FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2034155024> ?p ?o ?g. }

• W2034155024 endingPage "44975" @default.
• W2034155024 startingPage "44970" @default.
• W2034155024 abstract "The human facilitative transporter Glut1 is the major glucose transporter present in all human cells, has a central role in metabolism, and is an archetype of the superfamily of major protein facilitators. Here we describe a three-dimensional structure of Glut1 based on helical packing schemes proposed for lactose permease and Glut1 and predictions of secondary structure, and refined using energy minimization, molecular dynamics simulations, and quality and environmental scores. The Ramachandran scores and the stereochemical quality of the structure obtained were as good as those for the known structures of the KcsA K+ channel and aquaporin 1. We found two channels in Glut1. One of them traverses the structure completely, and is lined by many residues known to be solvent-accessible. Since it is delimited by the QLS motif and by several well conserved residues, it may serve as the substrate transport pathway. To validate our structure, we determined the distance between these channels and all the residues for which mutations are known. From the locations of sugar transporter signatures, motifs, and residues important to the transport function, we find that this Glut1 structure is consistent with mutagenesis and biochemical studies. It also accounts for functional deficits in seven pathogenic mutants. The human facilitative transporter Glut1 is the major glucose transporter present in all human cells, has a central role in metabolism, and is an archetype of the superfamily of major protein facilitators. Here we describe a three-dimensional structure of Glut1 based on helical packing schemes proposed for lactose permease and Glut1 and predictions of secondary structure, and refined using energy minimization, molecular dynamics simulations, and quality and environmental scores. The Ramachandran scores and the stereochemical quality of the structure obtained were as good as those for the known structures of the KcsA K+ channel and aquaporin 1. We found two channels in Glut1. One of them traverses the structure completely, and is lined by many residues known to be solvent-accessible. Since it is delimited by the QLS motif and by several well conserved residues, it may serve as the substrate transport pathway. To validate our structure, we determined the distance between these channels and all the residues for which mutations are known. From the locations of sugar transporter signatures, motifs, and residues important to the transport function, we find that this Glut1 structure is consistent with mutagenesis and biochemical studies. It also accounts for functional deficits in seven pathogenic mutants. lactose permease p-chloromercuribenzenesulfonate N-ethylmaleimide The facilitative glucose transporter Glut1 is perhaps the most extensively studied membrane transporter. Over the last 15 to 20 years a number of technologies have been developed which have allowed investigators to observe and describe glucose transporter structure and function. Purification and reconstitution of the erythrocyte glucose transporter have allowed investigators to analyze its secondary structure using spectroscopic techniques (1Alvarez J. Lee D.C. Baldwin S.A. Chapman D. J. Biol. Chem. 1987; 262: 3502-3509Abstract Full Text PDF PubMed Google Scholar, 2Pawagi A.B. Deber C.M. Biochemistry. 1990; 29: 950-955Crossref PubMed Scopus (53) Google Scholar). Concurrently, the use of affinity labels such as phloretin, forskolin (3Sergeant S. Kim H.D. J. Biol. Chem. 1985; 260: 14677-14682Abstract Full Text PDF PubMed Google Scholar), and cytochalasin B (4Krupka R.M. Deves R. Biochem. Cell Biol. 1986; 64: 1099-1107Crossref PubMed Scopus (15) Google Scholar, 5Basketter D.A. Widdas W.F. J. Physiol. 1978; 278: 389-401Crossref PubMed Scopus (97) Google Scholar), group-specific chemical reagents (6Rampal A.L. Jung C.Y. Biochim. Biophys. Acta. 1987; 896: 287-294Crossref PubMed Scopus (8) Google Scholar, 7May J.M. Biochim. Biophys. Acta. 1989; 986: 207-216Crossref PubMed Scopus (9) Google Scholar), proteases (8Takata K. Kasahara T. Kasahara M. Ezaki O. Hirano H. Cell Tissue Res. 1992; 267: 407-412Crossref PubMed Scopus (94) Google Scholar), and antibodies (8Takata K. Kasahara T. Kasahara M. Ezaki O. Hirano H. Cell Tissue Res. 1992; 267: 407-412Crossref PubMed Scopus (94) Google Scholar) have provided a topographical map of Glut1. cDNAs encoding the Glut1 protein have been isolated from human, rat, mouse, rabbit, and pig tissues (9Mueckler M. Caruso C. Baldwin S.A. Panico M. Blench I. Morris H.R. Allard W.J. Lienhard G.E. Lodish H.F. Science. 1985; 229: 941-945Crossref PubMed Scopus (1131) Google Scholar, 10Birnbaum M.J. Haspel H.C. Rosen O.M. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 5784-5788Crossref PubMed Scopus (436) Google Scholar, 11Kaestner K.H. Christy R.J. McLenithan J.C. Braiterman L.T. Cornelius P. Pekala P.H. Lane M.D. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 3150-3154Crossref PubMed Scopus (248) Google Scholar, 12Baldwin S.A. Biochim. Biophys. Acta. 1993; 1154: 17-49Crossref PubMed Scopus (279) Google Scholar, 13Asano T. Shibasaki Y. Kasuga M. Kanazawa Y. Takaku F. Akanuma Y. Oka Y. Biochem. Biophys. Res. Commun. 1988; 154: 1204-1211Crossref PubMed Scopus (74) Google Scholar). All encode proteins of 492 amino acids and all exhibit an extraordinarily high level of amino acid identity (∼97%). Using hydropathy analysis, Glut1 was predicted by Mueckler and colleagues (9Mueckler M. Caruso C. Baldwin S.A. Panico M. Blench I. Morris H.R. Allard W.J. Lienhard G.E. Lodish H.F. Science. 1985; 229: 941-945Crossref PubMed Scopus (1131) Google Scholar) to consist of 12 transmembrane-spanning α-helices with the N and C termini and a large loop between transmembrane helices 6 and 7 located on the cytoplasmic side of the membrane (9Mueckler M. Caruso C. Baldwin S.A. Panico M. Blench I. Morris H.R. Allard W.J. Lienhard G.E. Lodish H.F. Science. 1985; 229: 941-945Crossref PubMed Scopus (1131) Google Scholar, 14Cairns M.T. Alvarez J. Panico M. Gibbs A.F. Morris H.R. Chapman D. Baldwin S.A. Biochim. Biophys. Acta. 1987; 905: 295-310Crossref PubMed Scopus (61) Google Scholar, 15Davies A. Meeran K. Cairns M.T. Baldwin S.A. J. Biol. Chem. 1987; 262: 9347-9352Abstract Full Text PDF PubMed Google Scholar, 16Davies A. Ciardelli T.L. Lienhard G.E. Boyle J.M. Whetton A.D. Baldwin S.A. Biochem. J. 1990; 266: 799-808PubMed Google Scholar). A smaller loop between transmembrane helices 1 and 2 was predicted to be extracellular (17Asano T. Katagiri H. Takata K. Lin J.L. Ishihara H. Inukai K. Tsukuda K. Kikuchi M. Hirano H. Yazaki Y. J. Biol. Chem. 1991; 266: 24632-24636Abstract Full Text PDF PubMed Google Scholar). The bulk of experimental evidence to date supports this model. There are other Glut sugar transporter isoforms; of these, Glut2–5 have very high homology to Glut1, which suggests strong structural conservation between the different members of the family. By applying mutagenetic techniques to Glut1, selected conserved amino acids and whole domains have been altered, swapped, and deleted. The mutagenesis data have provided insight into locations, which are crucial for substrate binding and for conformational changes that result in d-glucose translocation (18Barrett M.P. Walmsley A.R. Gould G.W. Curr. Opin. Cell Biol. 1999; 11: 496-502Crossref PubMed Scopus (65) Google Scholar). Glut1 admits dehydroascorbic acid as a substrate (19Vera J.C. Rivas C.I. Fischbarg J. Golde D.W. Nature. 1993; 364: 79-82Crossref PubMed Scopus (446) Google Scholar), and also exhibits a modest water conductance (20Fischbarg J. Kuang K. Vera J.C. Arant S. Silverstein S.C. Loike J. Rosen O.M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3244-3247Crossref PubMed Scopus (165) Google Scholar), suggesting the possible presence of a pore through the protein (9Mueckler M. Caruso C. Baldwin S.A. Panico M. Blench I. Morris H.R. Allard W.J. Lienhard G.E. Lodish H.F. Science. 1985; 229: 941-945Crossref PubMed Scopus (1131) Google Scholar).The lactose permease (lac permease)1 ofEscherichia coli (21Kaback H.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5539-5543Crossref PubMed Scopus (74) Google Scholar, 22Kaback H.R. Voss J. Wu J. Curr. Opin. Struct. Biol. 1997; 7: 537-542Crossref PubMed Scopus (88) Google Scholar) and Glut1 are typical 12 transmembrane α-helical proteins of the major facilitator superfamily (23Pao S.S. Paulsen I.T. Saier M.H. Microbiol. Mol. Biol. Rev. 1998; 62: 1-34Crossref PubMed Google Scholar). Application to lac permease of cysteine scanning mutagenesis in conjunction with biochemical, biophysical, and immunological techniques has resulted in some 100 interactions mapped between residues in different helices. Based on the above, a helix-packing model of lac permease has been advanced (24Frillingos S. Sahin-Toth M. Wu J. Kaback H.R. FASEB J. 1998; 12: 1281-1299Crossref PubMed Scopus (318) Google Scholar, 25Venkatesan P. Liu Z. Hu Y. Kaback H.R. Biochemistry. 2000; 39: 10649-10655Crossref PubMed Scopus (41) Google Scholar). In addition, from cysteine scanning mutagenesis of Glut1, Hruz and Mueckler (26Hruz P.W. Mueckler M.M. J. Biol. Chem. 1999; 274: 36176-36180Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 27Hruz P.W. Mueckler M.M. Biochemistry. 2000; 39: 9367-9372Crossref PubMed Scopus (34) Google Scholar) and Keller and collegues (28Olsowski A. Monden I. Keller K. Biochemistry. 1998; 37: 10738-10745Crossref PubMed Scopus (20) Google Scholar, 29Olsowski A. Monden I. Krause G. Keller K. Biochemistry. 2000; 39: 2469-2474Crossref PubMed Scopus (48) Google Scholar) have described residues related to glucose transport, and solvent accessible residues in helices II, V, VII, and XI. From this, a helix-packing model of Glut1 similar to that of lac permease has been suggested (27Hruz P.W. Mueckler M.M. Biochemistry. 2000; 39: 9367-9372Crossref PubMed Scopus (34) Google Scholar).Our understanding of the sequence, biology, and biochemistry of Glut1 is increasing rapidly. However, given the attending difficulties in crystallizing membrane proteins, there is also growing interest in the development and application of molecular modeling techniques to understand the structure of Gluts and relate it to their function. The lack of crystallographic structures for most classes of membrane proteins (including Gluts) means that there are no suitable templates that can be used to generate structures by homology modeling. This creates a need for alternative modeling approaches in which the available experimental biological and biophysical data are used as a reference for the modeling process. The glucose transporter Glut1 is unique given the large amount of experimental data that are available. Therefore, starting from the helical packing schemes referred to above for lac permease and Glut1, we have been able to arrive at a three-dimensional structure of Glut1. We describe here the procedures utilized and offer validation for our structure using stereochemical analysis and mutagenesis data.EXPERIMENTAL PROCEDURESThere are no crystallographic or NMR structure of any proteins with significant overall sequence similarity to Glut1 in the Research Collaboratory for Structural Bioinformatics Protein Data Bank. Thus we could not construct Glut1 by homology modeling. Instead, we chose to use a piecemeal strategy.Helical AssignmentsWe used the GTR1_HUMAN sequence of Glut1 from Swiss Protein Data Base P11166. For the initial structure, α-helices were predicted using the consensus of the programs PHD (30Rost B. Methods Enzymol. 1996; 266: 525-539Crossref PubMed Google Scholar), TMPRED (31Pasquier C. Promponas V.J. Palaios G.A. Hamodrakas J.S. Hamodrakas S.J. Protein Eng. 1999; 12: 381-385Crossref PubMed Scopus (143) Google Scholar), and PSA (32Stultz C.M. White J.V. Smith T.F. Protein Sci. 1993; 2: 305-314Crossref PubMed Scopus (156) Google Scholar).ModelingFor molecular modeling we utilized a Silicon Graphics Octane work station with InsightII software (Molecular Simulations, Inc.). The predicted helices were given the tilt and the three-dimensional proximity depicted in Kaback's scheme for lactose permease (24Frillingos S. Sahin-Toth M. Wu J. Kaback H.R. FASEB J. 1998; 12: 1281-1299Crossref PubMed Scopus (318) Google Scholar, 25Venkatesan P. Liu Z. Hu Y. Kaback H.R. Biochemistry. 2000; 39: 10649-10655Crossref PubMed Scopus (41) Google Scholar). For the rotation around their z axis, we assumed that the helices were arranged with their hydrophilic sides facing a central channel. This was done based on deuterium exchange studies suggesting that 80% of the Glut1 backbone is accessible to water (1Alvarez J. Lee D.C. Baldwin S.A. Chapman D. J. Biol. Chem. 1987; 262: 3502-3509Abstract Full Text PDF PubMed Google Scholar, 33Jung E.K. Chin J.J. Jung C.Y. J. Biol. Chem. 1986; 261: 9155-9160Abstract Full Text PDF PubMed Google Scholar), and on evidence that Glut1 has a modest but finite permeability to water (20Fischbarg J. Kuang K. Vera J.C. Arant S. Silverstein S.C. Loike J. Rosen O.M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3244-3247Crossref PubMed Scopus (165) Google Scholar, 34Zhang R. Alper S. Thorens B. Verkman A.S. J. Clin. Invest. 1991; 88: 1553-1558Crossref PubMed Scopus (40) Google Scholar). For helices II, V, VII, and XI, the rotation was determined using the results of cysteine-scanning mutagenesis studies. For the rest of the helices, we used the results of Ducarme et al. (35Ducarme P. Rahman M. Lins L. Brasseur R. J. Mol. Modeling. 1996; 2: 27-45Google Scholar) to determine the solvent-accessible faces. Loops were generated and connected using the “filgap” command in the program Whatif (36Oliveira L. Paiva A.C. Vriend G. Protein Eng. 1999; 12: 1087-1095Crossref PubMed Scopus (64) Google Scholar).RefinementThe ensemble obtained was subject to energy minimization using the Discover module of InsightII (100 iterations with the steepest descent algorithm, and 1000 more with the conjugate gradients algorithm). By then, the root mean square derivative was <0.1 kcal mol−1 Å-1. We then used the program ProsaII (37Sippl M.J. Proteins. 1993; 17: 355-362Crossref PubMed Scopus (1750) Google Scholar) to assess and improve the locations of helical caps based on Prosa energy plots and Z-scores. The resulting residue assignments for the 12 α-helices are H1, 19–27; H2, 67–81; H3, 99–111; H4, 124–143; H5, 156–175; H6, 191–204; H7, 276–290; H8, 309–326; H9, 347–356; H10, 366–378; H11, 403–417; and H12, 424–445. At this stage of modeling, helical residues fell well into the most favored Ramachandran regions, but many loop residues fell in disallowed regions and exhibited close contacts (distances < 2.2 Å) and undesirable torsion angles. We therefore searched for homologous loops with better conformations using the program Swiss-Pdb Viewer (V3.5) (38Guex N. Peitsch M.C. Electrophoresis. 1997; 18: 2714-2723Crossref PubMed Scopus (9467) Google Scholar). The structure was then subjected to minimization as above, and to molecular dynamics (CVFF forcefield; 298 K; 5 ps of initial equilibration followed by 5 ps dynamics run) with the backbone fixed to optimize the position of the side chains.RESULTS AND DISCUSSIONA ribbon representation of the Glut1 structure we found is given in Fig. 1. Helices 1–5, 8, and 10–12 are arranged in a 9-member barrel-like manner, delimiting a hydrophilic central channel. Helix 7 projects itself into the channel, suggesting a central role in regulating putative transport of solutes through that channel. From the side view, the structure appears roughly symmetrical, as in other barrel proteins. The arrangement of the helices is conical, the shorter side facing exofacially. This may be related to the fact that, as a rule, the exofacial loops tend to be shorter than the endofacial ones.The quality of the Glut1 structure was ascertained using the program PROCHECK (version 3.3.2) (39Laskowki R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallog. 1993; 26: 283-291Crossref Google Scholar). For comparison, we also determined the quality of the structures reported for two α-helical membrane proteins solved by crystallography, the KcsA K+ channel (40Doyle D.A. Cabral J.M. Pfuetzner R.A. Kuo A. Gulbis J.M. Cohen S.L. Chait B.T. MacKinnon R. Science. 1998; 280: 69-77Crossref PubMed Scopus (5684) Google Scholar), and the aquaporin 1 (AQP1) water channel (41Ren G. Reddy S. Cheng A. Melnyk P. Mitra A.K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1398-1403Crossref PubMed Scopus (183) Google Scholar). TableI gives the distribution of the φ, ψ angles in the different regions of the Ramachandran plot. As can be seen, the Glut1 structure is at least as good as those of the other proteins. Several PROCHECK stereochemical parameters and the stereochemical quality are summarized in TableII. From this table, the quality of the Glut1 structure appears as good as (or better) that of two α-helical membrane proteins, the Kcsa and AQP1 channels, implying excellent structural quality for our model structure. To be noted, the PDB data base from which the loops were derived is composed overwhelmingly by globular proteins. It is unclear whether the standard quality factors for facilitator/channel membrane proteins would be precisely the same as those for globular ones, as facilitators/channels would be expected to include a water-filled internal pore in their fold. Hence, that the quality of our structure may appear “better” than those of the comparison channels may be simply related to the data base peculiarity noted.Table IPROCHECK Ramachandran scores for the structures of Glut1, the KcsA K+ channel, and the AQP1 water channelStructureRamachandran plot (%)CoreAllowedGenerousDisallowedGlut11-aProtein Data Bank number:1JA5.81.516.91.70.0KCSA1-bProtein Data Bank number: 1BL8.74.724.11.20.0AQP11-cProtein Data Bank number: 1IH5.70.720.98.40.01-a Protein Data Bank number:1JA5.1-b Protein Data Bank number: 1BL8.1-c Protein Data Bank number: 1IH5. Open table in a new tab Table IISummary of PROCHECK quality assessment (63Morris A.L. MacArthur M.W. Hutchinson E.G. Thornton J.M. Proteins. 1992; 12: 345-364Crossref PubMed Scopus (1383) Google Scholar) dataStructureH-bond energy S.D.Bad contacts per 100 residuesχ−1 pooled SDStereochemical quality index2-aQuality index: 1 highest.φ, ψ Distributionχ1 S.D.HB energyGlut10.871.220.9132KCSA1.06.723.2233AQP10.87.630.82432-a Quality index: 1 highest. Open table in a new tab We next sought to determine if our structure agrees with the functional and biochemical characteristics experimentally determined for Glut1 by many laboratories. Using the program HOLE (42Smart O.S. Goodfellow J.M. Wallace B.A. Biophys. J. 1993; 65: 2455-2460Abstract Full Text PDF PubMed Scopus (500) Google Scholar), we looked for solvent-accessible channels that might serve as transport pathways. We find two channels in our structure. One (the main one) traverses both α-helical and loop regions, and passes close to and curls around helix 7 (cf. Figs. 2 and3). This channel could serve as the glucose transport channel, as elaborated below. The second one (auxiliary channel) is delimited by helices 1, 2, 3, and 7; it is open only at the endofacial site, where it overlaps with the main channel. A study by Keller's (29Olsowski A. Monden I. Krause G. Keller K. Biochemistry. 2000; 39: 2469-2474Crossref PubMed Scopus (48) Google Scholar) laboratory concluded there is an exofacial solvent accessible cleft between helices 2 and 7, and that such a cleft could serve as a pathway for substrates other than sugars. The presence of the auxiliary channel in our structure is consistent with such conclusion. In addition, the existence of two solvent-filled channels in the Glut1 structure is also supported by the deuterium exchange studies cited above suggesting that 80% of the Glut1 backbone is accessible to water (1Alvarez J. Lee D.C. Baldwin S.A. Chapman D. J. Biol. Chem. 1987; 262: 3502-3509Abstract Full Text PDF PubMed Google Scholar, 33Jung E.K. Chin J.J. Jung C.Y. J. Biol. Chem. 1986; 261: 9155-9160Abstract Full Text PDF PubMed Google Scholar).Figure 2Ribbon representation of Glut1 with a space filling representation of the main channel (in yellow) and the auxiliary channel (in blue). Helices arecolored and loops are white, as in Fig. 1.a, side view, showing helices 2, 4, 5, 7, 8, and 11. All loops and the other helices are omitted for clarity. b, end-on view from the cytoplasmic surface; loops omitted. Residue representations are: large blue balls, sites of pathogenic mutations; medium blue balls, QLS motif; small blue balls, essential for glucose transport; green sticks, sensitive to mercurials NEM or/and pCMBS; gray sticks, both essential for glucose transport and mercurial sensitive.View Large Image Figure ViewerDownload (PPT)Figure 3Ribbon representation of Glut1 with a space-filling representation of the main channel (inyellow). Helices are colored and loops are white, as in Fig. 1. Residues in space-filling rendering correspond to several conserved motifs around the channel; Gln279, Leu280, Ser281 (QLS motif) are red, Tyr292 and Tyr293 arepurple; Gln282, green; and Trp412, cyan. Residues 388–412 implicated in the putative binding site for cytochalasin B are colored by atom. Last, all cysteines are shown as sticks inred.View Large Image Figure ViewerDownload (PPT)In Fig. 2 we show the relationships between main channel and auxiliary channel with several residues known to be pathogenic mutations (bold), or essential for glucose transport function (italics), or mercurial-sensitive (underlined), or both essential for transport and mercurial-accessible (italics underlined). The main channel enters the 12 α-helical domain close to residues Thr310 (43Klepper J. Wang D. Fischbarg J. Vera J.C. Jarjour I.T. O'Driscoll K.R. De Vivo D.C. Neurochem. Res. 1999; 24: 587-594Crossref PubMed Scopus (85) Google Scholar) (H8), Gly175 (H5), and to H7. The figure then shows the proximity of the channel to H7 and residues Gln172 , Val290 ,Phe416 , Ile287 , and Asn288. About midway, the main channel turns toward H2 (Fig. 2 b), passing close to Ser80,Leu280 ,Ser281, and Gln282. It then turns once more and goes toward the cytoplasmic side passing near Leu278 ,Val277 , andVal276 . The channel has two segments joined by a bottleneck region near the level of the QLS motif. As Fig. 2 shows, H7, H8, and H11 are all crucial components of the channel. Interestingly, this organization is strikingly reminiscent of the one in a Glut1 model suggested by Jung and colleagues (44Zeng H. Parthasarathy R. Rampal A.L. Jung C.Y. Biophys. J. 1996; 70: 14-21Abstract Full Text PDF PubMed Scopus (45) Google Scholar). Of the two Glut1 models offered there, both of them had H7, H8, and H11 forming part of the channel, as in this one. While on the subject of Glut models, importantly, D. S. Dwyer (45Dwyer D.S. Proteins. 2001; 42: 531-541Crossref PubMed Scopus (43) Google Scholar) has recently described a three-dimensional model for Glut3, which is the major glucose transporter of neuronal cells and is highly homologous to Glut1. Dwyer arrived at his structure differently than ourselves. He used homology modeling on the basis of structural data from the MscL protein, a mechanosensititive ion channel, and general insights from aquaporin 1. A detailed comparison of his model with our current one cannot be offered, as no three-dimensional coordinates for Dwyer's model have appeared in the PDB data base at this writing. In broad terms, in Dwyer's approach, functional correlations with Glut3 residues at equivalent positions of Glut1 mutants are unclear, the pore appears somewhat narrower than that in our model (perhaps as a result of his assumption that only 6 helices limit the pore), and the fold of his long intracellular segment is more extended than ours. On the other hand, in the molecular dynamics he presents, glucose does move 3.5 Å along the pore. In addition, although the helical tilts from the vertical appear less pronounced in his case, there is similarity with the overall packing of our own model (and Kaback's scheme) both in the relative positions of the helices and in the fact that his helix 7 also appears inside a large hydrophilic cavity and borders on the pore. Given the different starting points referred to, such similarity between the models appears quite significant.To return to our structure, the exofacial segment of the main channel is surrounded by the periplasmic ends of H5, H7, H8, H9, and H10 (Figs.1 b and 2), and the endofacial segment by the cytoplasmic ends of H1, H2, H3, H7, and H11. Fittingly, there is evidence (26Hruz P.W. Mueckler M.M. J. Biol. Chem. 1999; 274: 36176-36180Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) that the periplasmic segments of H5 and H7 were accessible to extracellular pCMBS while their cytoplasmic segments were not. From Fig. 2, the main channel curls around H7, away from the cytoplasmic portion of H5, and moving toward the opposite face of H7 as it forms its endofacial segment. Although the cytoplasmic portion of H7 is close to the endofacial segment of the channel, it is structurally not easily accessed by extracellular pCMBS because of the bottleneck. However, it can be accessed by NEM (26Hruz P.W. Mueckler M.M. J. Biol. Chem. 1999; 274: 36176-36180Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), as referred to below.To investigate the location of the exofacial-binding site, we positioned a glucose molecule in the widest part of the exofacial segment of the main channel (not shown). During a subsequent 10-ps molecular dynamics simulation, the glucose molecule remained in that general area and formed a hydrogen bond to Asn288. This is consistent with experimental evidence: the mutation N288C resulted in a 10-fold reduction in normalized glucose transport (27Hruz P.W. Mueckler M.M. Biochemistry. 2000; 39: 9367-9372Crossref PubMed Scopus (34) Google Scholar). It would appear that the minimized structure we have arrived at corresponds to or is near the so-called endofacial conformation. The exploration of putative intermediate conformations leading to an exofacial one will be the subject of future studies.We next determined the spatial relations between the main channel in our structure and the locations of conserved residues and motifs, which are characteristic of the family of hexose transporters. From mutagenesis experiments, the conserved QLS motif in helix 7 forms part of the exofacial substrate-binding site and acts as a selectivity filter allowing Glut1, -3, and -4 to transport glucose, but not fructose (46Seatter M.J. De la Rue S.A. Porter L.M. Gould G.W. Biochemistry. 1998; 37: 1322-1326Crossref PubMed Scopus (93) Google Scholar). In our structure (Fig. 3), the QLS motif delimits the main channel (herein after channel) and is in the vicinity of the putative exofacial substrate occupancy site. Similarly, it is known that substitutions of the conserved residues Tyr292/293, and Trp412 markedly affect transporter function (18Barrett M.P. Walmsley A.R. Gould G.W. Curr. Opin. Cell Biol. 1999; 11: 496-502Crossref PubMed Scopus (65) Google Scholar). Fittingly, in our structure (Fig. 3), Tyr292/293 delimit the channel, and Trp412 is in close proximity to it. Turning now to the cytochalasin B-binding site, evidence locates it somewhere near residues 388–412 (47Holman G.D. Rees W.D. Biochim. Biophys. Acta. 1987; 897: 395-405Crossref PubMed Scopus (64) Google Scholar). As highlighted in Fig. 3, those residues are very close to the endofacial end of both channels. Finally, it is known that none of the native Glut1 cysteine residues are essential for transport function (48Wellner M. Monden I. Keller K. FEBS Lett. 1995; 370: 19-22Crossref PubMed Scopus (22) Google Scholar). Once more, as Fig. 3 shows, the structure conforms to prior evidence: in our structure, all cysteines are far from the channel. From all this, our structure accounts very well for the experimental evidence this far.There are studies suggesting that Glut1 in erythrocytes can exist as an oligomer with a putative disulfide bond between cysteine residues 347 and 421 (49Zottola R.J. Cloherty E.K. Coderre P.E. Hansen A. Hebert D.N. Carruthers A. Biochemistry. 1995; 34: 9734-9747Crossref PubMed Scopus (114) Google Scholar). However, when the Glut1 construct with all the cysteine residues replaced (C-less Glut1 (48Wellner M. Monden I. Keller K. FEBS Lett. 1995; 370: 19-22Crossref PubMed Scopus (22) Google Scholar, 50Mueckler M. Makepeace C. J. Biol. Chem. 1997; 272: 30141-30146Crossref PubMed Scopus (46) Google Scholar)) is expressed in Xenopus laevis oocytes, glucose transport is practically unaffected. Moreover, other studies suggest the existence of monomeric functional Glut transporters (forms 1–3) with no alteration in the kinetic parameters when expressed in X. laevis oocytes (51Burant C.F. Bell G.I. Biochemistry. 1992; 31: 10414-10420Crossref PubMed Scopus (156) Google Scholar). The significance of the polymeric nature of Gluts is an interesting, still unclear question. In contrast, as detailed in the context, using our model for a monomer we can explain the results of almost all the mutagenic studies done so far.As a further test of our structure, we next sought to quantify the degree to which the locations of the channels in the structure correspond to those of all Glut1 94 residues which have been mutated (26Hruz P.W. Mueckler M.M. J. Biol. Chem. 1999; 274: 36176-36180Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 27Hruz P.W. Mueckler M.M. Biochemistry. 2000; 39: 9367-9372Crossref PubMed Scopus (34) Google Scholar, 28Olsowski A. Monden" @default.
• W2034155024 created "2016-06-24" @default.
• W2034155024 creator A5029007816 @default.
• W2034155024 creator A5032155879 @default.
• W2034155024 creator A5051750761 @default.
• W2034155024 creator A5073392172 @default.
• W2034155024 creator A5075565884 @default.
• W2034155024 creator A5078190387 @default.
• W2034155024 creator A5088001264 @default.
• W2034155024 date "2001-11-01" @default.
• W2034155024 modified "2023-09-26" @default.
• W2034155024 title "A Three-dimensional Model of the Human Facilitative Glucose Transporter Glut1" @default.
• W2034155024 cites W1483341493 @default.
• W2034155024 cites W1489461884 @default.
• W2034155024 cites W1499450468 @default.
• W2034155024 cites W1501783721 @default.
• W2034155024 cites W1506378995 @default.
• W2034155024 cites W1543885354 @default.
• W2034155024 cites W1556927657 @default.
• W2034155024 cites W1574505285 @default.
• W2034155024 cites W1583081035 @default.
• W2034155024 cites W1603195684 @default.
• W2034155024 cites W1668671560 @default.
• W2034155024 cites W1707091457 @default.
• W2034155024 cites W1721322204 @default.
• W2034155024 cites W1963644001 @default.
• W2034155024 cites W1967518399 @default.
• W2034155024 cites W1968200351 @default.
• W2034155024 cites W1981640620 @default.
• W2034155024 cites W1986191025 @default.
• W2034155024 cites W1992640002 @default.
• W2034155024 cites W1995272366 @default.
• W2034155024 cites W2000579634 @default.
• W2034155024 cites W2001689363 @default.
• W2034155024 cites W2006072955 @default.
• W2034155024 cites W2007199404 @default.
• W2034155024 cites W2015532430 @default.
• W2034155024 cites W2015642465 @default.
• W2034155024 cites W2015649064 @default.
• W2034155024 cites W2021167768 @default.
• W2034155024 cites W2021939059 @default.
• W2034155024 cites W2024875104 @default.
• W2034155024 cites W2037033695 @default.
• W2034155024 cites W2046847544 @default.
• W2034155024 cites W2048700689 @default.
• W2034155024 cites W2056202520 @default.
• W2034155024 cites W2064346749 @default.
• W2034155024 cites W2067401451 @default.
• W2034155024 cites W2069453488 @default.
• W2034155024 cites W2072850070 @default.
• W2034155024 cites W2072989980 @default.
• W2034155024 cites W2074783201 @default.
• W2034155024 cites W2078029627 @default.
• W2034155024 cites W2078499931 @default.
• W2034155024 cites W2078501767 @default.
• W2034155024 cites W2086833515 @default.
• W2034155024 cites W2089918014 @default.
• W2034155024 cites W2095081648 @default.
• W2034155024 cites W2096996169 @default.
• W2034155024 cites W2101532349 @default.
• W2034155024 cites W2106154817 @default.
• W2034155024 cites W2108442080 @default.
• W2034155024 cites W2125207055 @default.
• W2034155024 cites W2126405180 @default.
• W2034155024 cites W2130381741 @default.
• W2034155024 cites W2134836351 @default.
• W2034155024 cites W2143070723 @default.
• W2034155024 cites W2143266871 @default.
• W2034155024 cites W2152238111 @default.
• W2034155024 cites W2152982678 @default.
• W2034155024 cites W2157165549 @default.
• W2034155024 cites W2160589114 @default.
• W2034155024 cites W2411747497 @default.
• W2034155024 doi "https://doi.org/10.1074/jbc.m107350200" @default.
• W2034155024 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/11571301" @default.
• W2034155024 hasPublicationYear "2001" @default.
• W2034155024 type Work @default.
• W2034155024 sameAs 2034155024 @default.
• W2034155024 citedByCount "57" @default.
• W2034155024 countsByYear W20341550242013 @default.
• W2034155024 countsByYear W20341550242016 @default.
• W2034155024 countsByYear W20341550242020 @default.
• W2034155024 countsByYear W20341550242023 @default.
• W2034155024 crossrefType "journal-article" @default.
• W2034155024 hasAuthorship W2034155024A5029007816 @default.
• W2034155024 hasAuthorship W2034155024A5032155879 @default.
• W2034155024 hasAuthorship W2034155024A5051750761 @default.
• W2034155024 hasAuthorship W2034155024A5073392172 @default.
• W2034155024 hasAuthorship W2034155024A5075565884 @default.
• W2034155024 hasAuthorship W2034155024A5078190387 @default.
• W2034155024 hasAuthorship W2034155024A5088001264 @default.
• W2034155024 hasBestOaLocation W20341550241 @default.
• W2034155024 hasConcept C104317684 @default.
• W2034155024 hasConcept C134018914 @default.
• W2034155024 hasConcept C149011108 @default.
• W2034155024 hasConcept C161573976 @default.
• W2034155024 hasConcept C2777952866 @default.
• W2034155024 hasConcept C2779306644 @default.

• next