp was manipulated using the ionophores nigericin and valinomycin. Nigericin mediates the antiport of H⁺ and K⁺ down their concentration gradients, thereby selectively dissipating the ΔpH in an electroneutral manner. Valinomycin mediates electrogenic uniport of K⁺, allowing the electrophoretic uptake of K⁺ in cells with dissipation of the Δψ. In the presence of a ΔpH only (through the addition of valinomycin), the uptake of ⁴⁵Ca²⁺ was reduced slightly compared with Δp-dependent uptake, pointing to the ΔpH dependence of Ca²⁺ transport (Fig. 5A). In the presence of a Δψ only (through the addition of nigericin), ⁴⁵Ca²⁺ uptake was reduced more strongly but remained significantly above control uptake in the absence of the Δp (in the presence of both ionophores). This result points to the Δψ dependence of the transport reaction (Fig. 5A). The absence of robust ⁴⁵Ca²⁺ uptake in control inside-out membrane vesicles lacking LmrP (Fig. 5B) and in inside-out membrane vesicles containing DE mutant LmrP (see below) under these conditions indicates that the observed Ca²⁺ transport reaction is LmrP-dependent. Taken together, these data suggest that LmrP mediates calcium transport by a Δp-dependent, electrogenic Ca²⁺/nH⁺ exchange reaction in which n ≥ 3. To determine the affinity of LmrP for Ca²⁺ in the transport reaction in inside-out membrane vesicles, the rate of uptake of ⁴⁵Ca²⁺ in LmrP-containing inside-out membrane vesicles was followed at ⁴⁵Ca²⁺ concentrations ranging from 0.1 to 100.3 μm. The initial rates of linear uptake were determined between 20 and 90 s following ⁴⁵Ca²⁺ addition and were plotted against the ⁴⁵Ca²⁺ concentration (Fig. 5C). The data were fitted to a hyperbola from which an apparent affinity constant (K_t) of 8.6 ± 2.3 μm and a maximum rate (V_max) of 2.5 ± 0.3 pmol Ca²⁺/mg of protein/s was estimated. This K_t value corresponds well to the apparent K_d of 7.2 μm derived in the Ca²⁺ binding experiments (Fig. 3B). Compared with the GalP control, DE mutant LmrP did not mediate a significant transport of ⁴⁵Ca²⁺ in the kinetic experiments (Fig. 5C). In control experiments, the functionality of the LmrP proteins was tested in a Hoechst 33342 transport assay, in which the fluorescence quenching of the dye was detected following its transport from the membrane into the acidic, aqueous lumen of the inside-out membrane vesicles (Fig. 5D). Both WT LmrP and the DE mutant exhibited a substantial activity in this assay.

Substrate Transport by Purified LmrP in Proteoliposomes

To further exclude a significant role of endogenous Ca²⁺ transporters in the observed Ca²⁺ transport activity in LmrP-containing inside-out membrane vesicles, LmrP was purified and functionally reconstituted in proteoliposomes. To impose a reversed ΔpH (interior acidic) across the liposomal membrane that could drive LmrP-mediated ⁴⁵Ca²⁺ accumulation, proteoliposomes were prepared in NH₄Cl-containing buffer (pH 6.8) (see “Experimental Procedures”) and diluted in NH₄Cl-free buffer (pH 7.5) to allow the transmembrane diffusion of NH₃ from the (proteo)liposomes with concomitant acidification of the lumen. To test transport in the absence of the ΔpH, (proteo)liposomes were diluted in the buffer in which they were prepared, thus preventing the outward diffusion of NH₃. The functionality of the reconstituted LmrP was first confirmed in a Hoechst 33342 transport assay, as described previously (15), in which the (proteo)liposomes were diluted in buffers containing 0.25 μm Hoechst 33342. The transport-associated quenching of the dye relative to empty liposomes (control) was only obtained for WT LmrP and the DE mutant upon imposition of the ΔpH (Fig. 6A), but not in the absence of the ΔpH (Fig. 6B).

When the external buffer was supplemented with 1.1 μm ⁴⁵Ca²⁺ instead of Hoechst 33342, a significant ΔpH-dependent accumulation of the radioactive ion was obtained in proteoliposomes containing WT LmrP, whereas the accumulation for the DE mutant was at the background level in empty liposomes (Fig. 6C). In absence of the ΔpH, no ⁴⁵Ca²⁺ accumulation was detected in any of the samples (Fig. 6D). These experiments provide strong evidence for the ability of LmrP itself to mediate proton-coupled Ca²⁺ transport.

Ca²⁺ Inhibits the Interaction of LmrP with Hoechst 33342

Because LmrP can transport both Hoechst 33342 and Ca²⁺, we tested the potential interaction between these substrates on LmrP. Ca²⁺ at a 1 mm concentration inhibited the Δp-dependent transport of Hoechst 33342 by WT LmrP in inside-out membrane vesicles, whereas an equal concentration of Mn²⁺ had no inhibitory effect (Fig. 7A). Under identical conditions, neither of these cations influenced Hoechst 33342 fluorescence in control membrane vesicles lacking LmrP (Fig. 7B). The mechanism of inhibition by Ca²⁺ was further examined in a Hoechst 33342 binding assay (16). In this assay, the binding of Hoechst 33342 (0.7–6.0 μm) to purified WT LmrP and the DE mutant LmrP (0.28 μm) as a control was determined from the enhancement of fluorescence upon transfer of the probe from the aqueous buffer to nonpolar binding site(s) on the protein. Lineweaver-Burk plots for the data yielded an apparent K_d of 2.6 ± 0.3 μm Hoechst 33342 and apparent B_max of 107 ± 6 a.u. for WT LmrP (Fig. 7C), and an apparent K_d of 3.5 ± 0.2 μm Hoechst 33342 and apparent B_max of 102 ± 8 a.u. for the DE mutant (Fig. 7D). In the presence of 1 mm Ca²⁺, the apparent K_d for WT LmrP increased 1.3-fold (p < 0.02), whereas the apparent B_max reduced 1.7-fold (p < 0.03) (Fig. 7C). In contrast, no significant differences in the apparent K_d and B_max values for Hoechst 33342 binding were obtained for DE LmrP in the presence or absence of Ca²⁺ (Fig. 7D), consistent with the observations for this mutant of substantial Hoechst 33342 transport (Figs. 5D and 6A) but impaired Ca²⁺ binding (Fig. 3) and Ca²⁺ transport (Figs. 4–6). The Lineweaver-Burk plot for the inhibition of Hoechst 33342 binding to WT LmrP by Ca²⁺ (Fig. 7C) shows an increase in the apparent K_d and a reduction in the apparent B_max and suggests a noncompetitive mechanism of inhibition. These findings are reminiscent of those for the inhibition of Hoechst 33342 binding to LmrP by the calcium channel blocker nicardipine and the antimicrotubule drug vinblastine (15). Thus, Ca²⁺ has analogous effects on Hoechst 33342-LmrP interactions as known drugs.

DISCUSSION

Similar to eukaryotic cells, eubacterial cells are equipped with transporters to efflux Ca²⁺ and maintain Ca²⁺ homeostasis (17). The cytosolic Ca²⁺ concentration is usually significantly lower than in the extracellular environment, roughly between 0.1 and 1 μm in E. coli (18, 19). Early studies by Rosen and McClees (20) and Harold and co-workers (21) first identified a Ca²⁺/H⁺ antiporter in E. coli and an ATP-dependent P-type Ca²⁺ efflux pump in Streptococcus faecalis, and these types of systems were later shown to also exist in other bacteria (22, 23) including L. lactis (24, 25). However, Ca²⁺ transport has not been studied extensively in bacteria, and in addition to the bacterial transport systems listed above, others might exist.

Our data suggest that LmrP mediates the selective binding and transport of Ca²⁺ in cells, inside-out membrane vesicles, and proteoliposomes containing purified protein. No significant Ca²⁺ binding or transport activities were obtained in any of the experimental systems lacking LmrP (Figs. 2–4). Similar to published observations for Ca²⁺ channels, for which Ba²⁺ can pass through the channel apart from Ca²⁺ (26, 27), Ca²⁺ can be replaced by Ba²⁺ but not by Mg²⁺, Mn²⁺, or Co²⁺ in the binding experiments with LmrP (Fig. 3D). The determined affinities of LmrP for Ca²⁺ in our binding assay (apparent K_d of 7.2 ± 4.3 μm) (Fig. 3B) and transport assay (apparent K_t of 8.6 ± 2.3 μm) (Fig. 5C) are consistent with each other. For the designated calcium/proton antiporter from Bacillus subtilis, an apparent K_t of 40 μm Ca²⁺ was found (28), whereas for the Ca²⁺/H⁺ antiporter YfkE in this organism, an apparent K_t was determined of ∼12.5 μm (29). The apparent K_d of mammalian calmodulin for Ca²⁺ covers a range from 14 μm down to 0.5 μm in the presence of the activatable target enzyme (30, 31). Hence, the apparent affinity of LmrP for Ca²⁺ lies in a similar range as observed for proteins for which the interaction with Ca²⁺ is established. The affinity of LmrP for Ca²⁺ is also in a similar range as the affinity for a typical multidrug substrate such as Hoechst 33342, which is bound by LmrP with an apparent K_d of 2.6 ± 0.3 μm (Fig. 7C).

In many Ca²⁺-binding proteins, the Ca²⁺-binding pocket exhibits a pentagonal bipyramidal geometry (32), through positioning of side chains in a characteristic helix-loop-helix structural motif termed the EF hand (33). The end of the first helix and the start of the second helix often contain a carboxyl residue that is crucial for coordinating Ca²⁺ (see Fig. 1B for example). It is this pair of carboxyl residues that led us to Ca²⁺-binding proteins during a motif search in the SPASM data base with the coordinates of the Asp-235/Glu-327 side chains of LmrP as the bait. Mutations of acidic residues are known to significantly change (mainly decrease) the affinity of calcium binding to EF hand proteins (30, 34). The importance of Asp-235 and Glu-327 in Ca²⁺ binding was predicted in our docking studies (Fig. 1A) and was demonstrated in binding and transport assays using DE mutant LmrP (Figs. 3–6). In a study on the pH dependence of Ca²⁺ binding, a pH optimum was reached between pH 6.0 and 7.0 for WT LmrP but not for the DE mutant (Fig. 3C), suggesting that deprotonation of Asp-235 and Glu-327 in the binding pocket is crucial for Ca²⁺ binding. Very recently, a crystal structure of the liganded Na⁺/Ca²⁺ exchanger NCX from Methanococcus jannaschii was resolved at 1.9 Å resolution, showing the direct role of Glu-54 and Glu-213 in the coordination of Ca²⁺ in its binding pocket (35). It is important to note that although the DE mutant is inhibited in Ca²⁺ binding and transport (Figs. 3–7) and propidium²⁺ transport (4), electroneutral ethidium⁺-H⁺ antiport (4) and Hoechst 33342 transport (Figs. 5D and 6A) are retained. Hence, the DE mutant is strongly inhibited in Ca²⁺ transport activity but can still function as a multidrug transporter with a different selectivity compared with the wild-type protein.

Studies on the energetics of LmrP-mediated ⁴⁵Ca²⁺ transport in inside-out membrane vesicles demonstrated that this reaction is based on electrogenic Ca²⁺/nH⁺ antiport (n ≥ 3) (Fig. 5A), similar to published observations for the calium/proton antiporter in B. subtilis (28). The transport of Ca²⁺ by LmrP might therefore be analogous to the transport of divalent cationic propidium, which is based on electrogenic propidium²⁺/3H⁺ exchange (4). However, compared with Ca²⁺, propidium²⁺ has a much larger structure in which the two positive charges are separated by a C₃ alkyl chain. Although Asp-235 and Glu-327 are crucial for binding of each of these substrates (this work and Ref. 4), our docking studies on Ca²⁺ binding predict that the charge neutralization of Ca²⁺ is in part based on direct electrostatic interactions with these carboxylates, whereas for propidium²⁺, the charge neutralization of at least one positive charge is most likely achieved through interactions with residues located further away, e.g., aromatic and polar residues as observed for the binding of berberine to the crystallized multidrug-binding transcriptional regulator QacR (36). The binding sites in LmrP for Ca²⁺ and propidium²⁺ might therefore partially overlap. On the other hand, our findings of noncompetitive inhibition of Hoechst 33342 binding by Ca²⁺ (Fig. 7C) point to the binding of Ca²⁺ to unliganded LmrP and the Hoechst 33342-LmrP complex, and hence, to separate, communicating sites for these substrates. Inhibitory effects by Ca²⁺ on drug interactions in LmrP were also observed for ethidium⁺ as a substrate (data not shown). The mechanism of this inhibition was not further investigated.

In conclusion, we present compelling evidence that the multidrug transporter LmrP can mediate the selective binding and transport of Ca²⁺ and that this activity can inhibit multidrug transport. Ca²⁺ is implicated in a number of important bacterial functions including heat shock, pathogenicity, chemotaxis, differentiation, and cell cycle (17, 22). Our observations relate to previous findings of Na⁺(K⁺)/H⁺ antiport by the secondary active multidrug transporter MdfA (37) and tetracyline transporter Tet(L) (38, 39) in E. coli and of Na⁺-Cl⁻-H⁺ symport by the multidrug ATP-binding cassette transporter LmrA in L. lactis (40–42). These findings raise the paradox that multidrug transporters with a broad specificity for amphiphilic organic cations can, at the same time, be highly selective for certain inorganic ions. They suggest that these multidrug transporters fulfill additional physiological roles to that of multidrug transport and that such roles might promote the persistence of multidrug transporter even in the absence of exposure to antibiotics (43, 44). Indeed, for MdfA a role in alkali tolerance was demonstrated (37). Many molecular details of the “dual specificity” of LmrP and other systems are still unknown, and a systematic comparison with transporters with an established, dedicated role in inorganic ion translocation will be an important challenge. Clearly, there is a lot to be learned about substrate recognition and transport by multidrug transporters, which will be crucial for the generation of selective multidrug resistance inhibitors with a low general toxicity and for our understanding of the factors that can trigger transporter expression.