Melibiose Transport Research Articles

Atomic-Level Free Energy Landscape Reveals Cooperative Symport Mechanism of Melibiose Transporter.

The Major Facilitator Superfamily (MFS) transporters are an essential class of secondary active transporters involved in various physiological and pathological processes. The melibiose permease (MelB), which catalyzes the stoichiometric symport of the disaccharide melibiose and monovalent cations (e.g., Na+, H+, or Li+), is a key model for understanding the cation-coupled symport mechanisms. Extensive experimental data has established that positive cooperativity between the cargo melibiose and the coupling cation is central to the symport mechanism. However, the structural and energetic origins of this cooperativity remain unclear at the atomistic level for MelB and most other coupled transporters. Here, leveraging recently resolved structures in inward- and outward-facing conformations, we employed the string method and replica-exchange umbrella sampling simulation techniques to comprehensively map the all-atom free energy landscapes of the Na+-coupled melibiose translocation across the MelB in Salmonella enterica serovar Typhimurium (MelBSt), in comparison with the facilitated melibiose transport in a uniporter mutant. The simulation results unravel asymmetrical free energy profiles of melibiose translocation, which is tightly coupled to protein conformational changes in both the N- and C-terminal domains. Notably, the cytoplasmic release of the melibiose induces the simultaneous opening of an inner gate, resulting in a high-energy state of the system. Periplasmic sugar binding and cytoplasmic melibiose released are dynamically coupled with changes in the internal gating elements along the translocation pathway. The outward-facing sugar-bound state is thermodynamically most stable, while the occluded state is a transient state. The binding of Na+ facilitates melibiose translocation by increasing the melibiose-binding affinity and decreasing the overall free energy barrier and change. The cooperative binding of the two substrates results from the allosteric coupling between their binding sites instead of direct electrostatic interaction. These findings add substantial new atomic-level details into how Na+ binding facilitates melibiose translocation and deepen the fundamental understanding of the molecular basis underlying the symport mechanism of cation-coupled transporters.

Distinct roles of the major binding residues in the cation-binding pocket of the melibiose transporter MelB

Salmonella enterica serovar Typhimurium melibiose permease (MelBSt) is a prototype of the major facilitator superfamily (MFS) transporters, which play important roles in human health and diseases. MelBSt catalyzed the symport of galactosides with either Na+, Li+, or H+, but prefers the coupling with Na+. Previously, we determined the structures of the inward- and outward-facing conformation of MelBSt and the molecular recognition for galactoside and Na+. However, the molecular mechanisms for H+- and Na+-coupled symport still remain poorly understood. In this study, we solved two x-ray crystal structures of MelBSt, the cation-binding site mutants D59C at an unliganded apo-state and D55C at a ligand-bound state, and both structures display the outward-facing conformations virtually identical as published. We determined the energetic contributions of three major Na+-binding residues for the selection of Na+ and H+ by free energy simulations. Transport assays showed that the D55C mutant converted MelBSt to a solely H+-coupled symporter, and together with the free-energy perturbation calculation, Asp59 is affirmed to be the sole protonation site of MelBSt. Unexpectedly, the H+-coupled melibiose transport exhibited poor activities at greater bulky ΔpH and better activities at reversal ΔpH, supporting the novel theory of transmembrane-electrostatically localized protons and the associated membrane potential as the primary driving force for the H+-coupled symport mediated by MelBSt. This integrated study of crystal structure, bioenergetics, and free energy simulations, demonstrated the distinct roles of the major binding residues in the cation-binding pocket of MelBSt.

Mobile barrier mechanisms for Na+-coupled symport in an MFS sugar transporter

While many 3D structures of cation-coupled transporters have been determined, the mechanistic details governing the obligatory coupling and functional regulations still remain elusive. The bacterial melibiose transporter (MelB) is a prototype of major facilitator superfamily transporters. With a conformation-selective nanobody, we determined a low-sugar affinity inward-facing Na+-bound cryoEM structure. The available outward-facing sugar-bound structures showed that the N- and C-terminal residues of the inner barrier contribute to the sugar selectivity. The inward-open conformation shows that the sugar selectivity pocket is also broken when the inner barrier is broken. Isothermal titration calorimetry measurements revealed that this inward-facing conformation trapped by this nanobody exhibited a greatly decreased sugar-binding affinity, suggesting the mechanisms for substrate intracellular release and accumulation. While the inner/outer barrier shift directly regulates the sugar-binding affinity, it has little or no effect on the cation binding, which is supported by molecular dynamics simulations. Furthermore, the hydron/deuterium exchange mass spectrometry analyses allowed us to identify dynamic regions; some regions are involved in the functionally important inner barrier-specific salt-bridge network, which indicates their critical roles in the barrier switching mechanisms for transport. These complementary results provided structural and dynamic insights into the mobile barrier mechanism for cation-coupled symport.

Mobile barrier mechanisms for Na+-coupled symport in an MFS sugar transporter.

While many 3D structures of cation-coupled transporters have been determined, the mechanistic details governing the obligatory coupling and functional regulations still remain elusive. The bacterial melibiose transporter (MelB) is a prototype of major facilitator superfamily transporters. With a conformation-selective nanobody, we determined a low-sugar affinity inward-facing Na+-bound cryoEM structure. The available outward-facing sugar-bound structures showed that the N- and C-terminal residues of the inner barrier contribute to the sugar selectivity. The inward-open conformation shows that the sugar selectivity pocket is also broken when the inner barrier is broken. Isothermal titration calorimetry measurements revealed that this inward-facing conformation trapped by this nanobody exhibited a greatly decreased sugar-binding affinity, suggesting the mechanisms for substrate intracellular release and accumulation. While the inner/outer barrier shift directly regulates the sugar-binding affinity, it has little or no effect on the cation binding, which is supported by molecular dynamics simulations. Furthermore, the hydron/deuterium exchange mass spectrometry analyses allowed us to identify dynamic regions; some regions are involved in the functionally important inner barrier-specific salt-bridge network, which indicates their critical roles in the barrier switching mechanisms for transport. These complementary results provided structural and dynamic insights into the mobile barrier mechanism for cation-coupled symport.

In vivo and in vitro characterizations of melibiose permease (MelB) conformation-dependent nanobodies reveal sugar-binding mechanisms

Salmonella enterica serovar Typhimurium melibiose permease (MelBSt) is a prototype of the Na+-coupled major facilitator superfamily (MFS) transporters, which are important for the cellular uptake of molecules including sugars and small drugs. Although the symport mechanisms have been well studied, mechanisms of substrate binding and translocation remain enigmatic. We have previously determined the sugar-binding site of outward-facing MelBSt by crystallography. To obtain other key kinetic states, here we raised camelid single-domain nanobodies (Nbs) and carried out a screening against the WT MelBSt under four ligand conditions. We applied an in vivo cAMP-dependent two-hybrid assay to detect interactions of Nbs with MelBSt and melibiose transport assays to determine the effects on MelBSt functions. We found that all selected Nbs showed partial to complete inhibitions of MelBSt transport activities, confirming their intracellular interactions. A group of Nbs (714, 725, and 733) was purified, and isothermal titration calorimetry measurements showed that their binding affinities were significantly inhibited by the substrate melibiose. When titrating melibiose to the MelBSt/Nb complexes, Nb also inhibited the sugar-binding. However, the Nb733/MelBSt complex retained binding to the coupling cation Na+ and also to the regulatory enzyme EIIAGlc of the glucose-specific phosphoenolpyruvate/sugar phosphotransferase system. Further, EIIAGlc/MelBSt complex also retained binding to Nb733 and formed a stable super-complex. All data indicated that MelBSt trapped by Nbs retained its physiological functions and the trapped conformation is similar to that bound by the physiological regulator EIIAGlc. Therefore, these conformational Nbs can be useful tools for further structural, functional, and conformational analyses.

Production, homology modeling and mutagenesis studies on GlcH glucose transporter from Prochlorococcus sp. strain SS120

The marine cyanobacterium Prochlorococcus is one of the main primary producers on Earth, which can take up glucose by using the high affinity, multiphasic transporter GlcH. We report here the overexpression of glcH from Prochlorococcus marinus strain SS120 in Escherichia coli. Modeling studies of GlcH using the homologous MelB melibiose transporter from Salmonella enterica serovar Typhimurium showed high conservation at the overall fold. We observed that an important structural interaction, mediated by a strong hydrogen bond between D8 and R141, is conserved in Prochlorococcus, although the corresponding amino acids in MelB from Salmonella are different. Biased docking studies suggested that when glucose reaches the pocket of the transporter and interacts with D8 and R141, the hydrogen bond network in which these residues are involved could be disrupted, favoring a conformational change with the subsequent translocation of the glucose molecule towards the cytoplasmic region of the pmGlcH structure. Based on these theoretical predictions and on the conservation of N117 and W348 in other MelB structures, D8, N117, R141 and W348 were mutated to glycine residues. Their key role in glucose transport was evaluated by glucose uptake assays. N117G and W348G mutations led to 17 % decrease in glucose uptake, while D8G and R141G decreased the glucose transport by 66 % and 92 % respectively. Overall, our studies provide insights into the Prochlorococcus 3D-structure of GlcH, paving the way for further analysis to understand the features which are involved in the high affinity and multiphasic kinetics of this transporter.

Molecular recognition mechanism for the galactoside binding in a melibiose transporter MelB

Molecular recognition mechanism for the galactoside binding in a melibiose transporter MelB

X-ray crystallography reveals molecular recognition mechanism for sugar binding in a melibiose transporter MelB

Major facilitator superfamily_2 transporters are widely found from bacteria to mammals. The melibiose transporter MelB, which catalyzes melibiose symport with either Na+, Li+, or H+, is a prototype of the Na+-coupled MFS transporters, but its sugar recognition mechanism has been a long-unsolved puzzle. Two high-resolution X-ray crystal structures of a Salmonella typhimurium MelB mutant with a bound ligand, either nitrophenyl-α-d-galactoside or dodecyl-β-d-melibioside, were refined to a resolution of 3.05 or 3.15 Å, respectively. In the substrate-binding site, the interaction of both galactosyl moieties on the two ligands with MelBSt are virturally same, so the sugar specificity determinant pocket can be recognized, and hence the molecular recognition mechanism for sugar binding in MelB has been deciphered. The conserved cation-binding pocket is also proposed, which directly connects to the sugar specificity pocket. These key structural findings have laid a solid foundation for our understanding of the cooperative binding and symport mechanisms in Na+-coupled MFS transporters, including eukaryotic transporters such as MFSD2A.

Topological analysis of TMEM180, a newly identified membrane protein that is highly expressed in colorectal cancer cells

New target molecules for diagnosis of and drug development for colorectal cancer (CRC) are always in great demand. Previously, we identified a new colorectal cancer–specific protein, TMEM180, and successfully developed an anti-TMEM180 monoclonal antibody (mAb) for the diagnosis and treatment of CRC. Although TMEM180 is classified as a member of the cation symporter family and multi-pass membrane protein, little is known about its function. In this study, we examined topology of this membrane protein and analyzed its function. Using a homology model of human TMEM180, we experimentally determined that the protein has 12 transmembrane domains, and that its N-terminal and C-termini are exposed extracellularly. Moreover, we found that the putative cation-binding site of TMEM180 is conserved among orthologs, and that its position is similar to that of melibiose transporter MelB. These results suggest that TMEM180 acts as a cation symporter. Our topological analysis based on the homology model provides insight into functional and structural roles of TMEM180 that may help to elucidate the pathology of CRC.

Structural and functional characterization of protein\u2013lipid interactions of the Salmonella typhimurium melibiose transporter MelB

BackgroundMembrane lipids play critical roles in the structure and function of membrane-embedded transporters. Salmonella typhimurium MelB (MelBSt) is a symporter coupling melibiose translocation with a cation (Na+, Li+, or H+). We present an extensive study on the effects of specific phospholipids on the structure of MelBSt and the melibiose transport catalyzed by this protein.ResultsLipidomic analysis and thin-layer chromatography (TLC) experiments reveal that at least one phosphatidylethanolamine (PE) and one phosphatidylglycerol (PG) molecule associate with MelBSt at high affinities. Solid-state nuclear magnetic resonance (ssNMR) spectroscopy experiments confirmed the presence of lipid tails and glycerol backbones that co-purified with MelBSt; headgroups of PG were also observed. Studies with lipid-engineered strains, including PE-deficient, cardiolipin (CL)- and PG-deficient, or CL-deficient strains, show that lack of PE or PG, however not CL, largely inhibits both H+- and Na+-coupled melibiose active transport to different extents. Interestingly, neither the co-substrate binding (melibiose or Na+) nor MelBSt folding and stability are affected by changing lipid compositions. Remarkably, the delipidated MelBSt with only 2–3 bound lipids, regardless of the headgroup species, also exhibits unchanged melting temperature values as shown by circular dichroism spectroscopy.Conclusions(1) Lipid tails and glycerol backbones of interacting PE and PG may contribute to the stability of the structure of MelBSt. (2) The headgroups of PE and PG, but not of CL, play important roles in melibiose transport; however, lipid headgroups do not modulate the folding and stability of MelBSt.

Helical unwinding and side-chain unlocking unravel the outward open conformation of the melibiose transporter.

Molecular dynamics simulations have been used to study the alternate access mechanism of the melibiose transporter from Escherichia coli. Starting from the outward-facing partially occluded form, 2 out of 12 simulations produced an outward full open form and one partially open, whereas the rest yielded fully or partially occluded forms. The shape of the outward-open form resembles other outward-open conformations of secondary transporters. During the transporter opening, conformational changes in some loops are followed by changes in the periplasm region of transmembrane helix 7. Helical curvature relaxation and unlocking of hydrophobic and ionic locks promote the outward opening of the transporter making accessible the substrate binding site. In particular, FRET studies on mutants of conserved aromatic residues of extracellular loop 4 showed lack of substrate binding, emphasizing the importance of this loop for making crucial interactions that control the opening of the periplasmic side. This study indicates that the alternate access mechanism for the melibiose transporter fits better into a flexible gating mechanism rather than the archetypical helical rigid-body rocker-switch mechanism.

Suppression of conformation-compromised mutants of Salmonella enterica serovar Typhimurium MelB.

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.

Functional Inhibition of Sugar Transport by a Designed Novel Protein

Designed ankyrin repeat proteins have been shown to be useful for different applications such as structural, mechanistic, clinical studies, as well as drug development. We have constructed a combinatorial DNA library encoding ankyrin repeat proteins by PCR-based assembly. The ankyrin proteins have both N- and C-terminal caps and five internal repeats (called N5C). The estimated diversity of the library is higher than 1020. Applying circular dichroism spectroscopy, four purified proteins exhibit high melting temperature (> 95 °C). We have tested the constructed library for the selection of specific ankyrin proteins targeting the melibiose transport in Escherichia coli. The melibiose permease is one of few overexpressed proteins during bacterial infection for gaining energy. Using cells with plasmid-encoded constructed library and chromosomally-encoded MelA, the alpha-galactosidase, and MelB, the melibiose permease, we have collected a group of N5C candidates that alter the melibiose fermentation with no inhibition of glucose fermentation, indicating no effect on the downstream utilization pathways. Three candidates show significant inhibition of melibiose transport. Further tests, using sugar transport assay on different genetic backgrounds, MelA activity assay, and protein-protein interaction assay, have been applied to identify specific effect. These preliminary studies suggested that one N5C protein possibly inhibits mel operon. These bio-reagents could be potentially applied to study the sugar transport during bacterial infection and also facilitate transporter proteins crystallography.

Ionic Locks in Melibiose Permease: Scope towards GLUT1 Deficiency Syndrome

The recently solved 3D crystal structures reveal that the Na+-coupled melibiose permease of Salmonella typhimurium (MelBSt) is a member of the Major Facilitator Superfamily (MFS). These inside-closed conformations show that Arg residues at positions 295 (Helix IX), 141 (Helix V), and 363 (Loop10-11) govern three clusters of electrostatic interactions for stabilizing the sealed internal cavity between N- and C-terminal domains at the cytoplasmic side. The cluster of these electrostatic interactions is designated as ionic lock. Except Lys at position 141 or 363, the replacement of R295, R141, or R363 individually with Cys, Gln, Glu, Leu, or Lys shows inhibition of active transport of melibiose to a level of 2 - 25 % of the WT, with little effect on the binding affinities for both sugar and Na+. Interestingly, a suppressor D35E mutation at the periplasmic end of helix I was spontaneously isolated from the R363Q mutant. Remarkably, introduction of D35E mutation with each conformation-compromised mutant of R295, R141 (except R141E), or R363 significantly rescues the melibiose transport to a level up to 90 % of the WT. However, D35E mutation fails to restore the activity if two lock sites are mutated by Cys simultaneously at any combination. Previous threading model of MelBSt at inside-open conformation show that all the three lock sites are at unlocked state. We conclude that these ionic locks are cooperative and involved in the transport-required global conformational changes by an alteration of locking and unlocking processes. Strikingly, these ionic locks are conserved in MFS permeases. Furthermore, they link with human diseases; e.g., some patients with GLUT1 Deficiency Syndrome are due to a single mutation of Arg at predicted ionic locks of the glucose transporter GLUT1 expressed in the blood-brain barrier.

The substitution of Arg149 with Cys fixes the melibiose transporter in an inward-open conformation

The melibiose transporter from Escherichia coli (MelB) can use the electrochemical energy of either H+, Na+ or Li+ to transport the disaccharide melibiose to the cell interior. By using spectroscopic and biochemical methods, we have analyzed the role of Arg149 by mutagenesis. According to Fourier transform infrared difference and fluorescence spectroscopy studies, R149C, R149Q and R149K all bind substrates in proteoliposomes, where the protein is disposed inside-out. Analysis of right-side-out (RSO) and inside-out (ISO) membrane vesicles showed that the functionally active R149Q and R149K mutants could bind externally added fluorescent sugar analog in both types of vesicles. In contrast, the non-transporting R149C mutant does bind the fluorescent sugar analog as well as melibiose and Na+ in ISO, but not in RSO vesicles. Therefore, the mutation of Arg149 into cysteine restrains the orientation of transporter to an inward-open conformation, with the inherent consequences of a) reducing the frequency of access of outer substrates to the binding sites, and b) impairing active transport. It is concluded that Arg149, most likely located in the inner (cytoplasmic) half of transmembrane helix 5, is critically involved in the reorientation mechanism of the substrate-binding site accessibility in MelB.

Reduced Na + Affinity Increases Turnover of Salmonella enterica Serovar Typhimurium MelB

The melibiose permease of Salmonella enterica serovar Typhimurium (MelB(St)) catalyzes symport of melibiose with Na(+), Li(+), or H(+). Bioinformatics and mutational analyses indicate that a conserved Gly117 (helix IV) is a component of the Na(+)-binding site. In this study, Gly117 was mutated to Ser, Asn, or Cys. All three mutations increase the maximum rate (V(max)) for melibiose transport in Escherichia coli DW2 and greatly decrease Na(+) affinity, indicating that intracellular release of Na(+) is facilitated. Rapid melibiose transport, particularly by the G117N mutant, triggers osmotic lysis in the lag phase of growth. The findings support the previous conclusion that Gly117 plays an important role in cation binding and translocation. Furthermore, a spontaneous second-site mutation (P148L between loop(4-5) and helix V) in the G117C mutant prevents cell lysis. This mutation significantly decreases V(max) with little effect on cosubstrate binding in G117C, G117S, and G117N mutants. Thus, the P148L mutation specifically inhibits transport velocity and thereby blocks the lethal effect of elevated melibiose transport in the Gly117 mutants.

Inhibition of cell growth by an elevated turnover number of melibiose/Na+ symport catalyzed by melibiose permease of Salmonella typhimurium

Melibiose permease of Salmonella typhimurium (MelBSt) catalyzes melibiose/cation (H+, Na+ or Li+) symport. The transport mechanism is still poorly understood. Threading model indicates that a conserved Gly117 (helix IV) is part of the Na+‐binding site. Recently, mutation analyses suggested that Gly117 plays an important role in cation binding and translocation in MelBSt. In this study, we observed that Gly117→Cys MelBSt mutant drastically inhibited cell growth in the presence of melibiose in a concentration‐dependent fashion. The mutant G117C increased Vmax value for melibiose transport by >3 fold with little changes in Km and protein expression. A spontaneous mutation Pro148→Leu was isolated from mutant G117C. Strikingly, the double mutant G117C/P148L grew at a similar rate in the absence or presence of melibiose and also largely decreased the Vmax value, with 2‐fold lower than obtained from WT. A good correlation between the Vmax values and the cell‐growth rates in the presence of melibiose indicates that the Na+/melibiose symport dissipates cell membrane potential. With a largely elevated turnover number of melibiose transport, it is likely that the cotransported Na+ may collapse the membrane potential and inhibit cell growth.This work was supported by Texas Norman Hackerman Advanced Research Program 010674‐0034‐2009 to LG.

Role of Gly117 in the Cation/Melibiose Symport of MelB of Salmonella typhimurium

The melibiose permease of Salmonella typhimurium (MelB(St)) catalyzes symport of melibiose with Na(+), Li(+), or H(+), and bioinformatics analysis indicates that a conserved Gly117 (helix IV) is part of the Na(+)-binding site. We mutated Gly117 to Ala, Pro, Trp, or Arg; the effects on melibiose transport and binding of cosubstrates depended on the physical-chemical properties of the side chain. Compared with WT MelB(St), the Gly117 → Ala mutant exhibited little difference in either cosubstrate binding or stimulation of melibiose transport by Na(+) or Li(+), but all other mutations reduced melibiose active transport and efflux, and decreased the apparent affinity for Na(+). The bulky Trp at position 117 caused the greatest inhibition of melibiose binding, and Gly117 → Arg yielded less than a 4-fold decrease in the apparent affinity for melibiose at saturating Na(+) or Li(+) concentration. Remarkably, the mutant Gly117 → Arg catalyzed melibiose exchange in the presence of Na(+) or Li(+), but did not catalyze melibiose translocation involving net flux of the coupling cation, indicating that sugar is released prior to release of the coupling cation. Taken together, the findings are consistent with the notion that Gly117 plays an important role in cation binding and translocation.

Functional characterization of a eukaryotic melibiose transporter.

Pathogenic fungi drastically affect plant health and cause significant losses in crop yield and quality. In spite of their impact, little is known about the carbon sources used by these fungi in planta and about the fungal transporters importing sugars from the plant-fungus interface. Here, we report on the identification and characterization of MELIBIOSE TRANSPORTER1 (MBT1) from the hemibiotrophic fungus Colletotrichum graminicola (teleomorph Glomerella graminicola), the causal agent of leaf anthracnose and stalk rot disease in maize (Zea mays). Functional characterization of the MBT1 protein in baker's yeast (Saccharomyces cerevisiae) expressing the MBT1 cDNA revealed that α-D-galactopyranosyl compounds such as melibiose, galactinol, and raffinose are substrates of MBT1, with melibiose most likely being the preferred substrate. α-D-glucopyranosyl disaccharides like trehalose, isomaltose, or maltose are also accepted by MBT1, although with lower affinities. The MBT1 gene shows low and comparable expression levels in axenically grown C. graminicola and upon infection of maize leaves both during the initial biotrophic development of the fungus and during the subsequent necrotrophic phase. Despite these low levels of MBT1 expression, the MBT1 protein allows efficient growth of C. graminicola on melibiose as sole carbon source in axenic cultures. Although Δmbt1 mutants are unable to grow on melibiose, they do not show virulence defects on maize.

Mechanism of Melibiose/Cation Symport of the Melibiose Permease of Salmonella typhimurium

The MelB permease of Salmonella typhimurium (MelB-ST) catalyzes the coupled symport of melibiose and Na(+), Li(+), or H(+). In right-side-out membrane vesicles, melibiose efflux is inhibited by an inwardly directed gradient of Na(+) or Li(+) and stimulated by equimolar concentrations of internal and external Na(+) or Li(+). Melibiose exchange is faster than efflux in the presence of H(+) or Na(+) and stimulated by an inwardly directed Na(+) gradient. Thus, sugar is released from MelB-ST externally prior to the release of cation in agreement with current models proposed for MelB of Escherichia coli (MelB-EC) and LacY. Although Li(+) stimulates efflux, and an outwardly directed Li(+) gradient increases exchange, it is striking that internal and external Li(+) with no gradient inhibits exchange. Furthermore, Trp → dansyl FRET measurements with a fluorescent sugar (2'-(N-dansyl)aminoalkyl-1-thio-β-D-galactopyranoside) demonstrate that MelB-ST, in the presence of Na(+) or Li(+), exhibits (app)K(d) values of ∼1 mM for melibiose. Na(+) and Li(+) compete for a common binding pocket with activation constants for FRET of ∼1 mM, whereas Rb(+) or Cs(+) exhibits little or no effect. Taken together, the findings indicate that MelB-ST utilizes H(+) in addition to Na(+) and Li(+). FRET studies also show symmetrical emission maximum at ∼500 nm with MelB-ST in the presence of 2'-(N-dansyl)aminoalkyl-1-thio-β-D-galactopyranoside and Na(+), Li(+), or H(+), which implies a relatively homogeneous distribution of conformers of MelB-ST ternary complexes in the membrane.