title: Crystal structure of the membrane fusion protein CusB from Escherichia coli.
abstract: Gram-negative bacteria, such as Escherichia coli, frequently utilize tripartite efflux complexes belonging to the resistance-nodulation-division family to expel diverse toxic compounds from the cell. These systems contain a periplasmic membrane fusion protein (MFP) that is critical for substrate transport. We here present the x-ray structures of the CusB MFP from the copper/silver efflux system of E. coli. This is the first structure of any MFPs associated with heavy-metal efflux transporters. CusB bridges the inner-membrane efflux pump CusA and outer-membrane channel CusC to mediate resistance to Cu(+) and Ag(+) ions. Two distinct structures of the elongated molecules of CusB were found in the asymmetric unit of a single crystal, which suggests the flexible nature of this protein. Each protomer of CusB can be divided into four different domains, whereby the first three domains are mostly beta-strands and the last domain adopts an entirely helical architecture. Unlike other known structures of MFPs, the alpha-helical domain of CusB is folded into a three-helix bundle. This three-helix bundle presumably interacts with the periplasmic domain of CusC. The N- and C-termini of CusB form the first beta-strand domain, which is found to interact with the periplasmic domain of the CusA efflux pump. Atomic details of how this efflux protein binds Cu(+) and Ag(+) were revealed by the crystals of the CusB-Cu(I) and CusB-Ag(I) complexes. The structures indicate that CusB consists of multiple binding sites for these metal ions. These findings reveal novel structural features of an MFP in the resistance-nodulation-division efflux system and provide direct evidence that this protein specifically interacts with transported substrates.
authors.affiliation: Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, 50011, USA.
authors.forename: Chih-Chia
authors.initials: CC
authors.lastname: Su
authors.forename: Feng
authors.initials: F
authors.lastname: Yang
authors.forename: Feng
authors.initials: F
authors.lastname: Long
authors.forename: Deepak
authors.initials: D
authors.lastname: Reyon
authors.forename: Mathew D
authors.initials: MD
authors.lastname: Routh
authors.forename: Dennis W
authors.initials: DW
authors.lastname: Kuo
authors.forename: Adam K
authors.initials: AK
authors.lastname: Mokhtari
authors.forename: Jonathan D
authors.initials: JD
authors.lastname: Van Ornam
authors.forename: Katherine L
authors.initials: KL
authors.lastname: Rabe
authors.forename: Julie A
authors.initials: JA
authors.lastname: Hoy
authors.forename: Young Jin
authors.initials: YJ
authors.lastname: Lee
authors.forename: Kanagalaghatta R
authors.initials: KR
authors.lastname: Rajashankar
authors.forename: Edward W
authors.initials: EW
authors.lastname: Yu
mesh_terms: D001665:Binding Sites; D003300:Copper / Q000737:chemistry / Q000378:metabolism; D018360:Crystallography, X-Ray / Q000379:methods*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013058:Mass Spectrometry; D050576:Membrane Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012834:Silver / Q000737:chemistry / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D050576:Membrane Fusion Proteins; D012834:Silver; D003300:Copper
references.citation: Acta Crystallogr D Biol Crystallogr. 2001 Dec;57(Pt 12):1755-62
references.pmid: 11717487
references.citation: J Hosp Infect. 2005 May;60(1):1-7
references.pmid: 15823649
references.citation: Biochemistry. 2001 Feb 13;40(6):1528-39
references.pmid: 11327811
references.citation: Methods Enzymol. 1997;276:307-26
references.pmid: 27754618
references.citation: J Biol Chem. 2007 Dec 7;282(49):35695-702
references.pmid: 17893146
references.citation: J Bacteriol. 2003 Jul;185(13):3804-12
references.pmid: 12813074
references.citation: Nature. 2006 Sep 14;443(7108):173-9
references.pmid: 16915237
references.citation: Nat Chem Biol. 2008 Feb;4(2):107-9
references.pmid: 18157124
references.citation: Acta Crystallogr D Biol Crystallogr. 2002 Nov;58(Pt 11):1948-54
references.pmid: 12393927
references.citation: Structure. 2006 Mar;14(3):577-87
references.pmid: 16531241
references.citation: J Mol Biol. 2009 Apr 17;387(5):1286-97
references.pmid: 19254725
references.citation: Nature. 2000 Jun 22;405(6789):914-9
references.pmid: 10879525
references.citation: J Biol Chem. 2004 Dec 17;279(51):52816-9
references.pmid: 15507433
references.citation: Nat Struct Biol. 1999 Oct;6(10):976-83
references.pmid: 10504734
references.citation: Protein Sci. 2007 Oct;16(10):2287-93
references.pmid: 17893365
references.citation: Microbiology (Reading). 2001 Apr;147(Pt 4):965-972
references.pmid: 11283292
references.citation: Appl Microbiol Biotechnol. 1999 Jun;51(6):730-50
references.pmid: 10422221
references.citation: J Mol Microbiol Biotechnol. 1999 Aug;1(1):107-25
references.pmid: 10941792
references.citation: Proc Natl Acad Sci U S A. 2009 Apr 28;106(17):7173-8
references.pmid: 19342493
references.citation: Mol Microbiol. 1994 Mar;11(5):841-7
references.pmid: 8022262
references.citation: Acta Crystallogr D Biol Crystallogr. 1998 Sep 1;54(Pt 5):905-21
references.pmid: 9757107
references.citation: Science. 2003 May 9;300(5621):976-80
references.pmid: 12738864
references.citation: Acta Crystallogr D Biol Crystallogr. 2002 Oct;58(Pt 10 Pt 2):1772-9
references.pmid: 12351820
references.citation: J Proteome Res. 2007 Oct;6(10):3908-17
references.pmid: 17854217
references.citation: J Biol Chem. 2003 Apr 11;278(15):12903-12
references.pmid: 12482849
references.citation: Science. 2006 Sep 1;313(5791):1295-8
references.pmid: 16946072
references.citation: Nature. 2002 Oct 10;419(6907):587-93
references.pmid: 12374972
references.citation: Infect Control Hosp Epidemiol. 2003 Aug;24(8):563-8
references.pmid: 12940575
references.citation: Clin Infect Dis. 1999 Dec;29(6):1507-11
references.pmid: 10585804
references.citation: Science. 2003 Sep 5;301(5638):1383-7
references.pmid: 12958362
references.citation: J Antimicrob Chemother. 2007 Apr;59(4):587-90
references.pmid: 17307768
references.citation: Acta Crystallogr D Biol Crystallogr. 2004 Dec;60(Pt 12 Pt 1):2126-32
references.pmid: 15572765
references.citation: J Biol Chem. 2003 Dec 12;278(50):49691-8
references.pmid: 12966104
references.citation: FEMS Microbiol Rev. 2003 Jun;27(2-3):313-39
references.pmid: 12829273
references.citation: J Bacteriol. 1994 Jul;176(13):3825-31
references.pmid: 8021163
references.citation: Acta Crystallogr D Biol Crystallogr. 1999 Apr;55(Pt 4):849-61
references.pmid: 10089316
references.citation: Curr Top Med Chem. 2001 Dec;1(6):529-39
references.pmid: 11895129
references.citation: FEMS Microbiol Rev. 2003 Jun;27(2-3):341-53
references.pmid: 12829274
references.citation: J Ind Microbiol Biotechnol. 2006 Jul;33(7):627-34
references.pmid: 16761169
references.citation: Proc Natl Acad Sci U S A. 2004 Jul 6;101(27):9994-9
references.pmid: 15226509
references.citation: J Bacteriol. 1995 Feb;177(4):998-1007
references.pmid: 7860612
references.citation: J Mol Biol. 2009 May 29;389(1):134-45
references.pmid: 19361527
references.citation: J Biol Chem. 2004 Jun 18;279(25):25939-42
references.pmid: 15117957