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

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 (Ki = 2.4 μm) 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, Glu254 and Lys256 hydrogen-bond quercetin (Ki ≈ 10 μm) 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. β-d-Hexoses dock twice as frequently as their α-anomers to the 23 aromatic residues in the transport pathway, suggesting that endocyclic hexose hydrogens, as with maltosaccharides in maltoporins, form π-bonds with aromatic rings and slide between sites instead of being translocated via a single alternating site. 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 (Ki = 2.4 μm) 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, Glu254 and Lys256 hydrogen-bond quercetin (Ki ≈ 10 μm) 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. β-d-Hexoses dock twice as frequently as their α-anomers to the 23 aromatic residues in the transport pathway, suggesting that endocyclic hexose hydrogens, as with maltosaccharides in maltoporins, form π-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 (1Abramson J. Smirnova I. Kasho V. Verner G. Iwata S. Kaback H.R. Science. 2003; 301: 610-615Crossref PubMed Scopus (1218) Google Scholar, 2Hirai T. Heymann J.A. Shi D. Sarker R. Maloney P.C. Subramaniam S. Nat. Struct. Biol. 2002; 9: 597-600PubMed Google Scholar, 3Salas-Burgos A. Iserovich P. Zuniga F. Vera J.C. Fischbarg J. Biophys. J. 2004; 87: 2990-2999Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). The 12 transmembrane α-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 (1Abramson J. Smirnova I. Kasho V. Verner G. Iwata S. Kaback H.R. Science. 2003; 301: 610-615Crossref PubMed Scopus (1218) Google Scholar, 2Hirai T. Heymann J.A. Shi D. Sarker R. Maloney P.C. Subramaniam S. Nat. Struct. Biol. 2002; 9: 597-600PubMed Google Scholar). Molecular dynamic simulations show that glucose binds close to this position within the pore, as expected of lactose binding to Lac Y permease (2Hirai T. Heymann J.A. Shi D. Sarker R. Maloney P.C. Subramaniam S. Nat. Struct. Biol. 2002; 9: 597-600PubMed Google Scholar). Glucose docks additionally in a cavity at the external entrance of the pore (3Salas-Burgos A. Iserovich P. Zuniga F. Vera J.C. Fischbarg J. Biophys. J. 2004; 87: 2990-2999Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). GLUTs transport other substrates besides hexoses (e.g. dehydroascorbate (4Vera J.C Rivas C.I. Fischbarg J. Golde D.W. Nature. 2000; 364: 79-82Crossref Scopus (446) Google Scholar, 5Rumsey S.C. Daruwala R. Al-Hasani H. Zarnowski M.J. Simpson I.A. Levine M. J. Biol. Chem. 2000; 275: 28246-28253Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar) via GLUT1, -3, and -4 and glucosamine (6Uldry M. Ibbertson M. Hosokawa M. Thorens B. FEBS Lett. 2002; 524: 199-203Crossref PubMed Scopus (220) Google Scholar) via GLUT2). The flavonone, quercetin, is transported via GLUT4. Quercetin influx into GLUT4 is inhibited by high glucose or cytochalasin B concentrations (7Strobel P. Allard C. Perez-Acle T. Calderon R. Aldunate R. Leighton F. Biochem. J. 2005; 386: 471-478Crossref PubMed Scopus (193) Google Scholar). Conversely, quercetin inhibits glucose and ascorbate transport via GLUT1, -2, -3, and -4 (8Park J.B. Levine M. J. Nutr. 2000; 130: 1297-1302Crossref PubMed Scopus (63) Google Scholar, 9Song J. Kwon O. Chen S. Daruwala R. Eck P. Park J.B. Levine M. J. Biol. Chem. 2002; 277: 15252-15260Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, 10Naftalin R.J. Afzal I. Cunningham P. Halai M. 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Fischbarg J. 2005; PubMed Scopus Google Scholar). of a may this by the with an missense in amino at 12 sites have been observed in of the 10 of GLUT1 D. H. S. 2005; PubMed Scopus Google Scholar, J. A. Fischbarg J. 2005; PubMed Scopus Google Scholar). the three-dimensional GLUT1 these are close to all but of the hexose docking sites by AUTODOCK In this it is that of the glucose binding sites in the inner vestibule of GLUT1 that also binds uncharged quercetin is that this site is to Glu254 and Lys256 that hydrogen-bond to quercetin and glucose. The site is to for of net glucose and quercetin so that displacement of quercetin from it by intracellular glucose the observed of quercetin This model of transport via GLUT1 and (1Abramson J. Smirnova I. Kasho V. Verner G. Iwata S. Kaback H.R. Science. 2003; 301: 610-615Crossref PubMed Scopus (1218) Google Scholar, 2Hirai T. Heymann J.A. Shi D. Sarker R. Maloney P.C. Subramaniam S. Nat. Struct. 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This with the view that a single centrally located binding site for hexoses as and The cluster and site 3 are and from and binds with to d-glucose in all of the clusters clusters and in the external cavity and at the docking cluster in the The of docking from clusters and by with in for d-Glucose not dock in cluster The of GLUT1 a of amino with a high docking and affinity for d-glucose the of the external opening of the pore from and docking are absent In the internal vestibule, other sites to dock with of Glucose with in GLUT1 of the 10 missense sites GLUT1 the d-glucose cluster that five are five clusters adjacent clusters 3 and have two in and additionally is close to all of these glucose uptake into human erythrocytes by it be that at five of the nine docking sites within the central transport have for glucose other sites are from so these be with any at present to docking sites. site has with it The high affinity site cluster in the has Å form the sugar in the missense mutation, J. A. Fischbarg J. 2005; PubMed Scopus Google has been observed in close to cluster and a in glucose transport into the Quercetin and Quercetin and quercetin docking sites with d-glucose in quercetin not dock in a of docking for quercetin and quercetin fill the central hydrophilic pore hexose clusters with quercetin binding sites in the central pore and internal vestibule, but since quercetin not in the external vestibule, docking between glucose and quercetin at glucose clusters Quercetin is to Glu254 and Lys256 (Ki = μm) in the between and and to in the between 10 and and with glucose cluster 8 and a cytochalasin B binding site (3Salas-Burgos A. Iserovich P. Zuniga F. Vera J.C. Fischbarg J. Biophys. J. 2004; 87: 2990-2999Abstract Full Text Full Text PDF PubMed Scopus (162) Google its transport by cytochalasin B in GLUT4 (7Strobel P. Allard C. Perez-Acle T. Calderon R. Aldunate R. Leighton F. Biochem. J. 2005; 386: 471-478Crossref PubMed Scopus (193) Google Scholar). not hydrogen-bond here This quercetin binding site all of the by it is at the internal of to competitively inhibit glucose exit external glucose it with a anionic quercetin high intracellular glucose concentrations will quercetin from the site, to accelerated quercetin and the site is adjacent to the cytochalasin B binding site, the of quercetin uptake to cytochalasin B (3Salas-Burgos A. Iserovich P. Zuniga F. Vera J.C. Fischbarg J. Biophys. J. 2004; 87: 2990-2999Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, P. Allard C. Perez-Acle T. Calderon R. Aldunate R. Leighton F. Biochem. J. 2005; 386: 471-478Crossref PubMed Scopus (193) Google Scholar). The of quercetin binding in the external pore could for the of quercetin influx to phloretin binds in this cavity (3Salas-Burgos A. Iserovich P. Zuniga F. Vera J.C. Fischbarg J. Biophys. J. 2004; 87: 2990-2999Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). has high affinity docking is observed here that phloretin binding with the cluster in the external opening and the cluster in the pore of the vestibule as (3Salas-Burgos A. Iserovich P. Zuniga F. Vera J.C. Fischbarg J. Biophys. J. 2004; 87: 2990-2999Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). are also other high affinity phloretin binding sites in clusters 3, and and the high affinity site 10 in the The of all docking sites here that of the three-dimensional of GLUT1 (3Salas-Burgos A. Iserovich P. Zuniga F. Vera J.C. Fischbarg J. Biophys. J. 2004; 87: 2990-2999Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar) and the docking of AUTODOCK (11Morris G.M. Goodsell D.S. Halliday R.S. Huey R. Hart W.E. Belew R.K. Olson A.J. J. Comput. Chem. 1998; 19: 1639-1662Crossref Scopus (9045) Google Scholar). AUTODOCK than molecular dynamic simulations (3Salas-Burgos A. Iserovich P. Zuniga F. Vera J.C. Fischbarg J. Biophys. J. 2004; 87: 2990-2999Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar) a view of the ligand docking sites and additionally by an affinity to docking of the AUTODOCK program, to is that it that docking a protein in the AUTODOCK 3, of within the docking ligands is of the protein not from the of docking sites of of into the docking the for site The program has to the of the docking by from the within the the docking sites are all the of ligand that are determined from the of the energies from the between the ligand the and the adjacent protein and is of a cluster being by (11Morris G.M. Goodsell D.S. Halliday R.S. Huey R. Hart W.E. Belew R.K. Olson A.J. J. Comput. Chem. 1998; 19: 1639-1662Crossref Scopus (9045) Google Scholar). However, since AUTODOCK not its by dynamic by ligands the within the but by the the entire protein it docking sites in from the external this not with hexose or quercetin binding to GLUT1, it has a so all 10 hexose clusters and the quercetin sites within the hydrophilic pore are in with the external of the The docking of the hexose clusters are all as determined by the so their close to sites is sites are the entire central and the of quercetin transport is Five of the of the nine hexose clusters in the central pore of GLUT1 are by their with observed in and two are within of where protein these sites into of the cluster sites and transport The cluster site not adjacent to a site is the external site, also to l-glucose, or The are that quercetin and glucose are transported via GLUT1, that quercetin uptake is accelerated by intracellular and that cytochalasin B (7Strobel P. Allard C. Perez-Acle T. Calderon R. Aldunate R. Leighton F. Biochem. J. 2005; 386: 471-478Crossref PubMed Scopus (193) Google not inhibit quercetin This that the transport of glucose and quercetin are but not The and of quercetin uptake by extracellular and intracellular glucose is that these ligands at the same transport