ABSTRACT
The crystal structure of the Na+-coupled melibiose permease of Salmonella enterica serovar Typhimurium (MelBSt) demonstrates that MelB is a member of the major facilitator superfamily of transporters. Arg residues at positions 295, 141, and 363 are involved in interdomain interactions at the cytoplasmic side by governing three clusters of electrostatic/polar interactions. Insertion of (one at a time) Glu, Leu, Gln, or Cys at positions R295, R141, and R363, or Lys at position R295, inhibits active transport of melibiose to a level of 2 to 20% of the value for wild-type (WT) MelBSt, with little effect on binding affinities for both sugar and Na+. Interestingly, a spontaneous suppressor, D35E (periplasmic end of helix I), was isolated from the R363Q MelBSt mutant. Introduction of the D35E mutation in each of the mutants at R295, R141 (except R141E), or R363 rescues melibiose transport to up to 91% of the WT value. Single-site mutations for the pair of D35 and R175 (periplasmic end of helix VI) were constructed by replacing Asp with Glu, Gln, or Cys and R175 with Gln, Asn, or Cys. All mutants with mutations at R175 are active, indicating that a positive charge at R175 is not necessary. Mutant D35E shows reduced transport; D35Q and D35C are nearly inactivated. Surprisingly, the D35Q mutation partially rescues both R141C and R295Q mutations. The data support the idea that Arg at position 295 and a positive charge at positions 141 and 363 are required for melibiose transport catalyzed by MelBSt, and their mutation inhibits conformational cycling, which is suppressed by a minor modification at the opposite side of the membrane.
INTRODUCTION
The melibiose permease of Salmonella enterica serovar Typhimurium (MelBSt) belongs to the glycoside-pentoside-hexuronide/cation (GPH) family of membrane transport proteins (TC 2.A.2) (1, 2), a subgroup of the major facilitator superfamily (MFS) (3, 4). MelBSt catalyzes the coupled stoichiometric symport of a galactoside with a Na+, Li+, or H+ (5–10). Recently, we described three-dimensional (3-D) X-ray crystal structures of MelBSt captured in two conformations, an outward partially occluded state and an outward inactive state (3). These structures provide high-resolution structural information for this subgroup of permeases, some of which play important roles in human health and disease, such as the major facilitator superfamily domain 2A protein (11, 12).
Consistent with the previous prediction (4), the crystal structures reveal that MelB adopts a typical MFS fold with 12 transmembrane helices (Fig. 1a) (3). The observed partially occluded cavity allows us to localize the sugar-binding pocket that contains experimentally identified sugar-binding residues (13–15). Furthermore, in close proximity to this proposed sugar-binding site is the suggested cation-binding site for Na+, Li+, or H+. As proposed for other MFS permeases (16–20), MelBSt transports substrate by an alternating-access mechanism (4, 6, 21–23), which is involved in a large-scale movement of both N- and C-terminal helices.
Overall fold of MelBSt. The N-terminal and C-terminal helix bundles are colored in green and blue, respectively. The structural information on MelBSt in this paper is based only on Mol-A of the PDB entry 4M64. The cytoplasmic loop6-7, which contains two short helices (CH1 and CH2) and links the N- and C-terminal domains, is colored in yellow. The transmembrane helices are labeled with Roman numerals. (a) Side view. The positions involved in the interaction with Arg residues (295, 141, or 363) are shown by spheres (red, blue, and cyan for negative, positive, and other side chains, respectively). (b) Cytoplasmic view. The positions involved in the interaction with Arg residues (295, 141, or 363) are shown in sticks. All structure models are depicted using PyMOL.
Three cytoplasmic Arg residues, R295 in helix IX, R141 in helix V, and R363 in loop10-11, dictate three clusters of electrostatic/polar interactions between the N- and C-terminal domains (Fig. 1b). R295 holds helix V close to the C-terminal domain by forming multiple H-bonds; R141 stabilizes the interaction between helices V and X through ion pairs. Both interdomain interactions stabilize the hydrophobic patches that seal the N- and C-terminal domains at the cytoplasmic side by restricting the dynamics of helices V and X (3). Although it is not structurally well resolved, we proposed that R363 (loop10-11) could potentially have multiple interactions with the N-terminal loop2-3 and the N-terminal side of middle loop6-7, holding the N-terminal domain in an outward-facing conformation. Replacement of the three cytoplasmic Arg residues with Cys abolishes melibiose transport with little effect on protein expression and binding affinity for both melibiose and Na+, consistent with their structural role (3). On the periplasmic side of the N-terminal domain, D35 and R175 are in close proximity, which places the ends of helices I and VI together. R175 is located within a flexible region that contains multiple Gly residues (171GGADRGFG178). The role of this pair of charged residues has not been characterized. In the present communication, we characterize the role of the cytoplasmic Arg residues in MelBSt and identify a suppressor that is proposed to alter the rate of conformation change.
MATERIALS AND METHODS
Materials.[1-3H]melibiose was custom synthesized by PerkinElmer. The 2′-(N-dansyl)aminoalkyl-1-thio-β-d-galactopyranoside (D2G) was kindly provided by H. Ronald Kaback and Gérard Leblanc. Oligodeoxynucleotides were synthesized by Integrated DNA Technologies, Inc. MacConkey agar medium (lactose free) was from Difco. All other materials were reagent grade and obtained from commercial sources.
Bacterial strains and plasmids.Escherichia coli DW2 cells (melA+ melB lacZY) were used for functional studies. E. coli XL1-Blue cells were used for DNA manipulations. The expression plasmid pK95 AH/MelBSt/CHis10 (5, 24), which encodes the full-length MelBSt with L5M and a His10 tag at the C terminus (the wild type [WT]), was used for mutant construction with the QuikChange site-directed mutagenesis kit (Stratagene). All mutations were confirmed by DNA sequencing.
Protein overexpression.E. coli DW2 cells containing a given plasmid were grown in Luria-Bertani (LB) broth with 100 mg/liter of ampicillin in a 37°C shaker. The overnight cultures were diluted to 5% with LB broth supplemented with 0.5% glycerol and 100 mg/liter of ampicillin. Constitutive overexpression was obtained at 30°C for 5 h (6).
Preparation of crude membranes and Western blotting.Crude membranes from the 5-h cultures were prepared and analyzed as described previously (6). Twenty-five micrograms of crude membranes was loaded onto each lane of an SDS-16% PAGE gel. The Western blots were reacted with the penta-His horseradish peroxidase conjugate (Qiagen) and imaged by the ImageQuant LAS 4000 biomolecular imager (GE Healthcare Life Sciences). The protein concentration was measured by the micro-bicinchoninic acid (BCA) protein assay kit (Pierce).
Melibiose fermentation assay.The DW2 cells were transformed with a given plasmid, plated on a MacConkey agar plate supplemented with 30 mM melibiose (the sole carbohydrate source) and 100 mg/liter ampicillin, and incubated at 37°C for 18 h (6).
Isolation of second-site suppressor.Colonies showing yellow on the melibiose-containing MacConkey plate were kept on the bench. After 2 weeks, in one instance, the center of a yellow colony became red. The red colony was purified, and an additional mutation from GAT to GAG of the R363Q MelBSt mutant, which codes for Glu at the D35 position, was verified by DNA sequencing.
[1-3H]melibiose transport assay.E. coli DW2 cells expressing MelBSt were washed to remove Na+. Melibiose transport in the absence or presence of 20 mM NaCl or LiCl was assayed at 0.4 mM melibiose (10 mCi/mmol) unless otherwise defined (5).
Kinetics of melibiose transport.Initial rates of melibiose transport at a range of melibiose concentrations between 0.05 and 2.5 mM were obtained by a linear fit to the melibiose uptake at 0, 3, 4, 6, and 8 s, corrected by the rates obtained from nontransformed DW2 cells. The Km and Vmax values were determined by fitting a hyperbolic function to the data (OriginPro 9.0).
Preparation of RSO membrane vesicles.Right-side-out (RSO) membrane vesicles were prepared from E. coli DW2 cells by osmotic lysis (5, 25, 26); extensively washed; resuspended in 100 mM KPi, pH 7.5, at a protein concentration of 25 to 30 mg/ml; frozen in liquid N2; and stored at −80°C.
Na+-stimulation constant (K0.5Na+) for D2G fluorescence resonance energy transfer (FRET).Steady-state fluorescence measurements were performed with an Aminco-Bowman series 2 spectrometer with RSO membrane vesicles at a protein concentration of ∼1.0 mg/ml in 100 mM KPi, pH 7.5 (6, 27). With an excitation wavelength at 290 nm, the emission intensity was recorded at 490 nm. After the addition of 10 μM D2G, NaCl was consecutively added until no change in fluorescence emission occurred. An identical volume of water was used for the control. Increase in intensity (I, the difference before [I0] and after addition of NaCl) was expressed as the percentage of the I0, corrected by a dilution effect. The K0.5Na+ value was determined by hyperbolic fitting (OriginPro 9.0).
Melibiose concentration for the half-maximal displacement of bound D2G (IC50).Applying the same experimental setup (6, 27), melibiose was added stepwise to the RSO vesicle samples after addition of 10 μM D2G and 20 mM NaCl until no change in fluorescence emission occurred. An identical volume of water was added as a negative control. The decrease in intensity after each addition of melibiose was corrected by the dilution effect. The 50% inhibitory concentration (IC50) was determined by hyperbolic fitting (OriginPro 9.0).
RESULTS
Single-site mutations of Arg residues in the cytoplasmic side.As shown in the MelB of Escherichia coli (MelBEc) and MelBSt (3, 13, 21, 28, 29), mutations to Cys at position R295, R141, or R363 yield conformationally compromised proteins. Here, each position was further mutated to Lys, Glu, Gln, or Leu in a wild-type (WT) background. Most of the single-site mutation (Fig. 2a) has negligible effects on protein expression; only the mutants with the positively charged Lys at positions R141 and R363 can maintain H+-, Na+-, or Li+-coupled melibiose transport at initial rates of 33 to 66% and to the steady-state level of at least 80% of the WT value (Fig. 2b and c). Even the R363E mutant exhibits significant amounts of proteins; no transport was detected. Arg at position 295 and a positively charged residue at positions 141 and 363 are required for melibiose transport catalyzed by MelBSt.
Mutational analyses of the cytoplasmic Arg residues. (a) Twenty-five micrograms of crude membranes was loaded onto each lane of an SDS-16% PAGE gel. After transfer onto a polyvinylidene difluoride membrane, MelBSt proteins were detected with an anti-His tag antibody. (b and c) [3H]melibiose transport assays were carried out in E. coli DW2 intact cells expressing a given MelBSt mutant in 100 mM KPi (pH 7.5) and 10 mM MgSO4 at 0.7 mg/ml of protein. The transport assay was initiated by adding [3H]melibiose (0.4 mM, 10 mCi/mmol) in the absence or presence of 20 mM NaCl or LiCl. The initial rate (b), obtained by a linear fitting of melibiose uptake at 0, 5, and 10 s, and the level of melibiose accumulation (c) at 10 min, which represents the steady-state transport level, are expressed as percentages of the wild-type MelBSt values. Error bars show standard deviations (n = 2 to 3 for all mutants and n = 6 for the WT and DW2).
To test the mutational effect on the cosubstrate affinity, we quantitatively determined the Na+ activation constant (K0.5Na+) and IC50 for melibiose displacement of FRET from endogenous Trp residues to a fluorescent sugar substrate, 2′-(N-dansyl) aminoalkyl-1-thio-β-d-galactopyranoside (D2G) (5, 6, 30, 31). With right-side-out (RSO) membrane vesicles containing the WT or the R295C, R295L, R141C, R141Q, R363C, or R363Q mutant, both K0.5Na+ and IC50s are comparable to those of the WT (Table 1). These data support the previous conclusion that the primary role of R295, R141, and R363 is to facilitate conformational changes necessary for transport (3, 13, 21, 29).
Binding affinity for melibiose and Na+a
Second-site suppressor.On melibiose-containing MacConkey agar plates, DW2 cells containing WT MelBSt ferment melibiose and grow as red colonies (Fig. 3a). DW2 cells expressing the R363Q MelBSt yield yellow colonies, consistent with poor melibiose transport. Interestingly, from the R363Q mutant, a suppressor (D35→E) at the periplasmic end of helix I was isolated, and the yielded R363Q/D35E double mutant reproducibly ferments melibiose on MacConkey plates, like the WT does. The isolated or engineered R363Q/D35E mutant catalyzes Na+-coupled melibiose transport at 73% of the steady-state level of the WT (Fig. 3b, top panel). Similar recovery is observed when melibiose transport is coupled with H+ or Li+ (see Fig. S1 in the supplemental material). Furthermore, the D35→E suppressor was introduced in the inactive R363E, R363L, or R363C mutant. It is remarkable that the transport of all three mutants is significantly improved/rescued (Fig. 3b; see also Fig. S1). The steady-state level of accumulation in R363E/D35E, R363L/D35E, and R363C/D35E mutants is increased to 20%, 51%, and 91% of the WT value, respectively. Moreover, both R363C/D35E and R363Q/D35E mutants exhibit similar Km and Vmax values relative to the WT (Table 2).
Revertants. (a) Melibiose fermentation. E. coli DW2 (ΔlacY ΔlacZ melA+ ΔmelB) cells were transformed with a plasmid encoding the WT, mutant R363Q, or double mutant R363Q/D35E; plated on MacConkey agar (lactose free) containing melibiose at 30 mM; and incubated at 37°C for 18 h. (b) Melibiose transport assay of the double mutants in the presence of 20 mM NaCl. (c) Transport assay of the triple mutants. Time course of [3H]melibiose transport with intact cells expressing WT MelBSt or a given mutant carried out as described in the legend to Fig. 2 and Materials and Methods. [3H]melibiosein, intracellular melibiose. The single-site mutants are shown by green open diamonds, and corresponding second-site revertants are shown by filled blue triangles. Error bars show standard deviations (n = 2 for all mutants and n = 6 for the WT and DW2). (d) Membrane expression as described in the legend to Fig. 2.
Melibiose transport kineticsa
Suppressor D35E on other mutants.Since the D35→E mutation suppresses the R363 mutants, the D35 in all R295 and R141 mutants was mutated to Glu. The D35E mutation significantly rescues the melibiose transport of the mutants with Lys, Glu, Leu, Gln, or Cys at R295 to 50 to 81% of the WT value and those of the mutants with Cys and Leu at R141 to 28 to 40% of the WT value (Fig. 3b; see also Fig. S1 in the supplemental material). Because the R141 mutant forms salt-bridge interactions (Fig. 1b), the loss of the positive charge at R141 is expected to cause more severe effects on the protein conformational changes than mutations at the other two sites. Consistently, little or no rescue is observed with Gln or Glu at R141 (Fig. 3b). Including D351 and D354 (helix X), the three negative charges between helices V and X may prevent closure in the cytoplasmic surface, yielding the fully inactive R141E mutant. Furthermore, when combining mutations from any two of the three cytoplasmic Arg residues, the resulting triple mutants R295C/R141C/D35E, R141C/R363C/D35E, and R363C/R295C/D35E do not catalyze melibiose transport at all (Fig. 3c). It is noteworthy that the mutations have negligible effects on protein expression (Fig. 3d).
Periplasmic residues D35 and R175.D35 was replaced with Glu, Gln, or Cys in a WT background. The Gln or Cys replacement nearly abolishes transport (Fig. 4a) with significant protein expression (Fig. 4c). The Glu mutant also shows reduced activity, with 72% or 38% of the initial rate and 55% or 43% of the steady-state level of WT transport in the presence of Na+ or Li+, respectively, indicating that a negative charge at position 35 is required for active transport. The R175 at the periplasmic end of helix VI (Fig. 1a) was replaced with Lys, Gln, Asn, or Cys, but none of these mutations significantly inhibited the transport (Fig. 4b), indicating that the positive charge at position 175 is not necessary. It is surprising that Gln at position 35 with an R295K or R141C mutation ferments melibiose similarly to the WT (Fig. 4d) and also affords partial rescue in active melibiose transport (Fig. 4e).
Single D35 and R175 mutants. (a and b) Time courses of melibiose transport of D35 and R175 mutants. DW2 cells expressing either WT or a given MelBSt mutant were tested for Na+- and Li+-coupled melibiose transport as described in the legend to Fig. 2. [3H]melibiosein, intracellular melibiose. Error bars show standard deviations (n = 2). (c) Western blot. Twenty-five micrograms of crude membranes was loaded onto each lane of an SDS-16% PAGE gel. MelBSt proteins were detected with an anti-His tag antibody. (d) Melibiose fermentation. The picture was taken after a 24-h incubation. Effect of D35 mutations on R141C and R295K mutants. (e) The melibiose transport assay was carried out as described in the legend to Fig. 2. Error bars show standard deviations (n = 2). Note that the y axis is scaled differently.
DISCUSSION
The recently determined crystal structures of MelBSt suggest that three structural Arg residues (R295, R141, and R363) dictate three clusters of electrostatic/polar interactions between two domains (Fig. 1). The present extensive mutagenesis studies further support the idea that these Arg residues play critical roles in MelBSt's conformational changes, and their mutations to residues lacking a positive charge yield conformation-compromised mutant proteins (Fig. 2; Table 1). Since R295 is involved only in H-bonding interactions, even the mutation to Lys causes inactivation. The guanidinium group of Arg provides a greater number of electrostatic interactions than the amine group of Lys; thus, the Arg residue usually has a greater potential for a structural role (32) by fastening together backbones from different regions or stabilizing helix packing. It is possible that the Lys at R295 forms fewer H-bonds, affecting subsequent interactions at other sites (Fig. 1). The threading model of the inward-facing conformation of MelB (3, 4, 15) suggests that the interactions governed by the three Arg residues do not exist because the N- and C-terminal domains are separated in the cytoplasmic side. These Arg residues may interact with different positions within the domain at other conformational states. In MelBEc, it was concluded that a mutation in position R141 and a chemical modification at Cys364, a native Cys adjacent to R363, affect two different steps in the conformational transition subsequent to the sugar binding (13, 21, 29). Putting Cys in place of R141 inhibits the melibiose-induced fast electrogenic conformational transition; the alkylation of Cys364 with N-ethylmaleimide inhibits the following step. Taken all together, the consistent results from the mutational analyses, biophysical studies, and crystal structural information in both permeases support the notion that the three clusters of extramembrane electrostatic interactions play a major role in forming outward conformation and facilitating conformational cycling. It is likely that the reduced transport rate in the Arg mutants is due to the inhibition of the rate of conformational change.
It is remarkable that the periplasmic D35E mutation substantially rescues most of these cytoplasmic Arg mutations, and even D35Q partially suppresses the mutants R295K and R141C. The data imply that the H-bonding interaction may play an important role in the suppression mechanism; however, the charged Asp or polar Asn residue with a shorter side chain at position 35 does not suppress the conformational defect caused by the cytoplasmic Arg mutation. It is likely that an optimal geometry of the charged and/or polar groups between position 35 and its interacting partner is necessary. Although R175 is in close proximity to D35, replacement of R175 with Cys has no significant effect on transport, implying that the charge in R175 is not involved in the suppression. The backbone of helix VI might be the interacting site; unfortunately, this region is disordered.
D35 and the three Arg residues (R363, R295, and R141) are located on two opposite sides of the permease, and even in different domains (with respect to R363 and R295). The crystal structure reveals that the inner-layer helices I and V pack closely with outer-layer helix VI. The R295- and R141-organized multiple interactions hold the inner-layer helix V at an outward state, and R363 could also hold the outer-layer helix VI at an outward state through the connecting middle loop. Helix I, which contains the sugar-binding residue D19, has no direct interaction with a position involved in the three clusters of interactions. During the conformational change triggered by the binding of sugar, all these helices are expected to switch between the inward and outward states. It is likely that the transport-compromised mutant at position 141, 295, or 363 may hinder the helical movement at the cytoplasmic side, which is suppressed by the increased interaction at the periplasmic end through D35E by some mechanisms that are not fully understood.
It has been reported previously that some patients with GLUT1 deficiency syndrome have variants bearing a single Arg mutation of GLUT1 expressed in the brain-blood barrier, such as R153C or -H, R212C or -H, and R333W or -Q, which cause a decreased glucose uptake in the brain (33–35). In GLUT4, neutral mutation of R349 or R350 (36) has been reported to exhibit a normal ligand-binding affinity but inhibit conformational change. The mutational effects of these Arg residues in GLUT1 and GLUT4 are similar to what we found in MelBSt in this study. It is noteworthy that this type of mutation in MelBSt is significantly rescued at up to 91% of the WT value through a minor modification.
ACKNOWLEDGMENTS
This work was supported by the National Science Foundation (grant MCB-1158085), the Norman Hackerman Advanced Research Program (grant 010674-0034-2009), and the National Institutes of Health (grant R01 GM095538) to L.G.
FOOTNOTES
- Received 21 May 2014.
- Accepted 13 June 2014.
- Accepted manuscript posted online 23 June 2014.
- Address correspondence to Lan Guan, Lan.Guan{at}ttuhsc.edu.
Supplemental material for this article may be found at http://dx.doi.org/10.1128/JB.01868-14.
REFERENCES
- Copyright © 2014, American Society for Microbiology. All Rights Reserved.
