title: On the origin of large flexibility of P-glycoprotein in the inward-facing state.
abstract: P-glycoprotein (Pgp) is one of the most biomedically relevant transporters in the ATP binding cassette (ABC) superfamily due to its involvement in developing multidrug resistance in cancer cells. Employing molecular dynamics simulations and double electron-electron resonance spectroscopy, we have investigated the structural dynamics of membrane-bound Pgp in the inward-facing state and found that Pgp adopts an unexpectedly wide range of conformations, highlighted by the degree of separation between the two nucleotide-binding domains (NBDs). The distance between the two NBDs in the equilibrium simulations covers a range of at least 20 Å, including, both, more open and more closed NBD configurations than the crystal structure. The double electron-electron resonance measurements on spin-labeled Pgp mutants also show wide distributions covering both longer and shorter distances than those observed in the crystal structure. Based on structural and sequence analyses, we propose that the transmembrane domains of Pgp might be more flexible than other structurally known ABC exporters. The structural flexibility of Pgp demonstrated here is not only in close agreement with, but also helps rationalize, the reported high NBD fluctuations in several ABC exporters and possibly represents a fundamental difference in the transport mechanism between ABC exporters and ABC importers. In addition, during the simulations we have captured partial entrance of a lipid molecule from the bilayer into the lumen of Pgp, reaching the putative drug binding site. The location of the protruding lipid suggests a putative pathway for direct drug recruitment from the membrane.
authors: Center for Biophysics and Computational Biology, Department of Biochemistry, College of Medicine, and The Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, Illnois 61801, USA.
authors: Po-Chao
authors: PC
authors: Wen
authors: Brandy
authors: B
authors: Verhalen
authors: Stephan
authors: S
authors: Wilkens
authors: Hassane S
authors: HS
authors: Mchaourab
authors: Emad
authors: E
authors: Tajkhorshid
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry*; D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D008055:Lipids / Q000737:chemistry; D051379:Mice; D056004:Molecular Dynamics Simulation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D008055:Lipids; D000255:Adenosine Triphosphate
keywords: ABC Transporter; Double Electron Electron Resonance Spectroscopy (DEER); Electron Paramagnetic Resonance (EPR); Membrane Transport; Molecular Dynamics; P-glycoprotein; Protein Dynamics
references: J Chem Inf Model. 2012 Dec 21;52(12):3325-30
references: 23181365
references: Biochemistry. 2004 Oct 26;43(42):13242-7
references: 15491131
references: Biochemistry. 1997 May 13;36(19):5685-94
references: 9153408
references: EMBO J. 2005 Jun 1;24(11):1901-10
references: 15889153
references: Proteins. 2011 Jun;79(6):1977-90
references: 21488101
references: J Phys Chem B. 2012 Mar 8;116(9):2934-42
references: 22339391
references: FEBS Lett. 2012 Apr 5;586(7):972-6
references: 22569249
references: Biochim Biophys Acta. 2011 Dec;1808(12):2954-64
references: 21840296
references: Biochim Biophys Acta. 2009 May;1794(5):725-37
references: 19135557
references: Biochemistry. 2007 Mar 13;46(10):2767-78
references: 17302441
references: Mol Pharmacol. 2012 Jul;82(1):47-58
references: 22491935
references: J Gen Physiol. 2005 Sep;126(3):213-26
references: 16103276
references: BMC Struct Biol. 2009 Jan 22;9:3
references: 19159494
references: PLoS Comput Biol. 2011 Aug;7(8):e1002128
references: 21829343
references: PLoS Comput Biol. 2010 Apr 29;6(4):e1000762
references: 20454684
references: J Biomol Struct Dyn. 2013;31(6):612-29
references: 22888853
references: Naunyn Schmiedebergs Arch Pharmacol. 2006 Mar;372(6):385-99
references: 16541253
references: J Biol Chem. 2004 Nov 5;279(45):46518-26
references: 15326176
references: Biophys J. 2011 Aug 3;101(3):680-90
references: 21806936
references: Nat Rev Drug Discov. 2006 Mar;5(3):219-34
references: 16518375
references: Arch Biochem Biophys. 2006 Jan 1;445(1):124-8
references: 16343415
references: Bioinformation. 2012;8(22):1066-74
references: 23251040
references: J Phys Chem B. 2010 Dec 9;114(48):15948-57
references: 21069970
references: PLoS One. 2011;6(10):e25815
references: 21991360
references: Biochemistry. 2007 Sep 4;46(35):9899-910
references: 17696319
references: FEBS J. 2009 Dec;276(23):7016-26
references: 19878299
references: Eur J Pharm Sci. 2012 Dec 18;47(5):965-78
references: 23072916
references: Biochemistry. 2007 Mar 20;46(11):3075-83
references: 17302438
references: FEBS J. 2011 Sep;278(18):3226-45
references: 21740521
references: Nature. 2006 Sep 14;443(7108):180-5
references: 16943773
references: Biochemistry. 2004 Apr 6;43(13):3917-28
references: 15049699
references: Theor Biol Med Model. 2007 Sep 06;4:33
references: 17803828
references: PLoS Comput Biol. 2011 May;7(5):e1002036
references: 21589945
references: Mol Cell. 2006 Oct 6;24(1):51-62
references: 17018292
references: Biochem Biophys Res Commun. 2010 May 7;395(3):436-40
references: 20394729
references: BMC Struct Biol. 2005 Aug 16;5:14
references: 16105172
references: Biochemistry. 2000 Dec 5;39(48):14927-38
references: 11101309
references: J Mol Model. 2013 Jan;19(1):227-38
references: 22864626
references: J Biol Chem. 2011 Feb 4;286(5):3552-69
references: 21098040
references: PLoS Comput Biol. 2011 Jun;7(6):e1002083
references: 21731480
references: Proc Natl Acad Sci U S A. 2008 Sep 2;105(35):12837-42
references: 18725638
references: Proteins. 2003 Feb 15;50(3):437-50
references: 12557186
references: Biochemistry. 2001 Feb 6;40(5):1413-22
references: 11170469
references: Mol Cell. 2002 Jul;10(1):139-49
references: 12150914
references: Biochem J. 2005 Jul 15;389(Pt 2):517-26
references: 15799713
references: Neuron. 2004 Mar 25;41(6):859-65
references: 15046719
references: Mol Cell. 2003 Sep;12(3):651-61
references: 14527411
references: Biochemistry. 2012 Jun 26;51(25):5125-41
references: 22647192
references: Cell. 1996 Nov 1;87(3):507-17
references: 8898203
references: Biochem Pharmacol. 2010 Nov 15;80(10):1497-506
references: 20696137
references: Biochemistry. 2001 Jun 12;40(23):6937-47
references: 11389609
references: J Phys Chem A. 2012 Mar 22;116(11):3004-13
references: 22369471
references: Proc Natl Acad Sci U S A. 2011 Sep 13;108(37):15152-6
references: 21825153
references: Essays Biochem. 2011 Sep 7;50(1):161-78
references: 21967057
references: J Biol Chem. 2011 Mar 25;286(12):10476-82
references: 21278250
references: J Biol Chem. 2002 Nov 22;277(47):45688-94
references: 12244102
references: J Biol Chem. 2003 Mar 7;278(10):8294-9
references: 12501241
references: Mol Cell. 2009 Feb 27;33(4):528-36
references: 19250913
references: Biochem Biophys Res Commun. 1999 Aug 19;262(1):121-6
references: 10448079
references: FEBS Lett. 2007 Sep 4;581(22):4217-22
references: 17706648
references: J Biol Chem. 2004 Oct 22;279(43):45013-9
references: 15308647
references: Trends Biochem Sci. 2009 Oct;34(10):520-31
references: 19748784
references: Curr Opin Struct Biol. 2008 Dec;18(6):726-33
references: 18948194
references: Science. 2009 Mar 27;323(5922):1718-22
references: 19325113
references: Eur Biophys J. 2008 Apr;37(4):403-9
references: 17960373
references: ChemMedChem. 2008 Feb;3(2):280-95
references: 18175303
references: Nucleic Acids Res. 2012 Jan;40(Database issue):D290-301
references: 22127870
references: J Biol Chem. 2012 Jan 6;287(2):1112-27
references: 22086917
references: Biochem Pharmacol. 2012 Jan 1;83(1):57-68
references: 22044878
references: Proc Natl Acad Sci U S A. 2007 Nov 27;104(48):19005-10
references: 18024585
references: J Mol Biol. 2009 Oct 30;393(3):586-97
references: 19715702
references: Biochemistry. 2007 Dec 4;46(48):13787-99
references: 17988154
references: Proteins. 1995 Dec;23(4):566-79
references: 8749853
references: Essays Biochem. 2011 Sep 7;50(1):63-83
references: 21967052
references: Science. 2007 Sep 7;317(5843):1387-90
references: 17673622
references: FASEB J. 2009 May;23(5):1287-302
references: 19174475
references: Proc Natl Acad Sci U S A. 2012 Jul 3;109(27):10832-6
references: 22711831
references: J Bioenerg Biomembr. 2005 Dec;37(6):489-96
references: 16691488
references: Philos Trans R Soc Lond B Biol Sci. 2009 Jan 27;364(1514):239-45
references: 18957379
references: Proteins. 2010 Feb 15;78(3):738-53
references: 19847921
references: Nat Struct Mol Biol. 2012 Mar 25;19(4):395-402
references: 22447242
references: J Med Chem. 2012 Apr 12;55(7):3261-73
references: 22452412
references: PLoS Biol. 2007 Oct;5(10):e271
references: 17927448
references: Biophys J. 2007 Apr 15;92(8):2727-34
references: 17208973