title: Ligand binding and crystal structures of the substrate-binding domain of the ABC transporter OpuA.
abstract: BACKGROUND
  The ABC transporter OpuA from Lactococcus lactis transports glycine betaine upon activation by threshold values of ionic strength. In this study, the ligand binding characteristics of purified OpuA in a detergent-solubilized state and of its substrate-binding domain produced as soluble protein (OpuAC) was characterized.
  
  
  RESULTS
  The binding of glycine betaine to purified OpuA and OpuAC (K(D) = 4-6 microM) did not show any salt dependence or cooperative effects, in contrast to the transport activity. OpuAC is highly specific for glycine betaine and the related proline betaine. Other compatible solutes like proline and carnitine bound with affinities that were 3 to 4 orders of magnitude lower. The low affinity substrates were not noticeably transported by membrane-reconstituted OpuA. OpuAC was crystallized in an open (1.9 A) and closed-liganded (2.3 A) conformation. The binding pocket is formed by three tryptophans (Trp-prism) coordinating the quaternary ammonium group of glycine betaine in the closed-liganded structure. Even though the binding site of OpuAC is identical to that of its B. subtilis homolog, the affinity for glycine betaine is 4-fold higher.
  
  
  CONCLUSIONS
  Ionic strength did not affect substrate binding to OpuA, indicating that regulation of transport is not at the level of substrate binding, but rather at the level of translocation. The overlap between the crystal structures of OpuAC from L.lactis and B.subtilis, comprising the classical Trp-prism, show that the differences observed in the binding affinities originate from outside of the ligand binding site.
authors.affiliation: Biochemistry Department, Groningen Biomolecular Sciences and Biotechnology Institute, Netherlands Proteomics Centre, University of Groningen, Groningen, The Netherlands.
authors.forename: Justina C
authors.initials: JC
authors.lastname: Wolters
authors.forename: Ronnie P-A
authors.initials: RP
authors.lastname: Berntsson
authors.forename: Nadia
authors.initials: N
authors.lastname: Gul
authors.forename: Akira
authors.initials: A
authors.lastname: Karasawa
authors.forename: Andy-Mark W H
authors.initials: AM
authors.lastname: Thunnissen
authors.forename: Dirk-Jan
authors.initials: DJ
authors.lastname: Slotboom
authors.forename: Bert
authors.initials: B
authors.lastname: Poolman
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000737:chemistry*; D000251:Adenosine Triphosphatases / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry*; D001622:Betaine / Q000737:chemistry; D001665:Binding Sites; D018360:Crystallography, X-Ray; D008024:Ligands; D008074:Lipoproteins / Q000737:chemistry; D009994:Osmolar Concentration; D011485:Protein Binding; D011487:Protein Conformation; D012995:Solubility; D013379:Substrate Specificity
chemical_list: D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D008024:Ligands; D008074:Lipoproteins; C108613:OpuAC protein, Bacillus subtilis; D001622:Betaine; D000251:Adenosine Triphosphatases; C412743:OpuA protein, Lactococcus lactis
references.citation: Cell Mol Life Sci. 2003 Oct;60(10):2100-14
references.pmid: 14618258
references.citation: Biochemistry. 2003 Oct 14;42(40):11815-23
references.pmid: 14529293
references.citation: J Biol Chem. 2004 Feb 13;279(7):5588-96
references.pmid: 14612446
references.citation: J Biol Chem. 2004 Nov 12;279(46):48270-81
references.pmid: 15308642
references.citation: Nature. 2001 May 17;411(6835):269-76
references.pmid: 11357122
references.citation: Acta Crystallogr D Biol Crystallogr. 2002 Nov;58(Pt 11):1948-54
references.pmid: 12393927
references.citation: J Biol Chem. 2004 Mar 5;279(10):8747-52
references.pmid: 14672931
references.citation: Biochemistry. 2005 May 3;44(17):6597-608
references.pmid: 15850393
references.citation: J Bacteriol. 1994 Sep;176(17):5364-71
references.pmid: 8071213
references.citation: J Appl Crystallogr. 2007 Aug 1;40(Pt 4):658-674
references.pmid: 19461840
references.citation: J Bacteriol. 2007 Oct;189(19):6901-12
references.pmid: 17660277
references.citation: Biochim Biophys Acta. 1983 Jan 12;742(1):16-24
references.pmid: 6337632
references.citation: Proc Natl Acad Sci U S A. 2000 Jun 20;97(13):7102-6
references.pmid: 10860977
references.citation: Anal Biochem. 1989 May 15;179(1):131-7
references.pmid: 2757186
references.citation: EMBO J. 2003 Nov 17;22(22):5983-93
references.pmid: 14609945
references.citation: Neuron. 2004 Mar 25;41(6):907-14
references.pmid: 15046723
references.citation: Nature. 2009 Mar 5;458(7234):47-52
references.pmid: 19262666
references.citation: Biochemistry. 1999 Nov 2;38(44):14440-50
references.pmid: 10545166
references.citation: Methods. 2008 Oct;46(2):73-82
references.pmid: 18625320
references.citation: Acta Crystallogr D Biol Crystallogr. 1997 May 1;53(Pt 3):240-55
references.pmid: 15299926
references.citation: J Bacteriol. 2000 Jan;182(1):203-6
references.pmid: 10613881
references.citation: J Bacteriol. 1996 Sep;178(17):5229-34
references.pmid: 8752342
references.citation: Science. 2008 Jul 11;321(5886):250-3
references.pmid: 18621668
references.citation: J Biol Chem. 2006 Oct 6;281(40):29830-9
references.pmid: 16844687
references.citation: Nat Struct Biol. 2001 Sep;8(9):795-8
references.pmid: 11524684
references.citation: J Bacteriol. 2008 Aug;190(16):5663-71
references.pmid: 18567662
references.citation: Proc Natl Acad Sci U S A. 2006 Jul 11;103(28):10624-9
references.pmid: 16815971
references.citation: J Biomol Tech. 1999 Jun;10(2):51-63
references.pmid: 19499008
references.citation: J Mol Biol. 2006 Mar 24;357(2):592-606
references.pmid: 16445940
references.citation: J Biol Chem. 2008 Nov 21;283(47):32848-59
references.pmid: 18779321
references.citation: Biochemistry. 2008 Aug 5;47(31):8176-85
references.pmid: 18620422
references.citation: Mol Microbiol. 1996 Apr;20(1):17-25
references.pmid: 8861200
references.citation: J Biol Chem. 2003 Aug 8;278(32):29546-51
references.pmid: 12766159
references.citation: Protein Sci. 1995 Mar;4(3):337-60
references.pmid: 7795518
references.citation: Biochim Biophys Acta. 1999 Aug 20;1420(1-2):30-44
references.pmid: 10446288
references.citation: Science. 2008 Jul 11;321(5886):246-50
references.pmid: 18511655
references.citation: J Biol Chem. 1982 Feb 10;257(3):1131-3
references.pmid: 7035444
references.citation: FEBS Lett. 2007 Jul 24;581(18):3387-90
references.pmid: 17603047
references.citation: Nature. 2009 Jan 1;457(7225):115-8
references.pmid: 18987630
references.citation: Microbiology (Reading). 1998 Jan;144(1):83-90
references.pmid: 33757219
references.citation: Acta Crystallogr D Biol Crystallogr. 2004 Dec;60(Pt 12 Pt 1):2126-32
references.pmid: 15572765
references.citation: Nat Protoc. 2008;3(2):256-66
references.pmid: 18274528
references.citation: FEBS Lett. 2004 Apr 9;563(1-3):108-12
references.pmid: 15063732
references.citation: Biochemistry. 2008 Nov 18;47(46):12208-18
references.pmid: 18950194
references.citation: J Biol Chem. 2009 May 22;284(21):14368-76
references.pmid: 19329426
references.citation: Nature. 2007 Nov 22;450(7169):515-21
references.pmid: 18033289