Docking Studies Show That D-Glucose and Quercetin Slide through the Transporter GLUT1

Abstract

On a three-dimensional templated model of GLUT1 (Protein Data Bank code 1SUK), a molecular recognition program, AUTODOCK 3, reveals nine hexose-binding clusters spanning the entire "hydrophilic" channel. Five of these cluster sites are within 3-5 Å of 10 glucose transporter deficiency syndrome missense mutations. Another three sites are within 8 Å of two other missense mutations. D-Glucose binds to five sites in the external channel opening, with increasing affinity toward the pore center and then passes via a narrow channel into an internal vestibule containing four lower affinity sites. An external site, not adjacent to any mutation, also binding phloretin but recognizing neither D-fructose nor L-glucose, may be the main threading site for glucose uptake. Glucose exit from human erythrocytes is inhibited by quercetin (K i ‫؍‬ 2.4 M) but not anionic quercetin-semiquinone. Quercetin influx is retarded by extracellular D-glucose (50 mM) but not by phloretin and accelerated by intracellular D-glucose. Quercetin docking sites are absent from the external opening but fill the entire pore center. In the inner vestibule, Glu 254 and Lys 256 hydrogen-bond quercetin (K i ≈ 10 M) but not quercetin-semiquinone. Consistent with the kinetics, this site also binds D-glucose, so quercetin displacement by glucose could accelerate quercetin influx, whereas quercetin binding here will competitively inhibit glucose efflux. ␤-D-Hexoses dock twice as frequently as their ␣-anomers to the 23 aromatic residues in the transport pathway, suggesting that endocyclic hexose hydrogens, as with maltosaccharides in maltoporins, form -bonds with aromatic rings and slide between sites instead of being translocated via a single alternating site.The glucose uniporter GLUT1 (SLC2A1), a member of the major facilitator superfamily of solute transporters, has to date not been crystallized, but its three-dimensional structure has been modeled by templating it to that of Lac Y permease and glycerol 3-phosphate antiporter (GlpT) from Escherichia coli (1-3). The 12 transmembrane ␣-helical domains of the monomeric GLUT protein are arranged around a central water-filled pore lined predominantly with uncharged hydrophilic and hydrophobic amino acids. The 15-Å-long, 8-Å-wide channel narrows near its midpoint (1, 2). Molecular dynamic simulations show that glucose binds close to this position within the pore, as expected of lactose binding to Lac Y permease (2). Glucose docks additionally in a cavity at the external entrance of the pore (3).GLUTs transport other substrates besides hexoses (e.g. dehydroascorbate (4, 5) via GLUT1, -3, and -4 and glucosamine (6) via GLUT2). The flavonone, quercetin, is transported via GLUT4. Quercetin influx into GLUT4 is inhibited by high glucose or cytochalasin B concentrations (7). Conversely, quercetin inhibits glucose and ascorbate transport via GLUT1, -2, -3, and -4 (8 -10).This present study demonstrates that quercetin is also transported via GLUT1, and its uptake is accelerated by exchange with intracellular glucose. Our...

Functional Studies of the T295M Mutation Causing Glut1 Deficiency: Glucose Efflux Preferentially Affected by T295M

“…Substitutions at Y293 in this loop impair transport activity by reducing accessibility to the cytoplasmic substratebinding site, effectively locking the transporter into an outward facing conformation (11). The T295 amino acid residue is about 4.9 Å away from one of the nine clusters spanning the entire hydrophilic channel as revealed by docking studies (37) and locates along the extracellular opening of the transport channel (35). Mutagenesis studies of the T295A, T295S, and T295G Glut-4 mutants (amino acid residue corresponding to that of Glut1 in COS-7 cells) showed that T295A abolishes the glucose transport activity and markedly reduces the Cytochalasin B binding without affecting the photolabeling with ATB-BMPA.…”

Will the original glucose transporter isoform please stand up!

“…Recent docking studies have suggested that GLUT1 presents two exofacial and endofacial glucose-binding sites connected by a cavity sufficiently large to contain a glucose molecule (27,109). It should be noted, however, that homology modeling is not immune to overinterpretation.…”

“…The homology-modeled GLUT1 structure (109) closely resembles GlpT and LacY structures. (11); red, amino acids, which when mutagenized to cysteine are reactive with PC-MBS in the external solvent (94); green, amino acids, which when mutagenized to cysteine are reactive with p-chloromercuribenzylsulfonic (PCMBS) in the external solvent in a substrate-protected manner (94); yellow, cysteine residues that are accessible to iodoacetamide (11); purple, amino acids, which when mutagenized to cysteine result in Ն90% inhibition of GLUT1 (94); orange, putative substrate-binding sites predicted by docking studies (27,109); blue, amino acids implicated in substrate discrimination (87); black, sites at which mutations cause GLUT1 deficiency syndrome (68,124). Some amino acids fall into multiple categories.…”

“…These include adjustments to TM5, loops 5 and 6, and TM6 and TM12. Figure 1C summarizes the proposed modified GLUT1 topology and highlights seven different categories of amino acids: 1) residues that are accessible to trypsin, NHS-LC-biotin, and iodoacetamide (11), 2) residues that when mutagenized to cysteine allow sugar transport inhibition by extracellular p-chloromercuribenzylsulfonic acid (PCMBS) (94), 3) amino acids that when mutagenized to cysteine afford sugar-dependent protection against transport inhibition by PCMBS (94), 4) residues whose substitution by cysteine results in transport inhibition (94), 5) amino acids whose mutagenesis results in GLUT1 deficiency syndrome (68, 124), 6) amino acids whose positions are inferred glucose-binding sites in docking studies with Glpt-threaded GLUT1 structures (27,109), and 7) residues thought to discriminate between glucose and fructose (87).…”

Oral Bioavailability and Disposition of Phytochemicals

“…This transport process may be further complicated in that many PCs present in plants are linked to sugar moieties, which may have an impact on their oral absorption. For example, there is in vitro and in silico evidence that the human glucose transporter 1 (SLC2A1) and rat glucose transporter 4 (slc2a4) transports quercetin (Strobel et al, 2005;Cunningham et al, 2006). In addition, the pig but not human sodium-dependent glucose transporter-1 (SGLT1) appeared to be involved in the intestinal uptake of quercetin glucosides (Cermak et al, 2004;Kottra & Daniel, 2007).…”