Three-dimensional crystallization of the Escherichia coli glycerol-3-phosphate transporter: a member of the major facilitator superfamily

Here we report the successful three-dimensional crystallization of GlpT, the glycerol-3-phosphate transporter from Escherichia coli inner membrane. GlpT possesses 12 transmembrane alpha-helices and is a member of the major facilitator superfamily. It mediates the exchange of glycerol-3-phosphate for inorganic phosphate across the membrane. Approximately 20 phospholipid molecules per protein, identified as negatively charged phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin, were required for the monodispersity of purified GlpT. Analytical size-exclusion chromatography proved to be efficient in identifying detergents for GlpT monodispersity. Nine such detergents were later used for GlpT crystallization. Screening for crystal nucleation was carried out with a variety of polyethylene glycols as the precipitant over a wide pH range. Subsequent identification of a rigid protein core by limited proteolysis and mass spectroscopy resulted in better-ordered crystals. These crystals exhibited order to 3.7 A resolution in two dimensions. However, the stacking in the third dimension was partially disordered. This stacking problem was overcome by using a detergent mixture and manipulating the ionic interactions in the crystallization solution. The resulting GlpT crystals diffracted isotropically to 3.3 A resolution and were suitable for structure determination by X-ray crystallography.     

Structural Basis of Substrate Selectivity in the Glycerol-3-Phosphate: Phosphate Antiporter GlpT

Major facilitators represent the largest superfamily of secondary active transporter proteins and catalyze the transport of an enormous variety of small solute molecules across biological membranes. However, individual superfamily members, although they may be architecturally similar, exhibit strict specificity toward the substrates they transport. The structural basis of this specificity is poorly understood. A member of the major facilitator superfamily is the glycerol-3-phosphate (G3P) transporter (GlpT) from the Escherichia coli inner membrane. GlpT is an antiporter that transports G3P into the cell in exchange for inorganic phosphate (P_i). By combining large-scale molecular-dynamics simulations, mutagenesis, substrate-binding affinity, and transport activity assays on GlpT, we were able to identify key amino acid residues that confer substrate specificity upon this protein. Our studies suggest that only a few amino acid residues that line the transporter lumen act as specificity determinants. Whereas R45, K80, H165, and, to a lesser extent Y38, Y42, and Y76 contribute to recognition of both free P_i and the phosphate moiety of G3P, the residues N162, Y266, and Y393 function in recognition of only the glycerol moiety of G3P. It is the latter interactions that give the transporter a higher affinity to G3P over P_i.     

Salt-bridge dynamics control substrate-induced conformational change in the membrane transporter GlpT

Active transport of substrates across cytoplasmic membranes is of great physiological, medical and pharmaceutical importance. The glycerol-3-phosphate (G3P) transporter (GlpT) of the E. coli inner membrane is a secondary active antiporter from the ubiquitous major facilitator superfamily that couples the import of G3P to the efflux of inorganic phosphate (P_i) down its concentration gradient. Integrating information from a novel combination of structural, molecular dynamics simulations and biochemical studies, we identify the residues involved directly in binding of substrate to the inward-facing conformation of GlpT, thus defining the structural basis for the substrate-specificity of this transporter. The substrate binding mechanism involves protonation of a histidine residue at the binding site. Furthermore, our data suggest that the formation and breaking of inter- and intradomain salt bridges control the conformational change of the transporter that accompanies substrate translocation across the membrane. The mechanism we propose may be a paradigm for organophosphate:phosphate antiporters.     

Structural modeling of dual-affinity purified Pho84 phosphate transporter

The phosphate transporter Pho84 of Saccharomyces cerevisiae is predicted to contain 12 transmembrane (TM) regions, divided into two partially duplicated parts of 6 TM segments. The three-dimensional (3D) organization of the Pho84 protein has not yet been determined. However, the 3D crystal structure of the Escherichia coli MFS glycerol-3-phosphate/phosphate antiporter, GlpT, and lactose transporter, LacY, has recently been determined. On the basis of extensive prediction and fold recognition analyses (at the MetaServer), GlpT was proposed as the best structural template on which the arrangement of TM segments of the Pho84 transporter was fit, using the comparative structural modeling program MODELLER. To initiate an evaluation of the appropriateness of the Pho84 model, we have performed two direct tests by targeting spin labels to putative TM segments 8 and 12. Electron paramagnetic resonance spectroscopy was then applied on purified and spin labeled Pho84. The line shape from labels located at both positions is consistent with the structural environment predicted by the template-generated model, thus supporting the model.     

Glycerol-3-phosphate acquisition in spirochetes: distribution and biological activity of glycerophosphodiester phosphodiesterase (GlpQ) among Borrelia species

Relapsing-fever spirochetes achieve high cell densities (>10(8)/ml) in their host's blood, while Lyme disease spirochetes do not (<10(5)/ml). This striking contrast in pathogenicity of these two groups of bacteria suggests a fundamental difference in their ability to either exploit or survive in blood. Borrelia hermsii, a tick-borne relapsing-fever spirochete, contains orthologs to glpQ and glpT, genes that encode glycerophosphodiester phosphodiesterase (GlpQ) and glycerol-3-phosphate transporter (GlpT), respectively. In other bacteria, GlpQ hydrolyzes deacylated phospholipids to glycerol-3-phosphate (G3P) while GlpT transports G3P into the cytoplasm. Enzyme assays on 17 isolates of borreliae demonstrated GlpQ activity in relapsing-fever spirochetes but not in Lyme disease spirochetes. Southern blots demonstrated glpQ and glpT in all relapsing-fever spirochetes but not in the Lyme disease group. A Lyme disease spirochete, Borrelia burgdorferi, that was transformed with a shuttle vector containing glpTQ from B. hermsii produced active enzyme, which demonstrated the association of glpQ with the hydrolysis of phospholipids. Sequence analysis of B. hermsii identified glpF, glpK, and glpA, which encode the glycerol facilitator, glycerol kinase, and glycerol-3-phosphate dehydrogenase, respectively, all of which are present in B. burgdorferi. All spirochetes examined had gpsA, which encodes the enzyme that reduces dihydroxyacetone phosphate (DHAP) to G3P. Consequently, three pathways for the acquisition of G3P exist among borreliae: (i) hydrolysis of deacylated phospholipids, (ii) reduction of DHAP, and (iii) uptake and phosphorylation of glycerol. The unique ability of relapsing-fever spirochetes to hydrolyze phospholipids may contribute to their higher cell densities in blood than those of Lyme disease spirochetes.     

Helix dynamics in a membrane transport protein: comparative simulations of the glycerol-3-phosphate transporter and its constituent helices

The glycerol-3-phosphate transporter (GlpT) is a member of the major facilitator superfamily (MFS). GlpT is an organic phosphate/inorganic phosphate antiporter. It shares a similar fold with other MFS transporters (e.g. LacY and EmrD) consisting of 12 transmembrane (TM) helices which form two domains (each of six TM helices) surrounding a central ligand-binding cavity. The TM helices (especially the cavity-lining helices) contain a large number of proline and glycine residues, which may aid in the conformational changes believed to underline the transport mechanism. Molecular dynamics simulations in a phospholipid bilayer have been used to compare the conformational properties of the isolated TM helices with those in the intact GlpT protein. Analysis of these simulations focuses on the role of proline-induced flexibility in the TM helices. Our results are consistent with the proposed rocker switch mechanism for transport by GlpT. In particular, the simulations highlight the cavity-lining helices (H4, H5, H10 and H11) as being significantly flexible, suggesting that the transport mechanism may involve intra-helix motions in addition to pseudo-rigid body motions of the N- and C-terminal domains relative to one another.     

Pi exchange mediated by the GlpT-dependent sn-glycerol-3-phosphate transport system in Escherichia coli.

The GlpT system for sn-glycerol-3-phosphate transport in Escherichia coli is shown to catalyze a rapid efflux of Pi from the internal phosphate pools in response to externally added Pi or glycerol-3-phosphate. A glpR mutation, which results in constitutive expression of the GlpT system, is responsible for this rapid Pi efflux and the arsenate sensitivity of several laboratory strains, including the popular strain C600. Glucose and other phosphotransferase system sugars inhibit Pi efflux by repressing glpT expression.     

Kinetic evidence is consistent with the rocker-switch mechanism of membrane transport by GlpT

Secondary active transport of substrate across the cell membrane is crucial to many cellular and physiological processes. The crystal structure of one member of the secondary active transporter family, the sn-glycerol-3-phosphate (G3P) transporter (GlpT) of the inner membrane of Escherichia coli, suggests a mechanism for substrate translocation across the membrane that involves a rocker-switch-type movement of the protein. This rocker-switch mechanism makes two specific predictions with respect to kinetic behavior: the transport rate increases with the temperature, whereas the binding affinity of the transporter to a substrate is temperature-independent. In this work, we directly tested these two predictions by transport kinetics and substrate-binding experiments, integrating the data on this single system into a coherent set of observations. The transport kinetics of the physiologically relevant G3P-phosphate antiport reaction were characterized at different temperatures using both E. coli whole cells and GlpT reconstituted into proteoliposomes. Substrate-binding affinity of the transporter was measured using tryptophan fluorescence quenching in detergent solution. Indeed, the substrate transport velocity of GlpT increased dramatically with temperature. In contrast, neither the apparent Michaelis constant (Km) nor the apparent substrate-binding dissociation constant (Kd) showed temperature dependence. Moreover, GlpT-catalyzed G3P translocation exhibited a completely linear Arrhenius function with an activation energy of 35.2 kJ mol-1 for the transporter reconstituted into proteoliposomes, suggesting that the substrate-loaded transporter is delicately poised between the inward- and outward-facing conformations. When these results are taken together, they are in agreement with a rocker-switch mechanism for GlpT.     

Chemical and functional properties of the native and reconstituted forms of the membrane-bound, aerobic glycerol-3-phosphate dehydrogenase of Escherichia coli.

A simple purification for the membrane-associated, flavin-linked, glycerol-3-phosphate dehydrogenase of Escherichia coli has been developed which yields homogeneous enzyme in a detergent-solubilized state. 1. The dissociated form of the enzyme has a molecular weight of 58,000 and contains 0.5 mol of FAD/mol of protein monomer. 2. The solubilized enzyme-catalyzed reaction has a pH profile and temperature dependence similar to that observed for the membrane-bound enzyme. 3. The most efficient electron acceptor is potassium ferricyanide but phenazine methosulfate, methylene blue, menadione, and dichlorophenolindophenol can also be utilized. 4. The reaction is competitively inhibited by dihydroxyacetone phosphate, phosphoenolpyruvate, phosphoglycolic acid, glyceraldehyde-3-phosphate, and D-2- and D-3-phosphoglyceric acid. 5. The activity of the enzyme is regulated in a complex manner by ATP and GTP. 6. Detergent-depleted enzyme can be functionally reconstituted with Escherichia coli membrane vesicles to support glycerol-3-phosphate-dependent active transport of L-proline. 7. Detergent-depleted enzyme requires exogenous phospholipid or nondenaturing detergent for electron transfer activity.     

Interaction of fosfomycin with the Glycerol 3-phosphate Transporter of Escherichia coli

Background

Fosfomycin is widely used to treat urinary tract and pediatric gastrointestinal infections of bacteria. It is supposed that this antibiotic enters cells via two transport systems, including the bacterial Glycerol-3-phosphate Transporter (GlpT). Impaired function of GlpT is one mechanism for fosfomycin resistance.

Methods

The interaction of fosfomycin with the recombinant and purified GlpT of Escherichia coli reconstituted in liposomes has been studied. IC₅₀ and the half-saturation constant of the transporter for external fosfomycin (K_i) were determined by transport assay of [¹⁴C]glycerol-3-phosphate catalyzed by recombinant GlpT. Efficacy of fosfomycin on growth rates of GlpT defective bacteria strains transformed with recombinant GlpT was measured.

Results

Fosfomycin, externally added to the proteoliposomes, poorly inhibited the glycerol-3-phosphate/glycerol-3-phosphate antiport catalyzed by the reconstituted transporter with an IC₅₀ of 6.4 mM. A kinetic analysis revealed that the inhibition was completely competitive, that is, fosfomycin interacted with the substrate-binding site and the K_i measured was 1.65 mM. Transport assays performed with proteoliposomes containing internal fosfomycin indicate that it was not very well transported by GlpT. Complementation study, performed with GlpT defective bacteria strains, indicated that the fosfomycin resistance, beside deficiency in antibiotic transporter, could be due to other gene defects.

Conclusions

The poor transport observed in a reconstituted system together with the high value of K_i and the results of complementation study well explain the usual high dosage of this drug for the treatment of the urinary tract infections.

General significance

This is the first report regarding functional analysis of interaction between fosfomycin and GlpT.     

Isolation and properties of glycerol-3-phosphate oxidoreductase from human placenta

Glycerol-3-phosphate oxidoreductase (sn-glycerol 3-phosphate: NAD+ 2-oxidoreductase, EC 1.1.1.8) from human placenta has been purified by chromatography on 2,4,6-trinitrobenzenehexamethylenediamine-Sepharose, DEAE-Sephadex A-50 and 5'-AMP-Sepharose 4B approximately 15800-fold with an overall yield of about 19%. The final purified material displayed a specific activity of about 88 mumol NADH min-1 mg protein-1 and a single protein band on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The native molecular mass, determined by Ultrogel AcA 44 filtration, was 62000 +/- 2000 whereas the subunit molecular mass, established on polyacrylamide gel in the presence of 0.1% sodium dodecyl sulphate, was 38000 +/- 500. The isoelectric point of the enzyme protein, determined by column isoelectric focusing, was found to be 5.29 +/- 0.09. The pH optimum of the placental enzyme was in the range 7.4-8.1 for dihydroxyacetone phosphate reduction and 8.7-9.2 for sn-glycerol 3-phosphate oxidation. The apparent Michaelis constants (Km) for dihydroxyacetone phosphate, NADH, sn-glycerol 3-phosphate and NAD+ were 26 microM, 5 microM, 143 microM and 36 microM respectively. The activity ratio of cytoplasmic glycerol-3-phosphate oxidoreductase to mitochondrial glycerol-3-phosphate dehydrogenase in human placental tissue was 1:2. The consumption of oxygen by human placental mitochondria incubated with the purified glycerol-3-phosphate oxidoreductase, NADH and dihydroxyacetone phosphate was similar to that observed in the presence of sn-glycerol 3-phosphate. The possible physiological role of glycerol-3-phosphate oxidoreductase in placental metabolism is discussed.     

Purification and characterization of glycerol-3-phosphate dehydrogenase of Saccharomyces cerevisiae.

The NAD-dependent glycerol-3-phosphate dehydrogenase (glycerol-3-phosphate:NAD+ oxidoreductase; EC 1.1.1.8; G3P DHG) was purified 178-fold to homogeneity from Saccharomyces cerevisiae strain H44-3D by affinity- and ion-exchange chromatography. SDS-PAGE indicated that the enzyme had a molecular mass of approximately 42,000 (+/- 1,000) whereas a molecular mass of 68,000 was observed using gel filtration, implying that the enzyme may exist as a dimer. The pH optimum for the reduction of dihydroxyacetone phosphate (DHAP) was 7.6 and the enzyme had a pI of 7.4. NADPH will not substitute for NADH as coenzyme in the reduction of DHAP. The oxidation of glycerol-3-phosphate (G3P) occurs at 3% of the rate of DHAP reduction at pH 7.0. Apparent Km values obtained were 0.023 and 0.54 mM for NADH and DHAP, respectively. NAD, fructose-1,6-bisphosphate (FBP), ATP and ADP inhibited G3P DHG activity. Ki values obtained for NAD with NADH as variable substrate and FBP with DHAP as variable substrate were 0.93 and 4.8 mM, respectively.     

Kinetic properties of bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from spinach leaves.

A cDNA encoding 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase was isolated from a Spinacia oleracea leaf library and used to express a recombinant enzyme in Escherichia coli and Spodoptera frugiperda cells. The insoluble protein expressed in E. coli was purified and used to raise antibodies. Western blot analysis of a protein extract from spinach leaf showed a single band of 90.8 kDa. Soluble protein was purified to homogeneity from S. frugiperda cells infected with recombinant baculovirus harboring the isolated cDNA. The soluble protein had a molecular mass of 320 kDa, estimated by gel filtration chromatography, and a subunit size of 90.8 kDa. The purified protein had activity of both 6-phosphofructo-2-kinase specific activity 10.4-15.9 nmol min(-1) x mg protein (-1) and fructose-2,6-bisphosphatase (specific activity 1.65-1.75 nmol x mol(-1) mg protein(-1). The 6-phosphofructo-2-kinase activity was activated by inorganic phosphate, and inhibited by 3-carbon phosphorylated metabolites and pyrophosphate. In the presence of phosphate, 3-phosphoglycerate was a mixed inhibitor with respect to both fructose 6-phosphate and ATP. Fructose-2,6-bisphosphatase activity was sensitive to product inhibition; inhibition by inorganic phosphate was uncompetitive, whereas inhibition by fructose 6-phosphate was mixed. These kinetic properties support the view that the level of fructose 2,6-bisphosphate in leaves is determined by the relative concentrations of hexose phosphates, three-carbon phosphate esters and inorganic phosphate in the cytosol through reciprocal modulation of 6-phosphofructo-2-kinase and fructose-2,6-bisphosphatase activities of the bifunctional enzyme.     

Glycerol-3-phosphatase of Corynebacterium glutamicum

Formation of glycerol as by-product of amino acid production by Corynebacterium glutamicum has been observed under certain conditions, but the enzyme(s) involved in its synthesis from glycerol-3-phosphate were not known. It was shown here that cg1700 encodes an enzyme active as a glycerol-3-phosphatase (GPP) hydrolyzing glycerol-3-phosphate to inorganic phosphate and glycerol. GPP was found to be active as a homodimer. The enzyme preferred conditions of neutral pH and requires Mg2? or Mn2? for its activity. GPP dephosphorylated both L- and D-glycerol-3-phosphate with a preference for the D-enantiomer. The maximal activity of GPP was estimated to be 31.1 and 1.7 U mg?1 with K(M) values of 3.8 and 2.9 mM for DL- and L-glycerol-3-phosphate, respectively. For physiological analysis a gpp deletion mutant was constructed and shown to lack the ability to produce detectable glycerol concentrations. Vice versa, gpp overexpression increased glycerol accumulation during growth in fructose minimal medium. It has been demonstrated previously that intracellular accumulation of glycerol-3-phosphate is growth inhibitory as shown for a recombinant C. glutamicum strain overproducing glycerokinase and glycerol facilitator genes from E. coli in media containing glycerol. In this strain, overexpression of gpp restored growth in the presence of glycerol as intracellular glycerol-3-phosphate concentrations were reduced to wild-type levels. In C. glutamicum wild type, GPP was shown to be involved in utilization of DL-glycerol-3-phosphate as source of phosphorus, since growth with DL-glycerol-3-phosphate as sole phosphorus source was reduced in the gpp deletion strain whereas it was accelerated upon gpp overexpression. As GPP homologues were found to be encoded in the genomes of many other bacteria, the gpp homologues of Escherichia coli (b2293) and Bacillus subtilis (BSU09240, BSU34970) as well as gpp1 from the plant Arabidosis thaliana were overexpressed in E. coli MG1655 and shown to significantly increase GPP activity.     

Identification and characterization of a bacterial glycerol-1-phosphate dehydrogenase: Ni(2+)-dependent AraM from Bacillus subtilis

The exclusive presence of glycerol-1-phosphate dehydrogenases (G1PDH) has been postulated to be a key feature that distinguishes archaea from bacteria. However, homologues of G1PDH genes can be found in several bacterial species, among them the hitherto uncharacterized open reading frame araM from Bacillus subtilis. We produced recombinant AraM in Escherichia coli and demonstrate that the purified protein forms a homodimer that reversibly reduces dihydroxyacetone phosphate (DHAP) to glycerol-1-phosphate (G1P) in a NADH-dependent manner. AraM, which constitutes the first identified G1PDH from bacteria, has a similar catalytic efficiency as its archaeal homologues, but its activity is dependent on the presence of Ni (2+) instead of Zn (2+). On the basis of these findings and the analysis of an araM knockout mutant, we propose that AraM generates G1P for the synthesis of phosphoglycerolipids in Gram-positive bacterial species.     

Kinetic study of sn-glycerol-1-phosphate dehydrogenase from the aerobic hyperthermophilic archaeon, Aeropyrum pernix K1.

A gene having high sequence homology (45-49%) with the glycerol-1-phosphate dehydrogenase gene from Methanobacterium thermoautotrophicum was cloned from the aerobic hyperthermophilic archaeon Aeropyrum pernix K1 (JCM 9820). This gene expressed in Escherichia coli with the pET vector system consists of 1113 nucleotides with an ATG initiation codon and a TAG termination codon. The molecular mass of the purified enzyme was estimated to be 38 kDa by SDS/PAGE and 72.4 kDa by gel column chromatography, indicating presence as a dimer. The optimum reaction temperature of this enzyme was observed to be 94-96 degrees C at near neutral pH. This enzyme was subjected to two-substrate kinetic analysis. The enzyme showed substrate specificity for NAD(P)H-dependent dihydroxyacetone phosphate reduction and NAD(+)-dependent glycerol-1-phosphate (Gro1P) oxidation. NADP(+)-dependent Gro1P oxidation was not observed with this enzyme. For the production of Gro1P in A. pernix cells, NADPH is the preferred coenzyme rather than NADH. Gro1P acted as a noncompetitive inhibitor against dihydroxyacetone phosphate and NAD(P)H. However, NAD(P)(+) acted as a competitive inhibitor against NAD(P)H and as a noncompetitive inhibitor against dihydroxyacetone phosphate. This kinetic data indicates that the catalytic reaction by glycerol- 1-phosphate dehydrogenase from A. pernix follows a ordered bi-bi mechanism.     

Sequence analysis and characterization of a 40-kilodalton Borrelia hermsii glycerophosphodiester phosphodiesterase homolog.

We report the purification, molecular cloning, and characterization of a 40-kDa glycerophosphodiester phosphodiesterase homolog from Borrelia hermsii. The 40-kDa protein was solubilized from whole organisms with 0.1% Triton X-100, phase partitioned into the Triton X-114 detergent phase, and purified by fast-performance liquid chromatography (FPLC). The gene encoding the 40-kDa protein was cloned from a B. hermsii chromosomal DNA lambda EXlox expression library and identified by using affinity antibodies generated against the purified native protein. The deduced amino acid sequence included a 20-amino-acid signal peptide encoding a putative leader peptidase II cleavage site, indicating that the 40-kDa protein was a lipoprotein. Based on significant homology (31 to 52% identity) of the 40-kDa protein to glycerophosphodiester phosphodiesterases of Escherichia coli (GlpQ), Bacillus subtilis (GlpQ), and Haemophilus influenzae (Hpd; protein D), we have designated this B. hermsii 40-kDa lipoprotein a glycerophosphodiester phosphodiesterase (Gpd) homolog, the first B. hermsii lipoprotein to have a putative functional assignment. A nonlipidated form of the Gpd homolog was overproduced as a fusion protein in E. coli BL21(DE3)(pLysE) and was used to immunize rabbits to generate specific antiserum. Immunoblot analysis with anti-Gpd serum recognized recombinant H. influenzae protein D, and conversely, antiserum to H. influenzae protein D recognized recombinant B. hermsii Gpd (rGpd), indicating antigenic conservation between these proteins. Antiserum to rGpd also identified native Gpd as a constituent of purified outer membrane vesicles prepared from B. hermsii. Screening of other pathogenic spirochetes with anti-rGpd serum revealed the presence of antigenically related proteins in Borrelia burgdorferi, Treponema pallidum, and Leptospira kirschneri. Further sequence analysis both upstream and downstream of the Gpd homolog showed additional homologs of glycerol metabolism, including a glycerol-3-phosphate transporter (GlpT), a glycerol-3-phosphate dehydrogenase (GlpD), and a thioredoxin reductase (TrxB).     

Modeling of glycerol-3-phosphate transporter suggests a potential 'tilt' mechanism involved in its function

Many major facilitator superfamily (MFS) transporters have similar 12-transmembrane alpha-helical topologies with two six-helix halves connected by a long loop. In humans, these transporters participate in key physiological processes and are also, as in the case of members of the organic anion transporter (OAT) family, of pharmaceutical interest. Recently, crystal structures of two bacterial representatives of the MFS family--the glycerol-3-phosphate transporter (GlpT) and lac-permease (LacY)--have been solved and, because of assumptions regarding the high structural conservation of this family, there is hope that the results can be applied to mammalian transporters as well. Based on crystallography, it has been suggested that a major conformational "switching" mechanism accounts for ligand transport by MFS proteins. This conformational switch would then allow periodic changes in the overall transporter configuration, resulting in its cyclic opening to the periplasm or cytoplasm. Following this lead, we have modeled a possible "switch" mechanism in GlpT, using the concept of rotation of protein domains as in the DynDom program17 and membranephilic constraints predicted by the MAPAS program.(23) We found that the minima of energies of intersubunit interactions support two alternate positions consistent with their transport properties. Thus, for GlpT, a "tilt" of 9 degrees -10 degrees rotation had the most favorable energetics of electrostatic interaction between the two halves of the transporter; moreover, this confirmation was sufficient to suggest transport of the ligand across the membrane. We conducted steered molecular dynamics simulations of the GlpT-ligand system to explore how glycerol-3-phosphate would be handled by the "tilted" structure, and obtained results generally consistent with experimental mutagenesis data. While biochemical data remain most consistent with a single-site alternating access model, our results raise the possibility that, while the "rocker switch" may apply to certain MFS transporters, intermediate "tilted" states may exist under certain circumstances or as transitional structures. Although wet lab experimental confirmation is required, our results suggest that transport mechanisms in this transporter family should probably not be assumed to be conserved simply based on standard structural homology considerations. Furthermore, steered molecular dynamics elucidating energetic interactions of ligands with amino acid residues in an appropriately modeled transporter may have predictive value in understanding the impact of mutations and/or polymorphisms on transporter function.