Three-dimensional Structure of P-glycoprotein

Abstract

Multidrug resistance of cancer cells and pathogens is a serious clinical problem. A major factor contributing to drug resistance in cancer is the over-expression of P-glycoprotein, a plasma membrane ATP-binding cassette (ABC) drug efflux pump. Three-dimensional structural data with a resolution limit of ∼8 Å have been obtained from two-dimensional crystals of P-glycoprotein trapped in the nucleotide-bound state. Each of the two transmembrane domains of P-glycoprotein consists of six long α-helical segments. Five of the α-helices from each transmembrane domain are related by a pseudo-2-fold symmetry, whereas the sixth breaks the symmetry. The two α-helices positioned closest to the (pseudo-) symmetry axis at the center of the molecule appear to be kinked. A large loop of density at the extracellular surface of the transporter is likely to correspond to the glycosylated first extracellular loop, whereas two globular densities at the cytoplasmic side correspond to the hydrophilic, nucleotide-binding domains. This is the first three-dimensional structure for an intact eukaryotic ABC transporter. Comparison with the structures of two prokaryotic ABC transporters suggests significant differences in the packing of the transmembrane α-helices within this protein family. Multidrug resistance of cancer cells and pathogens is a serious clinical problem. A major factor contributing to drug resistance in cancer is the over-expression of P-glycoprotein, a plasma membrane ATP-binding cassette (ABC) drug efflux pump. Three-dimensional structural data with a resolution limit of ∼8 Å have been obtained from two-dimensional crystals of P-glycoprotein trapped in the nucleotide-bound state. Each of the two transmembrane domains of P-glycoprotein consists of six long α-helical segments. Five of the α-helices from each transmembrane domain are related by a pseudo-2-fold symmetry, whereas the sixth breaks the symmetry. The two α-helices positioned closest to the (pseudo-) symmetry axis at the center of the molecule appear to be kinked. A large loop of density at the extracellular surface of the transporter is likely to correspond to the glycosylated first extracellular loop, whereas two globular densities at the cytoplasmic side correspond to the hydrophilic, nucleotide-binding domains. This is the first three-dimensional structure for an intact eukaryotic ABC transporter. Comparison with the structures of two prokaryotic ABC transporters suggests significant differences in the packing of the transmembrane α-helices within this protein family. The ATP-binding cassette (ABC) 1The abbreviations used are: ABC, ATP-binding cassette; NBD, nucleotide-binding domain; TMD, transmembrane domain; P-gp, P-glycoprotein; ICD, intracytoplasmic domain.1The abbreviations used are: ABC, ATP-binding cassette; NBD, nucleotide-binding domain; TMD, transmembrane domain; P-gp, P-glycoprotein; ICD, intracytoplasmic domain. family of membrane protein transporters is associated with many genetic disorders as well as the resistance of pathogenic bacteria to antibiotics and cancer cells to chemotherapy. The minimal functional unit of an ABC transporter typically consists of four domains, two hydrophilic nucleotide-binding domains (NBDs) and two transmembrane domains (TMDs), each consisting of several (frequently six) putative membrane-spanning α-helices. X-ray crystallography-derived structures of two bacterial ABC transporters (MsbA and BtuCD) have recently been published (1Chang G. Roth C.B. Science. 2001; 293: 1793-1800Crossref PubMed Scopus (581) Google Scholar, 2Locher K.P. Lee A.T. Rees D.C. Science. 2002; 296: 1091-1098Crossref PubMed Scopus (920) Google Scholar, 3Chang G. J. Mol. Biol. 2003; 330: 419-430Crossref PubMed Scopus (247) Google Scholar). The structure of the hydrophilic energy-transducing NBD dimer of BtuCD is similar to the structures determined for isolated NBDs (see Ref. 4Kerr I.D. Biochim. Biophys. Acta. 2002; 1561: 47-64Crossref PubMed Scopus (115) Google Scholar for a review and Refs. 5Hung L.W. Wang I.X. Nikaido K. Liu P.Q. Ames G.F. Kim S.H. Nature. 1998; 396: 703-707Crossref PubMed Scopus (614) Google Scholar, 6Diederichs K. Diez J. Greller G. Muller C. Breed J. Schnell C. Vonrhein C. Boos W. Welte W. EMBO J. 2000; 19: 5951-5961Crossref PubMed Scopus (272) Google Scholar, 7Hopfner K-P. Karchner A. Shin D.S. Craig L. Arthur L.M. Carney J.P. Trainer J.A. Cell. 2000; 101: 789-800Abstract Full Text Full Text PDF PubMed Scopus (799) Google Scholar, 8Gaudet R. Wiley D.C. EMBO J. 2001; 20: 4964-4972Crossref PubMed Scopus (245) Google Scholar, 9Smith P.C. Karpowich N. Millen L. Moody J.E. Rosen J. Thomas P.J. Hunt J.F. Mol. Cell. 2002; 10: 139-149Abstract Full Text Full Text PDF PubMed Scopus (670) Google Scholar for examples). No significant structural homology between the TMDs of the two transporters was observed. Indeed BtuCD has 20 membrane-spanning α-helices, whereas MsbA has 12. Eukaryotic ABC transporters have been even more refractory to structural analysis than bacterial ABC transporters. Electron microscopy coupled with image analysis has revealed low resolution data for single particles (10Rosenberg M.F. Callaghan R. Ford R.C. Higgins C.F. J. Biol. Chem. 1997; 272: 10685-10694Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar) and two-dimensional crystals (11Rosenberg M.F. Velarde G.S. Ford R.C. Martin C. Berridge G. Kerr I.D. Callaghan R. Schmidlin A. Wooding C. Linton K.J. Higgins C.F. EMBO J. 2001; 20: 5615-5625Crossref PubMed Scopus (261) Google Scholar, 12Rosenberg M.F. Kamis A.B. Callaghan R. Higgins C.F. Ford R.C. J. Biol. Chem. 2003; 278: 8294-8299Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, 15Lee J-Y. Urbatsch I.L. Senior A.E. Wilkens S. J. Biol. Chem. 2002; 277: 40125-40131Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar) of P-glycoprotein (P-gp). These data demonstrated large scale conformational changes upon nucleotide binding (11Rosenberg M.F. Velarde G.S. Ford R.C. Martin C. Berridge G. Kerr I.D. Callaghan R. Schmidlin A. Wooding C. Linton K.J. Higgins C.F. EMBO J. 2001; 20: 5615-5625Crossref PubMed Scopus (261) Google Scholar, 12Rosenberg M.F. Kamis A.B. Callaghan R. Higgins C.F. Ford R.C. J. Biol. Chem. 2003; 278: 8294-8299Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar), reflecting coupling between ATP hydrolysis and drug binding. Low resolution structures from single particles of a peptide transporter, TAP (13Velarde G.S. Ford R.C. Rosenberg M.F. Powis S.J. J. Biol. Chem. 2001; 276: 46054-46063Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar), a yeast multidrug transporter, Pdr5p (14Ferreira-Pereira A. Marco S. Decottignies A. Nader J. Goffeau A. Rigaud J-L. J. Biol. Chem. 2003; 278: 11995-11999Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), and another mammalian multidrug transporter, MRP-1 (16Rosenberg M.F. Mao Q. Holzenburg A. Ford R.C. Deeley R.G. Cole S.P. J. Biol. Chem. 2001; 276: 16076-16082Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar), have also been reported. Most recently, the low resolution three-dimensional structure of the cystic fibrosis transmembrane conductance regulator protein (CFTR) was reported (17Rosenberg M.F. Kamis A.B. Aleksandrov L.A. Ford R.C. Riordan J.R. J. Biol. Chem. 2004; 279: 39051-39057Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar) and was demonstrated to show a strong structural homology to P-gp including conformational plasticity. In this study we present the three-dimensional structure for P-gp at ∼8Å resolution obtained by cryo-electron crystallography of two-dimensional crystals. These structural data are the highest resolution for any eukaryotic ABC transporter and the first to show the location and packing of the transmembrane α-helices. This structure was obtained in the of a conformational of an ABC transporter to be P-gp was and two-dimensional crystals by at the of and a ATP as (11Rosenberg M.F. Velarde G.S. Ford R.C. Martin C. Berridge G. Kerr I.D. Callaghan R. Schmidlin A. Wooding C. Linton K.J. Higgins C.F. EMBO J. 2001; 20: 5615-5625Crossref PubMed Scopus (261) Google Scholar, 12Rosenberg M.F. Kamis A.B. Callaghan R. Higgins C.F. Ford R.C. J. Biol. Chem. 2003; 278: 8294-8299Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, W. EMBO J. PubMed Scopus Google Scholar, R. Berridge G. Higgins C.F. Biochim. Biophys. Acta. 1997; PubMed Scopus Google Scholar). the surface of the was with a in and at a and at in a at with a low at a of and for with by of a with a a with an R. J. Biol. PubMed Scopus Google Scholar). of and for and data (see a and a large of to and three-dimensional density the S. Biol. PubMed Scopus Google Scholar) and the J. Mol. 10: Google Scholar). A with a low factor of was used for of the and location of the A with a factor of was for a more of α-helices to densities and the within the J. Mol. 10: Google Scholar). The NBDs to the three-dimensional the with ATP P.C. Karpowich N. Millen L. Moody J.E. Rosen J. Thomas P.J. Hunt J.F. Mol. Cell. 2002; 10: 139-149Abstract Full Text Full Text PDF PubMed Scopus (670) Google Scholar) the K.P. Lee A.T. Rees D.C. Science. 2002; 296: 1091-1098Crossref PubMed Scopus (920) Google Scholar) as of the image used for of of with and resolution of with and of of is of structure to Å of is of PubMed Scopus Google Scholar), and of the data in a resolution was by the of structure with of by the of structure in this the resolution of with and PubMed Scopus Google of is PubMed Scopus Google of the data in a resolution was by the of structure with of by the of structure in this in a crystals of P-gp in the nucleotide-bound and data by cryo-electron microscopy and by image The crystals well with strong to Å resolution for and crystals whereas be to Å The of the crystals was as determined by for any of the for several large The resolution limit of the data was to be Å in the to the and Å to the These of and of resolution as in with a of the of of in the in The three-dimensional of is within the to Å resolution and to to Å resolution of data is this between and Å resolution of structure are with a of the resolution limit of three-dimensional A resolution of Å was used for of the in and Å resolution for of the three-dimensional in data from P-gp crystals with The the the data at a resolution of 20 Å for with The the the data to a resolution of The in the two are by the The scale image data The three-dimensional density for P-gp in the with a resolution of Å at a of the density and a factor of of α-helices and NBDs is by the A and show side of the scale 20 is related to A by a the axis as at the of each are in the with the axis of the protein at an of from the with to the by the in each The of the and NBDs are by the and The putative extracellular loop is at the of the show the to the of the in The is to the from the A is in and with two of densities by the The scale in 20 Å also to image obtained from the two-dimensional crystals of P-gp a low resolution with a resolution at 20 Å to with low resolution of P-gp (10Rosenberg M.F. Callaghan R. Ford R.C. Higgins C.F. J. Biol. Chem. 1997; 272: 10685-10694Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar, M.F. Velarde G.S. Ford R.C. Martin C. Berridge G. Kerr I.D. Callaghan R. Schmidlin A. Wooding C. Linton K.J. Higgins C.F. EMBO J. 2001; 20: 5615-5625Crossref PubMed Scopus (261) Google Scholar, 12Rosenberg M.F. Kamis A.B. Callaghan R. Higgins C.F. Ford R.C. J. Biol. Chem. 2003; 278: 8294-8299Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). a unit consisting of a single molecule with a similar to obtained from crystals of P-gp in the nucleotide-bound (11Rosenberg M.F. Velarde G.S. Ford R.C. Martin C. Berridge G. Kerr I.D. Callaghan R. Schmidlin A. Wooding C. Linton K.J. Higgins C.F. EMBO J. 2001; 20: 5615-5625Crossref PubMed Scopus (261) Google Scholar, 12Rosenberg M.F. Kamis A.B. Callaghan R. Higgins C.F. Ford R.C. J. Biol. Chem. 2003; 278: 8294-8299Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). A has also been with two-dimensional crystals of with nucleotide (17Rosenberg M.F. Kamis A.B. Aleksandrov L.A. Ford R.C. Riordan J.R. J. Biol. Chem. 2004; 279: 39051-39057Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). the P-gp with a resolution at the Å resolution limit of strong of density of Å be with densities for transmembrane α-helices. The three-dimensional density of P-gp is in from a to the long axis are in A and from a to the are in Each to a of density Å The three-dimensional at a of the the density strong protein The of P-gp molecule within the density is by a The P-gp in crystals long an of to the to the of the is by the in A and The molecule is Å long and Å are similar to obtained for P-gp and for ABC transporters and obtained by low resolution structural (10Rosenberg M.F. Callaghan R. Ford R.C. Higgins C.F. J. Biol. Chem. 1997; 272: 10685-10694Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar, M.F. Velarde G.S. Ford R.C. Martin C. Berridge G. Kerr I.D. Callaghan R. Schmidlin A. Wooding C. Linton K.J. Higgins C.F. EMBO J. 2001; 20: 5615-5625Crossref PubMed Scopus (261) Google Scholar, 12Rosenberg M.F. Kamis A.B. Callaghan R. Higgins C.F. Ford R.C. J. Biol. Chem. 2003; 278: 8294-8299Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, A. Marco S. Decottignies A. Nader J. Goffeau A. Rigaud J-L. J. Biol. Chem. 2003; 278: 11995-11999Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, M.F. Kamis A.B. Aleksandrov L.A. Ford R.C. Riordan J.R. J. Biol. Chem. 2004; 279: 39051-39057Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, C. A. Rigaud Marco S. J. Mol. Biol. 2002; PubMed Scopus Google Scholar). densities in the of the three-dimensional each Å long and Å in A and These at to two the density in the and be as α-helical within the transmembrane domains (1Chang G. Roth C.B. Science. 2001; 293: 1793-1800Crossref PubMed Scopus (581) Google Scholar, 2Locher K.P. Lee A.T. Rees D.C. Science. 2002; 296: 1091-1098Crossref PubMed Scopus (920) Google Scholar, 3Chang G. J. Mol. Biol. 2003; 330: 419-430Crossref PubMed Scopus (247) Google Scholar, Higgins C.F. Kerr I.D. Linton K.J. J. 2003; PubMed Scopus Google Scholar, C. R. PubMed Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The of of densities was Å than to the intracytoplasmic of the TMDs intracytoplasmic domains as of the membrane-spanning α-helices in MsbA (1Chang G. Roth C.B. Science. 2001; 293: 1793-1800Crossref PubMed Scopus (581) Google Scholar, 3Chang G. J. Mol. Biol. 2003; 330: 419-430Crossref PubMed Scopus (247) Google Scholar), with the In BtuCD α-helical and the between transmembrane domains and NBDs are by K.P. Lee A.T. Rees D.C. Science. 2002; 296: 1091-1098Crossref PubMed Scopus (920) Google Scholar). The of the densities in P-gp has similar to MsbA (1Chang G. Roth C.B. Science. 2001; 293: 1793-1800Crossref PubMed Scopus (581) Google Scholar, 3Chang G. J. Mol. Biol. 2003; 330: 419-430Crossref PubMed Scopus (247) Google Scholar). The long of the putative α-helices to the long axis of the molecule and a center with a of Å putative was from the long This density was to from of the molecule to the (see by The densities of to the membrane are to be two of to the two the long axis of the protein in packing is in more The surface in with the has been to the glycosylated of the P-gp molecule (10Rosenberg M.F. Callaghan R. Ford R.C. Higgins C.F. J. Biol. Chem. 1997; 272: 10685-10694Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar, M.F. Velarde G.S. Ford R.C. Martin C. Berridge G. Kerr I.D. Callaghan R. Schmidlin A. Wooding C. Linton K.J. Higgins C.F. EMBO J. 2001; 20: 5615-5625Crossref PubMed Scopus (261) Google Scholar, 12Rosenberg M.F. Kamis A.B. Callaghan R. Higgins C.F. Ford R.C. J. Biol. Chem. 2003; 278: 8294-8299Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). This side of the and the in A and densities are the of the molecule to the single large extracellular loop of P-gp and be as two α-helices likely also to this The from the in the three-dimensional crystals to the cytoplasmic side of the molecule (10Rosenberg M.F. Callaghan R. Ford R.C. Higgins C.F. J. Biol. Chem. 1997; 272: 10685-10694Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar, M.F. Velarde G.S. Ford R.C. Martin C. Berridge G. Kerr I.D. Callaghan R. Schmidlin A. Wooding C. Linton K.J. Higgins C.F. EMBO J. 2001; 20: 5615-5625Crossref PubMed Scopus (261) Google Scholar, 12Rosenberg M.F. Kamis A.B. Callaghan R. Higgins C.F. Ford R.C. J. Biol. Chem. 2003; 278: 8294-8299Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar) in A and consists of two large domains related by a symmetry and has the axis to the long These densities correspond to the two NBDs K.P. Lee A.T. Rees D.C. Science. 2002; 296: 1091-1098Crossref PubMed Scopus (920) Google Scholar, 3Chang G. J. Mol. Biol. 2003; 330: 419-430Crossref PubMed Scopus (247) Google Scholar, 4Kerr I.D. Biochim. Biophys. Acta. 2002; 1561: 47-64Crossref PubMed Scopus (115) Google Scholar, 5Hung L.W. Wang I.X. Nikaido K. Liu P.Q. Ames G.F. Kim S.H. Nature. 1998; 396: 703-707Crossref PubMed Scopus (614) Google Scholar, 6Diederichs K. Diez J. Greller G. Muller C. Breed J. Schnell C. Vonrhein C. Boos W. Welte W. EMBO J. 2000; 19: 5951-5961Crossref PubMed Scopus (272) Google Scholar, 7Hopfner K-P. Karchner A. Shin D.S. Craig L. Arthur L.M. Carney J.P. Trainer J.A. Cell. 2000; 101: 789-800Abstract Full Text Full Text PDF PubMed Scopus (799) Google Scholar, 8Gaudet R. Wiley D.C. EMBO J. 2001; 20: 4964-4972Crossref PubMed Scopus (245) Google Scholar, 9Smith P.C. Karpowich N. Millen L. Moody J.E. Rosen J. Thomas P.J. Hunt J.F. Mol. Cell. 2002; 10: 139-149Abstract Full Text Full Text PDF PubMed Scopus (670) Google Scholar). The and α-helices of the NBDs be in an Å resolution and also be by the of density the of the of the associated with two-dimensional crystallography R. J. Biol. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). The NBD structure with ATP P.C. Karpowich N. Millen L. Moody J.E. Rosen J. Thomas P.J. Hunt J.F. Mol. Cell. 2002; 10: 139-149Abstract Full Text Full Text PDF PubMed Scopus (670) Google Scholar) and the NBD structure G. J. Mol. Biol. 2003; 330: 419-430Crossref PubMed Scopus (247) Google Scholar) be the P-gp density in a and in the of the NBDs with to the TMDs in P-gp is from of at this The for the NBDs in and is the dimer in the P.C. Karpowich N. Millen L. Moody J.E. Rosen J. Thomas P.J. Hunt J.F. Mol. Cell. 2002; 10: 139-149Abstract Full Text Full Text PDF PubMed Scopus (670) Google Scholar). have the first three-dimensional structure for P-gp at Å and is in The is in whereas show the transmembrane of the transporter with α-helices by In the molecule has an similar to the low resolution determined by (11Rosenberg M.F. Velarde G.S. Ford R.C. Martin C. Berridge G. Kerr I.D. Callaghan R. Schmidlin A. Wooding C. Linton K.J. Higgins C.F. EMBO J. 2001; 20: 5615-5625Crossref PubMed Scopus (261) Google Scholar, 12Rosenberg M.F. Kamis A.B. Callaghan R. Higgins C.F. Ford R.C. J. Biol. Chem. 2003; 278: 8294-8299Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). transmembrane α-helices of to the membrane are in two of six a pseudo-2-fold symmetry as the homology between the two TMDs of the transporter. α-helices are in the whereas α-helices with are The of the TMDs are in and of by the pseudo-2-fold Five α-helices from each are in each of the two of the transporter. The sixth of α-helices the transmembrane α-helices show a is to the TMD, whereas to the long α-helical and as α-helices, are by the symmetry of the transporter and and are of densities likely correspond to the major extracellular loop in P-gp, whereas is well from the long axis of the molecule and is to the The density and at the between unit the of unit and the extracellular surface of P-gp of the unit are two of this density by be as of the is at the resolution The from symmetry in the is likely to be a of conformational changes by nucleotide binding (11Rosenberg M.F. Velarde G.S. Ford R.C. Martin C. Berridge G. Kerr I.D. Callaghan R. Schmidlin A. Wooding C. Linton K.J. Higgins C.F. EMBO J. 2001; 20: 5615-5625Crossref PubMed Scopus (261) Google Scholar, 12Rosenberg M.F. Kamis A.B. Callaghan R. Higgins C.F. Ford R.C. J. Biol. Chem. 2003; 278: 8294-8299Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). Low resolution of P-gp in the have a more and a in the membrane (11Rosenberg M.F. Velarde G.S. Ford R.C. Martin C. Berridge G. Kerr I.D. Callaghan R. Schmidlin A. Wooding C. Linton K.J. Higgins C.F. EMBO J. 2001; 20: 5615-5625Crossref PubMed Scopus (261) Google Scholar, 12Rosenberg M.F. Kamis A.B. Callaghan R. Higgins C.F. Ford R.C. J. Biol. Chem. 2003; 278: 8294-8299Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). conformational have been in a related ABC transporter, (17Rosenberg M.F. Kamis A.B. Aleksandrov L.A. Ford R.C. Riordan J.R. J. Biol. Chem. 2004; 279: 39051-39057Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar), the for The present structure well with the structure of P-gp in crystals in the of microscopy of P-gp in the are to with the and of the conformational changes by nucleotide binding. the a of 20 and structures for the and with the (11Rosenberg M.F. Velarde G.S. Ford R.C. Martin C. Berridge G. Kerr I.D. Callaghan R. Schmidlin A. Wooding C. Linton K.J. Higgins C.F. EMBO J. 2001; 20: 5615-5625Crossref PubMed Scopus (261) Google Scholar) the conformational a in the of the P-gp These and have the conformational changes of by transporters K.P. Lee A.T. Rees D.C. Science. 2002; 296: 1091-1098Crossref PubMed Scopus (920) Google Scholar, 3Chang G. J. Mol. Biol. 2003; 330: 419-430Crossref PubMed Scopus (247) Google Scholar, M.F. Velarde G.S. Ford R.C. Martin C. Berridge G. Kerr I.D. Callaghan R. Schmidlin A. Wooding C. Linton K.J. Higgins C.F. EMBO J. 2001; 20: 5615-5625Crossref PubMed Scopus (261) Google Scholar, 12Rosenberg M.F. Kamis A.B. Callaghan R. Higgins C.F. Ford R.C. J. Biol. Chem. 2003; 278: 8294-8299Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, R. J. 1997; PubMed Scopus Google Scholar, P.C. 2003; PubMed Scopus Google Scholar, 2001; PubMed Scopus Google Scholar, 2000; PubMed Scopus Google Scholar, J. Linton K.J. Kerr I.D. Callaghan R. 2003; PubMed Scopus Google Ref. C.F. Linton K.J. Mol. Biol. 2004; PubMed Scopus Google Scholar for a P-gp has been the of and Higgins C.F. Kerr I.D. Linton K.J. J. 2003; PubMed Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, P.C. 2003; PubMed Scopus Google Scholar, C. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar, C. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar). These transmembrane α-helices and and to each at the cytoplasmic side of the and have been in drug binding C. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). The two α-helices at the center of the protein are for and These are at the are in in with data Higgins C.F. Kerr I.D. Linton K.J. J. 2003; PubMed Scopus Google Scholar). These also a in the density the in a similar in and significant of in the transporter J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). data have upon ATP binding with to A. J. J. Linton K.J. Kerr I.D. Callaghan R. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar) with the the at the center of the structure is and have also been by to be to each as have α-helices and C. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar, C. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar). is at this resolution to of the α-helices in the P-gp structure the and from the homology between P-gp and the bacterial ABC transporter the packing of the transmembrane for P-gp is with reported for the MsbA (1Chang G. Roth C.B. Science. 2001; 293: 1793-1800Crossref PubMed Scopus (581) Google Scholar, 3Chang G. J. Mol. Biol. 2003; 330: 419-430Crossref PubMed Scopus (247) Google Scholar, Higgins C.F. Kerr I.D. Linton K.J. J. 2003; PubMed Scopus Google Scholar, P.C. 2003; PubMed Scopus Google Scholar). are likely to be a of the the P-gp structure has to significant conformational changes in the transmembrane domains (11Rosenberg M.F. Velarde G.S. Ford R.C. Martin C. Berridge G. Kerr I.D. Callaghan R. Schmidlin A. Wooding C. Linton K.J. Higgins C.F. EMBO J. 2001; 20: 5615-5625Crossref PubMed Scopus (261) Google Scholar, 12Rosenberg M.F. Kamis A.B. Callaghan R. Higgins C.F. Ford R.C. J. Biol. Chem. 2003; 278: 8294-8299Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). the packing of the two of the molecule in P-gp with to each is from the reported for the MsbA (1Chang G. Roth C.B. Science. 2001; 293: 1793-1800Crossref PubMed Scopus (581) Google Scholar, 3Chang G. J. Mol. Biol. 2003; 330: 419-430Crossref PubMed Scopus (247) Google Scholar). the between the two TMDs in P-gp is more than in the MsbA of MsbA P.C. 2003; PubMed Scopus Google Scholar) this dimer whereas the P-gp structure to be in and S. the of P-gp is similar to the BtuCD K.P. Lee A.T. Rees D.C. Science. 2002; 296: 1091-1098Crossref PubMed Scopus (920) Google Scholar) the between the is and the long of each side by also the P-gp NBDs in the are to in the dimer K.P. Lee A.T. Rees D.C. Science. 2002; 296: 1091-1098Crossref PubMed Scopus (920) Google Scholar) and the dimer with ATP P.C. Karpowich N. Millen L. Moody J.E. Rosen J. Thomas P.J. Hunt J.F. Mol. Cell. 2002; 10: 139-149Abstract Full Text Full Text PDF PubMed Scopus (670) Google Scholar) are from the NBDs reported for MsbA (1Chang G. Roth C.B. Science. 2001; 293: 1793-1800Crossref PubMed Scopus (581) Google Scholar). The NBDs in the MsbA structure G. J. Mol. Biol. 2003; 330: 419-430Crossref PubMed Scopus (247) Google Scholar) are in and are to the in the P-gp major differences in structure with to the isolated NBD structures I.D. Biochim. Biophys. Acta. 2002; 1561: 47-64Crossref PubMed Scopus (115) Google Scholar, 5Hung L.W. Wang I.X. Nikaido K. Liu P.Q. Ames G.F. Kim S.H. Nature. 1998; 396: 703-707Crossref PubMed Scopus (614) Google Scholar, 6Diederichs K. Diez J. Greller G. Muller C. Breed J. Schnell C. Vonrhein C. Boos W. Welte W. EMBO J. 2000; 19: 5951-5961Crossref PubMed Scopus (272) Google Scholar, 7Hopfner K-P. Karchner A. Shin D.S. Craig L. Arthur L.M. Carney J.P. Trainer J.A. Cell. 2000; 101: 789-800Abstract Full Text Full Text PDF PubMed Scopus (799) Google Scholar, 8Gaudet R. Wiley D.C. EMBO J. 2001; 20: 4964-4972Crossref PubMed Scopus (245) Google Scholar, 9Smith P.C. Karpowich N. Millen L. Moody J.E. Rosen J. Thomas P.J. Hunt J.F. Mol. Cell. 2002; 10: 139-149Abstract Full Text Full Text PDF PubMed Scopus (670) Google Scholar) and with to the dimer and are for the P-gp In the structural data for P-gp the Higgins C.F. Kerr I.D. Linton K.J. J. 2003; PubMed Scopus Google Scholar) the MsbA structure the domain for this of ABC transporters. The structure a the packing of transmembrane α-helices in ABC transporters and a conformational be for of P-gp and ABC transporter of Higgins C.F. Kerr I.D. Linton K.J. J. 2003; PubMed Scopus Google Scholar, P.C. 2003; PubMed Scopus Google Scholar). This the first three-dimensional structure for a eukaryotic ABC transporter and the structure of any ABC transporter at a resolution to the packing of the transmembrane α-helices (1Chang G. Roth C.B. Science. 2001; 293: 1793-1800Crossref PubMed Scopus (581) Google Scholar, 2Locher K.P. Lee A.T. Rees D.C. Science. 2002; 296: 1091-1098Crossref PubMed Scopus (920) Google Scholar, 3Chang G. J. Mol. Biol. 2003; 330: 419-430Crossref PubMed Scopus (247) Google Scholar). In and of two of ABC the packing of the transmembrane α-helices is to be ABC transporters a of for the TMDs structural and for and the and for to at the of and and and for with