Red wine and green tea flavonoids are cis-allosteric activators and competitive inhibitors of glucose transporter 1 (GLUT1)-mediated sugar uptake.

The antioxidant- and flavonoid-rich contents of red wine and green tea are reported to offer protection against cancer, cardiovascular disease, and diabetes. Some studies, however, show that flavonoids inhibit GLUT1-mediated, facilitative glucose transport, raising the possibility that their interaction with GLUT1 and subsequent downstream effects on carbohydrate metabolism may also impact health. The present study explores the structure-function relationships of flavonoid-GLUT1 interactions. We find that low concentrations of flavonoids act as cis-allosteric activators of sugar uptake, whereas higher concentrations competitively inhibit sugar uptake and noncompetitively inhibit sugar exit. Studies with heterologously expressed human GLUT1, -3, or -4 reveal that quercetin-GLUT1 and -GLUT4 interactions are stronger than quercetin-GLUT3 interactions, that epicatechin gallate (ECG) is more selective for GLUT1, and that epigallocatechin gallate (EGCG) is less GLUT isoform-selective. Docking studies suggest that only one flavonoid can bind to GLUT1 at any instant, but sugar transport and ligand-binding studies indicate that human erythrocyte GLUT1 can bind at least two flavonoid molecules simultaneously. Quercetin and EGCG are each characterized by positive, cooperative binding, whereas ECG shows negative cooperative binding. These findings support recent studies suggesting that GLUT1 forms an oligomeric complex of interacting, allosteric, alternating access transporters. We discuss how modulation of facilitative glucose transporters could contribute to the protective actions of the flavonoids against diabetes and Alzheimer's disease.

Kinetic Basis of Cis- and Trans-Allostery in GLUT1-Mediated Sugar Transport.

A growing body of evidence demonstrates that GLUT1-mediated erythrocyte sugar transport is more complex than widely assumed and that contemporary interpretations of emergent GLUT1 structural data are incompatible with the available transport and biochemical data. This study examines the kinetic basis of one such incompatibility-transport allostery-and in doing so suggests how the results of studies examining GLUT1 structure and function may be reconciled. Three types of allostery are observed in GLUT1-mediated, human erythrocyte sugar transport: (1) exofacial cis-allostery in which low concentrations of extracellular inhibitors stimulate sugar uptake while high concentrations inhibit transport; (2) endofacial cis-allostery in which low concentrations of intracellular inhibitors enhance cytochalasin B binding to GLUT1 while high concentrations inhibit binding, and (3) trans-allostery in which low concentrations of ligands acting at one cell surface stimulate ligand binding at or sugar transport from the other surface while high concentrations inhibit these processes. We consider several kinetic models to account for these phenomena. Our results show that an inhibitor can only stimulate then inhibit sugar uptake if (1) the transporter binds two or more molecules of inhibitor; (2) high-affinity binding to the first site stimulates transport, and (3) low-affinity binding to the second site inhibits transport. Reviewing the available structural, transport, and ligand binding data, we propose that exofacial cis-allostery results from cross-talk between multiple, co-existent ligand interaction sites present in the exofacial cavity of each GLUT1 protein, whereas trans-allostery and endofacial cis-allostery require ligand-induced subunit-subunit interactions.

Reconciling contradictory findings: Glucose transporter 1 (GLUT1) functions as an oligomer of allosteric, alternating access transporters.

Recent structural studies suggest that GLUT1 (glucose transporter 1)-mediated sugar transport is mediated by an alternating access transporter that successively presents exofacial (e2) and endofacial (e1) substrate-binding sites. Transport studies, however, indicate multiple, interacting (allosteric), and co-existent, exo- and endofacial GLUT1 ligand-binding sites. The present study asks whether these contradictory conclusions result from systematic analytical error or reveal a more fundamental relationship between transporter structure and function. Here, homology modeling supported the alternating access transporter model for sugar transport by confirming at least four GLUT1 conformations, the so-called outward, outward-occluded, inward-occluded, and inward GLUT1 conformations. Results from docking analysis suggested that outward and outward-occluded conformations present multiple ß-d-glucose and maltose interaction sites, whereas inward-occluded and inward conformations present only a single ß-d-glucose interaction site. Gln-282 contributed to sugar binding in all GLUT1 conformations via hydrogen bonding. Mutating Gln-282 to alanine (Q282A) doubled the Km(app) for 2-deoxy-d-glucose uptake and eliminated cis-allostery (stimulation of sugar uptake by subsaturating extracellular maltose) but not trans-allostery (uptake stimulation by subsaturating cytochalasin B). cis-Allostery persisted, but trans-allostery was lost in an oligomerization-deficient GLUT1 variant in which we substituted membrane helix 9 with the equivalent GLUT3 sequence. Moreover, Q282A eliminated cis-allostery in the oligomerization variant. These findings reconcile contradictory conclusions from structural and transport studies by suggesting that GLUT1 is an oligomer of allosteric, alternating access transporters in which 1) cis-allostery is mediated by intrasubunit interactions and 2) trans-allostery requires intersubunit interactions.

WZB117 (2-Fluoro-6-(m-hydroxybenzoyloxy) Phenyl m-Hydroxybenzoate) Inhibits GLUT1-mediated Sugar Transport by Binding Reversibly at the Exofacial Sugar Binding Site.

WZB117 (2-fluoro-6-(m-hydroxybenzoyloxy) phenyl m-hydroxybenzoate) inhibits passive sugar transport in human erythrocytes and cancer cell lines and, by limiting glycolysis, inhibits tumor growth in mice. This study explores how WZB117 inhibits the erythrocyte sugar transporter glucose transport protein 1 (GLUT1) and examines the transporter isoform specificity of inhibition. WZB117 reversibly and competitively inhibits erythrocyte 3-O-methylglucose (3MG) uptake with Ki(app) = 6 µm but is a noncompetitive inhibitor of sugar exit. Cytochalasin B (CB) is a reversible, noncompetitive inhibitor of 3MG uptake with Ki(app) = 0.3 µm but is a competitive inhibitor of sugar exit indicating that WZB117 and CB bind at exofacial and endofacial sugar binding sites, respectively. WZB117 inhibition of GLUTs expressed in HEK293 cells follows the order of potency: insulin-regulated GLUT4 ≫ GLUT1 ≈ neuronal GLUT3. This may explain WZB117-induced murine lipodystrophy. Molecular docking suggests the following. 1) The WZB117 binding envelopes of exofacial GLUT1 and GLUT4 conformers differ significantly. 2) GLUT1 and GLUT4 exofacial conformers present multiple, adjacent glucose binding sites that overlap with WZB117 binding envelopes. 3) The GLUT1 exofacial conformer lacks a CB binding site. 4) The inward GLUT1 conformer presents overlapping endofacial WZB117, d-glucose, and CB binding envelopes. Interrogating the GLUT1 mechanism using WZB117 reveals that subsaturating WZB117 and CB stimulate erythrocyte 3MG uptake. Extracellular WZB117 does not affect CB binding to GLUT1, but intracellular WZB117 inhibits CB binding. These findings are incompatible with the alternating conformer carrier for glucose transport but are consistent with either a multisubunit, allosteric transporter, or a transporter in which each subunit presents multiple, interacting ligand binding sites.

Caffeine inhibits glucose transport by binding at the GLUT1 nucleotide-binding site.

Glucose transporter 1 (GLUT1) is the primary glucose transport protein of the cardiovascular system and astroglia. A recent study proposes that caffeine uncompetitive inhibition of GLUT1 results from interactions at an exofacial GLUT1 site. Intracellular ATP is also an uncompetitive GLUT1 inhibitor and shares structural similarities with caffeine, suggesting that caffeine acts at the previously characterized endofacial GLUT1 nucleotide-binding site. We tested this by confirming that caffeine uncompetitively inhibits GLUT1-mediated 3-O-methylglucose uptake in human erythrocytes [Vmax and Km for transport are reduced fourfold; Ki(app) = 3.5 mM caffeine]. ATP and AMP antagonize caffeine inhibition of 3-O-methylglucose uptake in erythrocyte ghosts by increasing Ki(app) for caffeine inhibition of transport from 0.9 ± 0.3 mM in the absence of intracellular nucleotides to 2.6 ± 0.6 and 2.4 ± 0.5 mM in the presence of 5 mM intracellular ATP or AMP, respectively. Extracellular ATP has no effect on sugar uptake or its inhibition by caffeine. Caffeine and ATP displace the fluorescent ATP derivative, trinitrophenyl-ATP, from the GLUT1 nucleotide-binding site, but d-glucose and the transport inhibitor cytochalasin B do not. Caffeine, but not ATP, inhibits cytochalasin B binding to GLUT1. Like ATP, caffeine renders the GLUT1 carboxy-terminus less accessible to peptide-directed antibodies, but cytochalasin B and d-glucose do not. These results suggest that the caffeine-binding site bridges two nonoverlapping GLUT1 endofacial sites-the regulatory, nucleotide-binding site and the cytochalasin B-binding site. Caffeine binding to GLUT1 mimics the action of ATP but not cytochalasin B on sugar transport. Molecular docking studies support this hypothesis.

Portability of the GN(R)A hairpin loop motif between RNA and DNA.

Hairpins are common nucleic acid secondary structures that serve many structural and functional roles. Recently, we reported that r(UNCG) and r(GNRA) hairpin families use molecular mimicry and electrostatic factors to attain exceptional thermodynamic stability with a CG closing base pair (cbp). Despite having very different overall folds, these tetraloops present the same functionalities and partial charges to the major groove edge of the CG cbp to achieve stability. Herein, we compare the r(GNRA) tetraloop family to the DNA triloop family d(GNA), which is also exceptionally stable with a CG cbp and possesses the same base pairing between the first and last positions of the loop. Nucleobase and functional group modifications were used to investigate interactions of d(GNA) loops with the cbp, which provided for comparison with similar substitutions in r(GNRA) hairpins. Interruption or deletion of loop-cbp interactions in d(GNA) was consistent with electrostatic interactions identified through nonlinear Poisson-Boltzmann (NLPB) calculations, and loop stability changed in a manner consistent with similar loop-cbp interactions for d(GNA) and r(GNRA) loops. We also compared the relationship of DeltaG degrees (37) and log[Na+] for d(GNA) and r(GNRA) loops and found a decreased dependence of stability on salt for both loop families when a CG cbp was present. The similarity of the loop-cbp interactions shows portability of this loop-cbp motif across polymer type and loop size and indicates convergence on similar molecular solutions for stability in RNA and DNA.