title: MacA, a periplasmic membrane fusion protein of the macrolide transporter MacAB-TolC, binds lipopolysaccharide core specifically and with high affinity.
abstract: The Escherichia coli MacAB-TolC transporter has been implicated in efflux of macrolide antibiotics and secretion of enterotoxin STII. In this study, we found that purified MacA, a periplasmic membrane fusion protein, contains one tightly bound rough core lipopolysaccharide (R-LPS) molecule per MacA molecule. R-LPS was bound specifically to MacA protein with affinity exceeding that of polymyxin B. Sequence analyses showed that MacA contains two high-density clusters of positively charged amino acid residues located in the cytoplasmic N-terminal domain and the periplasmic C-terminal domain. Substitutions in the C-terminal cluster reducing the positive-charge density completely abolished binding of R-LPS. At the same time, these substitutions significantly reduced the functionality of MacA in the protection of E. coli against macrolides in vivo and in the in vitro MacB ATPase stimulation assays. Taken together, our results suggest that R-LPS or a similar glycolipid is a physiological substrate of MacAB-TolC.
authors: Department of Chemistry and Biochemistry, University of Oklahoma, Stephenson Life Sciences Research Center, Norman, Oklahoma, USA.
authors: Shuo
authors: S
authors: Lu
authors: Helen I
authors: HI
authors: Zgurskaya
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D008070:Lipopolysaccharides / Q000737:chemistry / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017434:Protein Structure, Tertiary
chemical_list: D018528:ATP-Binding Cassette Transporters; D029968:Escherichia coli Proteins; D008070:Lipopolysaccharides; C436312:MacA protein, E coli
references: J Biol Chem. 2004 Jul 30;279(31):32116-24
references: 15155734
references: Cold Spring Harb Perspect Biol. 2010 May;2(5):a000414
references: 20452953
references: Proc Natl Acad Sci U S A. 2009 Dec 1;106(48):20440-5
references: 19906986
references: Mol Microbiol. 2007 Feb;63(3):895-910
references: 17214741
references: J Bacteriol. 2005 Sep;187(18):6363-9
references: 16159769
references: J Immunol Methods. 1990 Jan 24;126(1):109-17
references: 2106001
references: J Bacteriol. 2008 Dec;190(23):7693-8
references: 18805970
references: J Mol Biol. 2009 Oct 23;393(2):342-55
references: 19695261
references: Future Microbiol. 2009 Sep;4(7):919-32
references: 19722844
references: J Mol Biol. 2001 Oct 26;313(3):501-10
references: 11676535
references: Mol Microbiol. 2007 Oct;66(1):100-9
references: 17725563
references: J Bacteriol. 2005 Nov;187(21):7471-80
references: 16237030
references: Structure. 2000 Jun 15;8(6):585-92
references: 10873859
references: Mol Microbiol. 2011 Aug;81(4):937-51
references: 21696464
references: Proc Natl Acad Sci U S A. 2010 Jun 15;107(24):11038-43
references: 20534468
references: Biochim Biophys Acta. 2009 May;1794(5):834-43
references: 19230852
references: Proc Natl Acad Sci U S A. 2009 Apr 28;106(17):7173-8
references: 19342493
references: J Biol Chem. 1959 Apr;234(4):705-9
references: 13654246
references: Mol Microbiol. 2006 Jan;59(1):126-41
references: 16359323
references: Annu Rev Biochem. 2009;78:119-46
references: 19231985
references: J Bacteriol. 2001 Oct;183(19):5639-44
references: 11544226
references: J Bacteriol. 2001 Oct;183(19):5554-61
references: 11544217
references: Nat Struct Mol Biol. 2006 Sep;13(9):806-14
references: 16892065
references: Structure. 2006 Mar;14(3):577-87
references: 16531241
references: Microbiol Mol Biol Rev. 2008 Sep;72(3):545-54
references: 18772288
references: J Mol Biol. 2009 Apr 17;387(5):1286-97
references: 19254725
references: J Bacteriol. 1994 Jul;176(13):4144-56
references: 7517391
references: J Mol Microbiol Biotechnol. 2001 Apr;3(2):215-8
references: 11321576
references: Mol Microbiol. 2012 Dec;86(5):1132-43
references: 23057817
references: Anal Biochem. 1987 Nov 1;166(2):368-79
references: 2449095
references: J Bacteriol. 2002 Dec;184(23):6499-507
references: 12426337