title: Structures of P-glycoprotein reveal its conformational flexibility and an epitope on the nucleotide-binding domain.
abstract: P-glycoprotein (P-gp) is one of the best-known mediators of drug efflux-based multidrug resistance in many cancers. This validated therapeutic target is a prototypic, plasma membrane resident ATP-Binding Cassette transporter that pumps xenobiotic compounds out of cells. The large, polyspecific drug-binding pocket of P-gp recognizes a variety of structurally unrelated compounds. The transport of these drugs across the membrane is coincident with changes in the size and shape of this pocket during the course of the transport cycle. Here, we present the crystal structures of three inward-facing conformations of mouse P-gp derived from two different crystal forms. One structure has a nanobody bound to the C-terminal side of the first nucleotide-binding domain. This nanobody strongly inhibits the ATP hydrolysis activity of mouse P-gp by hindering the formation of a dimeric complex between the ATP-binding domains, which is essential for nucleotide hydrolysis. Together, these inward-facing conformational snapshots of P-gp demonstrate a range of flexibility exhibited by this transporter, which is likely an essential feature for the binding and transport of large, diverse substrates. The nanobody-bound structure also reveals a unique epitope on P-gp.
authors.affiliation: Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
authors.forename: Andrew B
authors.initials: AB
authors.lastname: Ward
authors.forename: Paul
authors.initials: P
authors.lastname: Szewczyk
authors.forename: Vinciane
authors.initials: V
authors.lastname: Grimard
authors.forename: Chang-Wook
authors.initials: CW
authors.lastname: Lee
authors.forename: Lorena
authors.initials: L
authors.lastname: Martinez
authors.forename: Rupak
authors.initials: R
authors.lastname: Doshi
authors.forename: Alexandra
authors.initials: A
authors.lastname: Caya
authors.forename: Mark
authors.initials: M
authors.lastname: Villaluz
authors.forename: Els
authors.initials: E
authors.lastname: Pardon
authors.forename: Cristina
authors.initials: C
authors.lastname: Cregger
authors.forename: Douglas J
authors.initials: DJ
authors.lastname: Swartz
authors.forename: Pierre Guy
authors.initials: PG
authors.lastname: Falson
authors.forename: Ina L
authors.initials: IL
authors.lastname: Urbatsch
authors.forename: Cedric
authors.initials: C
authors.lastname: Govaerts
authors.forename: Jan
authors.initials: J
authors.lastname: Steyaert
authors.forename: Geoffrey
authors.initials: G
authors.lastname: Chang
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry*; D000818:Animals; D016503:Drug Delivery Systems / Q000379:methods*; D018604:Epitope Mapping; D051379:Mice; D008958:Models, Molecular; D011487:Protein Conformation; D061905:Single-Domain Antibodies / Q000737:chemistry
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D061905:Single-Domain Antibodies
keywords: ABC transporter; membrane protein structure; nanobody-transporter complex
references.citation: Acta Crystallogr D Biol Crystallogr. 2011 Apr;67(Pt 4):235-42
references.pmid: 21460441
references.citation: PLoS One. 2011;6(8):e22577
references.pmid: 21826197
references.citation: J Am Chem Soc. 2012 Apr 4;134(13):5857-62
references.pmid: 22397466
references.citation: J Biol Chem. 2004 Dec 17;279(51):53593-601
references.pmid: 15383540
references.citation: Biochim Biophys Acta. 2010 Dec;1798(12):2304-14
references.pmid: 20541523
references.citation: FEBS Lett. 2006 Feb 13;580(4):998-1009
references.pmid: 16405967
references.citation: Brain Pathol. 2007 Oct;17(4):347-53
references.pmid: 17610523
references.citation: Handb Exp Pharmacol. 2011;(201):261-83
references.pmid: 21103972
references.citation: Proc Natl Acad Sci U S A. 2001 Feb 13;98(4):1525-30
references.pmid: 11171984
references.citation: Proc Natl Acad Sci U S A. 2012 Jul 3;109(27):10832-6
references.pmid: 22711831
references.citation: Biochim Biophys Acta. 2009 May;1794(5):725-37
references.pmid: 19135557
references.citation: Chem Rev. 2009 Jul;109(7):2989-3011
references.pmid: 19583429
references.citation: Br J Pharmacol. 1999 Sep;128(2):403-11
references.pmid: 10510451
references.citation: J Biol Chem. 2000 Aug 11;275(32):25031-8
references.pmid: 10831598
references.citation: Expert Opin Drug Metab Toxicol. 2010 May;6(5):603-19
references.pmid: 20397967
references.citation: Arch Biochem Biophys. 2006 Jan 1;445(1):124-8
references.pmid: 16343415
references.citation: Methods Mol Biol. 2010;596:405-32
references.pmid: 19949934
references.citation: Essays Biochem. 2011 Sep 7;50(1):209-32
references.pmid: 21967059
references.citation: Acta Crystallogr D Biol Crystallogr. 2010 Jan;66(Pt 1):12-21
references.pmid: 20057044
references.citation: Proc Natl Acad Sci U S A. 2013 Jun 11;110(24):9710-5
references.pmid: 23716676
references.citation: Nucleic Acids Res. 2003 Jul 1;31(13):3381-5
references.pmid: 12824332
references.citation: Nucleic Acids Res. 2012 Feb;40(4):1828-40
references.pmid: 22039098
references.citation: Acta Crystallogr D Biol Crystallogr. 2010 Apr;66(Pt 4):486-501
references.pmid: 20383002
references.citation: Vet Immunol Immunopathol. 2009 Mar 15;128(1-3):178-83
references.pmid: 19026455
references.citation: Nature. 2012 Oct 25;490(7421):566-9
references.pmid: 23000902
references.citation: Curr Drug Targets. 2011 May;12(5):600-20
references.pmid: 21039335
references.citation: Science. 2009 Mar 27;323(5922):1718-22
references.pmid: 19325113
references.citation: Trends Biochem Sci. 2010 Jan;35(1):36-42
references.pmid: 19819701
references.citation: Acta Crystallogr D Biol Crystallogr. 2010 Feb;66(Pt 2):213-21
references.pmid: 20124702
references.citation: Methods Enzymol. 1997;276:171-9
references.pmid: 9048376
references.citation: Antimicrob Agents Chemother. 2001 Oct;45(10):2807-12
references.pmid: 11557473
references.citation: J Biol Chem. 1995 Nov 10;270(45):26956-61
references.pmid: 7592942
references.citation: Proc Natl Acad Sci U S A. 1997 Nov 25;94(24):12908-13
references.pmid: 9371774
references.citation: Proc Natl Acad Sci U S A. 2007 Nov 27;104(48):19005-10
references.pmid: 18024585
references.citation: J Mol Biol. 2009 Oct 30;393(3):586-97
references.pmid: 19715702
references.citation: Curr Drug Targets. 2011 May;12(5):621-30
references.pmid: 21039338
references.citation: J Biol Chem. 2013 Jun 28;288(26):19211-20
references.pmid: 23658020
references.citation: Nat Struct Mol Biol. 2012 Mar 25;19(4):395-402
references.pmid: 22447242
references.citation: Proteins. 2010 Nov 1;78(14):2867-72
references.pmid: 20715055
references.citation: J Struct Biol. 2009 Apr;166(1):8-15
references.pmid: 19118632
references.citation: Adv Drug Deliv Rev. 2003 Jan 21;55(1):3-29
references.pmid: 12535572