Eukaryotic major facilitator superfamily transporter modeling based on the prokaryotic GlpT crystal structure

The major facilitator superfamily (MFS) of transporters represents the largest family of secondary active transporters and has a diverse range of substrates. With structural information for four MFS transporters, we can see a strong structural commonality suggesting, as predicted, a common architecture for MFS transporters. The rate for crystal structure determination of MFS transporters is slow, making modeling of both prokaryotic and eukaryotic transporters more enticing. In this review, models of eukaryotic transporters Glut1, G6PT, OCT1, OCT2 and Pho84, based on the crystal structures of the prokaryotic GlpT, based on the crystal structure of LacY are discussed. The techniques used to generate the different models are compared. In addition, the validity of these models and the strategy of using prokaryotic crystal structures to model eukaryotic proteins are discussed. For comparison, E. coli GlpT was modeled based on the E. coli LacY structure and compared to the crystal structure of GlpT demonstrating that experimental evidence is essential for accurate modeling of membrane proteins.     

Eukaryotic major facilitator superfamily transporter modeling based on the prokaryotic GlpT crystal structure (Review)

The major facilitator superfamily (MFS) of transporters represents the largest family of secondary active transporters and has a diverse range of substrates. With structural information for four MFS transporters, we can see a strong structural commonality suggesting, as predicted, a common architecture for MFS transporters. The rate for crystal structure determination of MFS transporters is slow, making modeling of both prokaryotic and eukaryotic transporters more enticing. In this review, models of eukaryotic transporters Glut1, G6PT, OCT1, OCT2 and Pho84, based on the crystal structures of the prokaryotic GlpT, based on the crystal structure of LacY are discussed. The techniques used to generate the different models are compared. In addition, the validity of these models and the strategy of using prokaryotic crystal structures to model eukaryotic proteins are discussed. For comparison, E. coli GlpT was modeled based on the E. coli LacY structure and compared to the crystal structure of GlpT demonstrating that experimental evidence is essential for accurate modeling of membrane proteins.     

Integration of Evolutionary Features for the Identification of Functionally Important Residues in Major Facilitator Superfamily Transporters

The identification of functionally important residues is an important challenge for understanding the molecular mechanisms of proteins. Membrane protein transporters operate two-state allosteric conformational changes using functionally important cooperative residues that mediate long-range communication from the substrate binding site to the translocation pathway. In this study, we identified functionally important cooperative residues of membrane protein transporters by integrating sequence conservation and co-evolutionary information. A newly derived evolutionary feature, the co-evolutionary coupling number, was introduced to measure the connectivity of co-evolving residue pairs and was integrated with the sequence conservation score. We tested this method on three Major Facilitator Superfamily (MFS) transporters, LacY, GlpT, and EmrD. MFS transporters are an important family of membrane protein transporters, which utilize diverse substrates, catalyze different modes of transport using unique combinations of functional residues, and have enough characterized functional residues to validate the performance of our method. We found that the conserved cores of evolutionarily coupled residues are involved in specific substrate recognition and translocation of MFS transporters. Furthermore, a subset of the residues forms an interaction network connecting functional sites in the protein structure. We also confirmed that our method is effective on other membrane protein transporters. Our results provide insight into the location of functional residues important for the molecular mechanisms of membrane protein transporters.     

Molecular mechanisms of sugar transport across mammalian and microbial cell membranes

Recent DNA cloning studies have revealed the existence of a large family of homologous sugar transporters in both prokaryotic and eukaryotic organisms. The family includes passive transporters typical of mammalian tissues and active, H(+)-linked sugar transporters from bacteria. Each of these transporters characteristically contains two groups of six putative membrane-spanning alpha-helices separated by a large, hydrophilic, cytoplasmic region. Both the N-terminal and C-terminal regions of the sequence are also predicted to be cytoplasmic. Biophysical and other studies on the human erythrocyte glucose transporter, the only member of the family so far isolated in functional form, suggest that the membrane-spanning alpha-helices associate to form a hydrophilic channel or a substrate-binding cleft extending across the membrane. It is likely that the mechanism of substrate translocation involves alternate exposure of the substrate-binding site to each face of the membrane via a conformational change. Studies in progress on the erythrocyte transporter are beginning to identify regions of the protein involved in substrate binding and the conformational change, and should throw light on the mechanism of sugar translocation in the sugar transporter family as a whole.     

Conserved cytoplasmic loops are important for both the transport and chemotaxis functions of PcaK, a protein from Pseudomonas putida with 12 membrane-spanning regions.

Chemotaxis to the aromatic acid 4-hydroxybenzoate (4-HBA) by Pseudomonas putida is mediated by PcaK, a membrane-bound protein that also functions as a 4-HBA transporter. PcaK belongs to the major facilitator superfamily (MFS) of transport proteins, none of which have so far been implicated in chemotaxis. Work with two well-studied MFS transporters, LacY (the lactose permease) and TetA (a tetracycline efflux protein), has revealed two stretches of amino acids located between the second and third (2-3 loop) and the eighth and ninth (8-9 loop) transmembrane regions that are required for substrate transport. These sequences are conserved among most MFS transporters, including PcaK. To determine if PcaK has functional requirements similar to those of other MFS transport proteins and to analyze the relationship between the transport and chemotaxis functions of PcaK, we generated strains with mutations in amino acid residues located in the 2-3 and 8-9 loops of PcaK. The mutant proteins were analyzed in 4-HBA transport and chemotaxis assays. Cells expressing mutant PcaK proteins had a range of phenotypes. Some transported at wild-type levels, while others were partially or completely defective in 4-HBA transport. An aspartate residue in the 8-9 loop that has no counterpart in LacY and TetA, but is conserved among members of the aromatic acid/H(+) symporter family of the MFS, was found to be critical for 4-HBA transport. These results indicate that conserved amino acids in the 2-3 and 8-9 loops of PcaK are required for 4-HBA transport. Amino acid changes that decreased 4-HBA transport also caused a decrease in 4-HBA chemotaxis, but the effect on chemotaxis was sometimes slightly more severe. The requirement of PcaK for both 4-HBA transport and chemotaxis demonstrates that P. putida has a chemoreceptor that differs from the classical chemoreceptors described for Escherichia coli and Salmonella typhimurium.     

Sugar recognition by CscB and LacY

The sucrose permease (CscB) and lactose permease (LacY) of Escherichia coli belong to the oligosaccharide/H(+) symporter subfamily of the major facilitator superfamily, and both catalyze sugar/H(+) symport across the cytoplasmic membrane. Thus far, there is no common substrate for the two permeases; CscB transports sucrose, and LacY is highly specific for galactopyranosides. Determinants for CscB sugar specificity are unclear, but the structural organization of key residues involved in sugar binding appears to be similar in CscB and LacY. In this study, several sugars containing galactopyranosyl, glucopyranosyl, or fructofuranosyl moieties were tested for transport with cells overexpressing either CscB or LacY. CscB recognizes not only sucrose but also fructose and lactulose, but glucopyranosides are not transported and do not inhibit sucrose transport. The findings indicate that CscB exhibits practically no specificity with respect to the glucopyranosyl moiety of sucrose. Inhibition of sucrose transport by CscB tested with various fructofuranosides suggests that the C(3)-OH group of the fructofuranosyl ring may be important for recognition by CscB. Lactulose is readily transported by LacY, where specificity is directed toward the galactopyranosyl ring, and the affinity of LacY for lactulose is similar to that observed for lactose. The studies demonstrate that the substrate specificity of CscB is directed toward the fructofuranosyl moiety of the substrate, while the specificity of LacY is directed toward the galactopyranosyl moiety.     

Lactose permease and the alternating access mechanism

Crystal structures of the lactose permease of Escherichia coli (LacY) reveal 12, mostly irregular transmembrane α-helices surrounding a large cavity open to the cytoplasm and a tightly sealed periplasmic side (inward-facing conformation) with the sugar-binding site at the apex of the cavity and inaccessible from the periplasm. However, LacY is highly dynamic, and binding of a galactopyranoside causes closing of the inward-facing cavity with opening of a complementary outward-facing cavity. Therefore, the coupled, electrogenic translocation of a sugar and a proton across the cytoplasmic membrane via LacY very likely involves a global conformational change that allows alternating access of sugar- and H(+)-binding sites to either side of the membrane. Here the various biochemical and biophysical approaches that provide strong support for the alternating access mechanism are reviewed. Evidence is also presented indicating that opening of the periplasmic cavity is probably the limiting step for binding and perhaps transport.     

A model for coupling of H(+) and substrate fluxes based on "time-sharing" of a common binding site

Both prokaryotic and eukaryotic cells contain an array of membrane transport systems maintaining the cellular homeostasis. Some of them (primary pumps) derive energy from redox reactions, ATP hydrolysis, or light absorption, whereas others (ion-coupled transporters) utilize ion electrochemical gradients for active transport. Remarkable progress has been made in understanding the molecular mechanism of coupling in some of these systems. In many cases carboxylic residues are essential for either binding or coupling. Here we suggest a model for the molecular mechanism of coupling in EmrE, an Escherichia coli 12-kDa multidrug transporter. EmrE confers resistance to a variety of toxic cations by removing them from the cell interior in exchange for two protons. EmrE has only one membrane-embedded charged residue, Glu-14, which is conserved in more than 50 homologous proteins. We have used mutagenesis and chemical modification to show that Glu-14 is part of the substrate-binding site. Its role in proton binding and translocation was shown by a study of the effect of pH on ligand binding, uptake, efflux, and exchange reactions. The studies suggest that Glu-14 is an essential part of a binding site, which is common to substrates and protons. The occupancy of this site by H(+) and substrate is mutually exclusive and provides the basis of the simplest coupling for two fluxes.     

The Alternating Access Transport Mechanism in LacY

Lactose permease of Escherichia coli (LacY) is highly dynamic, and sugar binding causes closing of a large inward-facing cavity with opening of a wide outward-facing hydrophilic cavity. Therefore, lactose/H(+) symport via LacY very likely involves a global conformational change that allows alternating access of single sugar- and H(+)-binding sites to either side of the membrane. Here, in honor of Stephan H. White's seventieth birthday, we review in camera the various biochemical/biophysical approaches that provide experimental evidence for the alternating access mechanism.     

Conservation of residues involved in sugar/H(+) symport by the sucrose permease of Escherichia coli relative to lactose permease

Building a three-dimensional model of the sucrose permease of Escherichia coli (CscB) with the X-ray crystal structure lactose permease (LacY) as template reveals a similar overall fold for CscB. Moreover, despite only 28% sequence identity and a marked difference in substrate specificity, the structural organization of the residues involved in sugar-binding and H(+) translocation is conserved in CscB. Functional analyses of mutants in the homologous key residues provide strong evidence that they play a similar critical role in the mechanisms of CscB and LacY.     

NhaA of Escherichia coli, as a model of a pH-regulated Na+/H+antiporter

Na(+)/H(+) antiporters are ubiquitous membrane proteins that are involved in homeostasis of H(+) and Na(+) throughout the biological kingdom. Corroborating their role in pH homeostasis, many of the Na(+)/H(+) antiporter proteins are regulated directly by pH. The pH regulation of NhaA, the Escherichia coli Na(+)/H(+) antiporter (EcNhaA), as of other, both eukaryotic and prokaryotic Na(+)/H(+) antiporters, involves a pH sensor and conformational changes in different parts of the protein that transduce the pH signal into a change in activity. Thus, residues that affect the pH response, the translocation or both activities cluster in separate domains along the antiporter molecules. Importantly, in the NhaA family, these domains are conserved. Helix-packing model of EcNhaA based on cross-linking data suggests, that in the three dimensional structure of NhaA, residues that affect the pH response may be in close proximity, forming a single pH sensitive domain. Therefore, it is suggested that, despite considerable differences in the primary structure of the antiporters from the bacterial NhaA to the mammalian NHEs, their three-dimensional architectures are conserved. Test of this possibility awaits the atomic resolution of the 3D structure of the antiporters.     

Homologous sugar transport proteins in Escherichia coli and their relatives in both prokaryotes and eukaryotes

Separate proteins for proton-linked transport of D-xylose, L-arabinose, D-galactose, L-rhamnose and L-fucose into Escherichia coli are being studied. By cloning and sequencing the appropriate genes, the amino acid sequences of proteins for D-xylose/H+ symport (XylE), L-arabinose/H+ symport (AraE), and part of the protein for D-galactose/H+ symport (GalP) have been determined. These are homologous, with at least 28% identical amino acid residues conserved in the aligned sequences, although their primary sequences are not similar to those of other E. coli transport proteins for lactose, melibiose, or D-glucose. However, they are equally homologous to the passive D-glucose transport proteins from yeast, rat brain, rat adipocytes, human erythrocytes, human liver, and a human hepatoma cell line. The substrate specificity of GalP from E. coli is similar to that of the mammalian glucose transporters. Furthermore, the activities of GalP, AraE and the mammalian glucose transporters are all inhibited by cytochalasin B and N-ethylmaleimide. Conserved residues in the aligned sequences of the bacterial and mammalian transporters are identified, and the possible roles of some in sugar binding, cation binding, cytochalasin binding, and reaction with N-ethylmaleimide are discussed. Each protein is independently predicted to form 12 hydrophobic, membrane-spanning alpha-helices with a central hydrophilic segment, also comprised of alpha-helix. This unifying structural model of the sugar transporters shares features with other ion-linked transport proteins for citrate or tetracycline.     

ATP binding cassette systems: structures, mechanisms, and functions

ATP-binding cassette (ABC) systems are found in all three domains of life and in some giant viruses and form one of the largest protein superfamilies. Most family members are transport proteins that couple the free energy of ATP hydrolysis to the translocation of solutes across a biological membrane. The energizing module is also used to drive non-transport processes associated, e.g., with DNA repair and protein translation. Many ABC proteins are of considerable medical importance. In humans, dysfunction of at least eighteen out of 49 ABC transporters is associated with disease, such as cystic fibrosis, Tangier disease, adrenoleukodystrophy or Stargardt’s macular degeneration. In prokaryotes, ABC proteins confer resistance to antibiotics, secrete virulence factors and envelope components, or mediate the uptake of a large variety of nutrients. Canonical ABC transporters share a common structural organization comprising two transmembrane domains (TMDs) that form the translocation pore and two nucleotide-binding domains (NBDs) that bind and hydrolyze ATP. In this Mini-Review, we summarize recent structural and biochemical data obtained from both prokaryotic and eukaryotic model systems.     

Protein exporter function and in vitro ATPase activity are correlated in ABC-domain mutants of HlyB

The Escherichia coli toxin exporter HlyB comprises an integral membrane domain fused to a cytoplasmic domain of the ATP-binding casette (ABC) super-family, and it directs translocation of the 110kDa haemolysin protein out of the bacterial cell without using an N-terminal secretion signal peptide. We have exploited the ability to purify the soluble HlyB ABC domain as a fusion with glutathione S-transferase to obtain a direct correlation of the in vivo export of protein by HlyB with the degree of ATP binding and hydrolysis measured in vitro. Mutations in residues that are invariant or highly conserved in the ATP-binding fold and glycine-rich linker peptide of prokaryotic and eukaryotic ABC transporters caused a complete less of both HlyB exporter function and ATPase activity in proteins still able to bind ATP effectively and undergo ATP-induced conformational change. Mutation of less-conserved residues caused reduced export and ATP hydrolysis, but not ATP binding, whereas substitutions of poorly conserved residues did not impair activity either in vivo or in vitro. The data show that protein export by HlyB has an absolute requirement for the hydrolysis of ATP bound by its cytoplasmic domain and indicate that comparable mutations that disable other prokaryotic and eukaryotic ABC transporters also cause a specific loss of enzymatic activity.     

3D model of the Escherichia coli multidrug transporter MdfA reveals an essential membrane-embedded positive charge

MdfA is an Escherichia coli multidrug transporter of the major facilitator superfamily (MFS) of secondary transporters. Although several aspects of multidrug recognition by MdfA have been characterized, better understanding the detailed mechanism of its function requires structural information. Previous studies have modeled the 3D structures of MFS proteins, based on the X-ray structure of LacY and GlpT. However, because of poor sequence homology, between LacY, GlpT, and MdfA additional constraints were required for a reliable homology modeling. Using an algorithm that predicts the angular orientation of each transmembrane helix (TM) (kPROT), we obtained a remarkably similar pattern for the 12 TMs of MdfA and those of GlpT and LacY, suggesting that they all have similar helix packing. Consequently, a 3D model was constructed for MdfA by structural alignment with LacY and GlpT, using the kPROT results as an additional constraint. Further refinement and a preliminary evaluation of the model were achieved by correlated mutation analysis and the available experimental data. Surprisingly, in addition to the previously characterized membrane-embedded glutamate at position 26, the model suggests that Asp34 and Arg112 are located within the membrane, on the same face of the cavity as Glu26. Importantly, Arg112 is evolutionarily conserved in secondary drug transporters, and here we show that a positive charge at this position is absolutely essential for multidrug transport by MdfA.     

Reconstructing a chloride-binding site in a bacterial neurotransmitter transporter homologue

In ion-coupled transport proteins, occupation of selective ion-binding sites is required to trigger conformational changes that lead to substrate translocation. Neurotransmitter transporters, targets of abused and therapeutic drugs, require Na(+) and Cl(-) for function. We recently proposed a chloride-binding site in these proteins not present in Cl(-)-independent prokaryotic homologues. Here we describe conversion of the Cl(-)-independent prokaryotic tryptophan transporter TnaT to a fully functional Cl(-)-dependent form by a single point mutation, D268S. Mutations in TnaT-D268S, in wild type TnaT and in serotonin transporter provide direct evidence for the involvement of each of the proposed residues in Cl(-) coordination. In both SERT and TnaT-D268S, Cl(-) and Na(+) mutually increased each other's potency, consistent with electrostatic interaction through adjacent binding sites. These studies establish the site where Cl(-) binds to trigger conformational change during neurotransmitter transport.     

Roles of K+, H+, H2O, and DeltaPsi in solute transport mediated by major facilitator superfamily members ProP and LacY

H (+)-solute symporters ProP and LacY are members of the major facilitator superfamily. ProP mediates osmoprotectant (e.g., proline) accumulation, whereas LacY transports the nutrient lactose. The roles of K (+), H (+), H 2O, and DeltaPsi in H (+)-proline and H (+)-lactose symport were compared using right-side-out cytoplasmic membrane vesicles (MVs) from bacteria expressing both transporters and proteoliposomes (PRLs) reconstituted with pure ProP-His 6. ProP activity increased as LacY activity decreased when osmotic stress (increasing osmolality) was imposed on MVs. The activities of both transporters decreased to similar extents when Na (+) replaced K (+) in MV preparations. Thus, K (+) did not specifically control ProP activity. As with LacY, an increasing extravesicular pH stimulated ProP-mediated proline efflux much more than ProP-mediated proline exchange from de-energized MVs. In contrast to that of LacY, ProP-mediated exchange was only 2-fold faster than ProP-mediated efflux and was inhibited by respiration. In the absence of the protonmotive force (Deltamu H (+) ), efflux of lactose from MVs was much more sensitive to increasing osmolality than lactose exchange. Thus, H 2O may be directly involved in proton transport via LacY. In the absence of Deltamu H (+) , proline efflux and exchange from MVs were osmolality-independent. In PRLs with a DeltapH of 1 (lumen alkaline), ProP-His 6 was inactive when the membrane potential (DeltaPsi) was zero, was active but insensitive to osmolality when DeltaPsi was -100 mV, and became osmolality-sensitive as DeltaPsi increased further to -137 mV. ProP-His 6 had the same membrane orientation in PRLs as in cells and MVs. ProP switches among "off", "on", and "osmolality-sensitive" states as the membrane potential increases. Kinetic parameters determined in the absence of Deltamu H (+) represent a ProP population that is predominantly off.     

Functionally important amino acids in rice sucrose transporter OsSUT1

Six conserved, charged amino acids within membrane spans in rice sucrose transporter OsSUT1 were identified using a three-dimensional structural model based on the crystal structures of three major facilitator superfamily (MFS) proteins: LacY, GlpT, and EmrD. These positions in OsSUT1 were selected for mutagenesis and biochemical assays. Among the six mutants, D177N completely lost transport function, D331N retained only a small fraction of sucrose uptake activity (2.3% of that of the wild type), and R335H and E336Q also displayed a substantial decrease in transport activity. D329N functioned as well as wild-type OsSUT1. R188K did not transport sucrose but showed a H(+) leak that was inhibited by sucrose, indicating that R188K had uncoupled sucrose and H(+) translocation. This demonstrates that charged amino acids within membrane spans are important for the transport mechanism of OsSUT1 as they are in lactose permease.     

Ion binding and internal hydration in the multidrug resistance secondary active transporter NorM investigated by molecular dynamics simulations

Recently, a 3.65 ? resolution structure of the transporter NorM from the multidrug and toxic compound extrusion family has been determined in the outward-facing conformation. This antiporter uses electrochemical gradients to drive substrate export of a large class of antibiotic and toxic compounds in exchange for small monovalent cations (H(+) and Na(+)), but the molecular details of this mechanism are still largely unknown. Here we report all-atom molecular dynamics simulations of NorM, with and without the bound Na(+) cation and at different ion concentrations. Spontaneous binding of Na(+) is observed in several independent simulations with transient ion binding to D36 being necessary to reach the final binding site for which two competitive binding modes occur. Finally, the simulations indicate that the extracellular vestibule of the transporter invariably loses its characteristic V shape indicated by the crystallographic data, and it is reduced to a narrow permeation pathway lined by polar residues that can act as a specific pore for the transport of small cations. This event, together with the available structures of evolutionarily related transporters of the major facilitator superfamily (MFS), suggests that differences in the hydrophobic content of the extracellular vestibule may be characteristic of multidrug resistance transporters in contrast to substrate-selective members of the MFS.