|Gene:||cusF||Accession Numbers: G6321 (EcoCyc), b0573, ECK0565|
Synonyms: agrF, cusX, ylcC, ORF110
Component of: copper / silver efflux system (extended summary available)
CusF is a periplasmic binding protein involved in the detoxification of copper and silver ions in E. coli as part of the CusCFBA copper/silver efflux system.
CusF forms a five-stranded β-barrel and has been crystallized in its apo form as well as with bound Ag(I) or Cu(I) [Loftin05, Loftin07, Xue08]. CusF is a metallochaperone that specifically binds Ag(I) and Cu(I), but not Cu(II) [Kittleson06] despite earlier evidence regarding binding of Cu(II) [Astashkin05]. CusF transfers metal directly to CusB for export [Bagai08]. CusF is a pink copper-binding protein and binds one copper ion per monomer [Franke03]. The histidine residue at position 58 and the two methionine residues at positions 69 and 71 are essential for copper/silver binding [Franke03, Kittleson06]. Cu(I) binding also involves a strong interaction between the metal ion and the aromatic ring of a tryptophan residue at position 44 [Xue08, Loftin09, Chakravorty11]. The UV-vis spectrum of copper-containing CusF showed an absorption-maximum around 510 nm, which has not been reported for any other copper protein [Franke03].
Gene Citations: [Franke01]
Locations: periplasmic space
|Map Position: [596,354 -> 596,686] (12.85 centisomes)||Length: 333 bp / 110 aa|
Molecular Weight of Polypeptide: 12.251 kD (from nucleotide sequence), 10.0 kD (experimental) [Franke03 ]
Unification Links: ASAP:ABE-0001967 , DIP:DIP-9350N , EchoBASE:EB3985 , EcoGene:EG14234 , EcoliWiki:b0573 , OU-Microarray:b0573 , PortEco:cusF , PR:PRO_000022352 , Protein Model Portal:P77214 , RefSeq:NP_415105 , RegulonDB:G6321 , SMR:P77214 , String:511145.b0573 , Swiss-Model:P77214 , UniProt:P77214
|Biological Process:||GO:0006878 - cellular copper ion homeostasis
GO:0010043 - response to zinc ion [Lee05a]
GO:0010272 - response to silver ion [Franke01, Lok08]
GO:0010273 - detoxification of copper ion [Franke03]
GO:0046688 - response to copper ion [Franke01, Yamamoto05a]
|Molecular Function:||GO:0005507 - copper ion binding
[Franke03, Astashkin05, Loftin05, Kittleson06, Loftin07, Xue08]
GO:0005515 - protein binding [Franke03]
GO:0016530 - metallochaperone activity [Bagai08]
GO:0016531 - copper chaperone activity [Bagai08]
GO:0046914 - transition metal ion binding [Bagai08, Kittleson06, Loftin07]
GO:0046872 - metal ion binding [UniProtGOA11a]
|Cellular Component:||GO:0030288 - outer membrane-bounded periplasmic space
GO:0042597 - periplasmic space [Franke03, UniProtGOA11, UniProtGOA11a]
|MultiFun Terms:||cell processes → adaptations|
|Growth Medium||Growth?||T (°C)||O2||pH||Osm/L||Growth Observations|
|LB enriched||Yes||37||Aerobic||6.95||Yes [Gerdes03, Comment 1]|
|LB Lennox||Yes||37||Aerobic||7||Yes [Baba06, Comment 2]|
|M9 medium with 1% glycerol||Yes||37||Aerobic||7.2||0.35||Yes [Joyce06, Comment 3]|
|MOPS medium with 0.4% glucose||Yes||37||Aerobic||7.2||0.22||Yes [Baba06, Comment 2] |
Yes [Feist07, Comment 4]
Subunit of: copper / silver efflux system
Subunit composition of
copper / silver efflux system = [CusC]3[CusB]6[CusF][CusA]3
copper / silver efflux system - outer membrane porin = CusC (summary available)
copper / silver efflux system - membrane fusion protein = CusB (summary available)
copper / silver efflux system - periplasmic binding protein and metallochaperone = CusF (extended summary available)
copper / silver efflux system - membrane subunit = CusA (summary available)
The cusCFBA operon in E. coli K-12 encodes proteins that function together as a copper/silver efflux system. CusCBA is a tripartite complex that spans both the inner and outer membrane and, along with the periplasmic chaperone CusF, functions to export copper and silver ions from both the the cytoplasm and the periplasm to the extracellular environment. CusC forms a channel in the outer membrane, CusB is a member of the membrane fusion protein (MFP) family and CusA is a resistance-nodulation-division (RND) permease. CusF is the periplasmic copper binding protein [Franke01, Grass01, Franke03, Bagai08]. The function of a membrane fusion protein like CusB may be to bring the outer membrane factor, CusC, closer to the resistance-nodulation-division permease, CusA.
Through it's efflux function, CusCFBA helps to protect E. coli K-12 from high levels of exogenous copper and silver however its primary physiological role may be to export endogenous copper(I) ions that accumulate in the periplasm under anaerobic amino acid limitation (a host-relevant environment). Free Cu(I) accumulates in the periplasmic space of E. coli grown under anaerobic amino acid limitation largely due to lack of methionine which is the principal intracellular Cu(I) chelator, although anaerobiosis will also favor the accumulation of Cu(I) over Cu(II). ΔcopA and/or ΔcusC mutants show compromised growth during fumarate respiration under anaerobic and amino acid-limited conditions possibly due to Cu(I) induced damage to the Fe-S clusters of fumarate reductase [Fung13].
CusA contains 12 transmembrane (TM) segments and a large periplasmic domain formed from two loops located between TM1 and 2 and TM7 and 8 [Nishino01, Su11]. As an RND transporter, CusA probably forms trimers in vivo, but it forms a mixture of oligomers in detergent solution [Stroebel07, Su11]. The amino acids M573, M623, M672, D405, E412, and A399 of CusA are essential for copper tolerance [Franke03].
CusB is monomeric in solution [Bagai07]. The amino acids M21, M36, and M38 of CusB are involved in metal binding - mutations at these sites result in reduced Ag(I) binding affinity and show reduced copper tolerance [Bagai07].
A co-crystal structure of the CusBA complex has been resolved at 2.9Å. The trimeric CusA permease directly contacts with 6 CusB molecules which form a hexameric funnel-like structure [Su11]. Crystal structures of CusBA in complex with copper(I) are also available [Su12].
Copper and silver extrusion through CusCFBA is dependent upon the proton-motive-force [Li97]. Selectable silver resistance is mediated by the CusCFBA system [Lok08]. An in-frame chromosomal deletion mutant of cusA yielded a silver-sensitive E. coli mutant strain which did not differ in copper resistance to its isogenic parent [Franke01, Gupta01a]. When combined with a mutation in cueO which is responsible for copper tolerance under aerobic conditions, cusA, cusB, cusC, and cusF mutants are copper-sensitive [Franke03]. Overexpression of cusA reversed the L-cysteine-induced growth inhibition of a tnaA mutant [Yamada06]. Overexpression also reduced levels of intracellular L-cysteine [Yamada06]. Induced expression of cusAB from a plasmid results in resistance to fosfomycin [Nishino01]. The phenotypes of cusA and copA mutants are not additive, so it has been suggested that the CusCFBA system may only be involved in export of copper and silver from the periplasm [Grass01].
The cusCFBA operon is induced upon addition of CuSO4 [Yamamoto05a] or ZnSO4 [Lee05a]. Induction is dependent upon the CusRS two-component system [Munson00, Yamamoto05a]. Northern hybridization, RT-PCR, primer extension analyses, and expression of an operon fusion indicate that cusCFBA mRNA is induced by the presence of silver and copper ions [Franke01]. CusF appears in the periplasmic space after induction with CuCl2 [Franke03]. cusB expression is induced by indole [Hirakawa05].
Relationship Links: PDB:Structure:3NE5
|Molecular Function:||GO:0005375 - copper ion transmembrane transporter activity
GO:0015080 - silver ion transmembrane transporter activity [Lok08]
|Cellular Component:||GO:0030313 - cell envelope [Su11]|
Enzymatic reaction of: export of Cu+ (copper / silver efflux system)
Enzymatic reaction of: Export of Ag+ (copper / silver efflux system)
|Feature Class||Location||Common Name||Citations||Comment|
|Signal-Sequence||1 -> 21||CusF signal sequence|
|Chain||22 -> 110|
|Mutagenesis-Variant||25 -> 27|
|Mutagenesis-Variant||57 -> 58|
|Metal-Binding-Site||58, 69, 71||CusF metal binding site|
Markus Krummenacker on Tue Oct 14, 1997:
Gene object created from Blattner lab Genbank (v. M52) entry.
Peter D. Karp on Thu Jan 16, 2003:
Predicted gene function revised as a result of E. coli genome reannotation by Serres et al. [Serres01 ].
Astashkin05: Astashkin AV, Raitsimring AM, Walker FA, Rensing C, McEvoy MM (2005). "Characterization of the copper(II) binding site in the pink copper binding protein CusF by electron paramagnetic resonance spectroscopy." J Biol Inorg Chem 10(3);221-30. PMID: 15770503
Baba06: Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H (2006). "Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection." Mol Syst Biol 2;2006.0008. PMID: 16738554
Bagai07: Bagai I, Liu W, Rensing C, Blackburn NJ, McEvoy MM (2007). "Substrate-linked conformational change in the periplasmic component of a Cu(I)/Ag(I) efflux system." J Biol Chem 282(49);35695-702. PMID: 17893146
Bleuel05: Bleuel C, Grosse C, Taudte N, Scherer J, Wesenberg D, Krauss GJ, Nies DH, Grass G (2005). "TolC is involved in enterobactin efflux across the outer membrane of Escherichia coli." J Bacteriol 187(19);6701-7. PMID: 16166532
Chakravorty11: Chakravorty DK, Wang B, Ucisik MN, Merz KM (2011). "Insight into the cation-π interaction at the metal binding site of the copper metallochaperone CusF." J Am Chem Soc 133(48);19330-3. PMID: 22029374
DiazMejia09: Diaz-Mejia JJ, Babu M, Emili A (2009). "Computational and experimental approaches to chart the Escherichia coli cell-envelope-associated proteome and interactome." FEMS Microbiol Rev 33(1);66-97. PMID: 19054114
Feist07: Feist AM, Henry CS, Reed JL, Krummenacker M, Joyce AR, Karp PD, Broadbelt LJ, Hatzimanikatis V, Palsson BO (2007). "A genome-scale metabolic reconstruction for Escherichia coli K-12 MG1655 that accounts for 1260 ORFs and thermodynamic information." Mol Syst Biol 3;121. PMID: 17593909
Franke01: Franke S, Grass G, Nies DH (2001). "The product of the ybdE gene of the Escherichia coli chromosome is involved in detoxification of silver ions." Microbiology 2001;147(Pt 4);965-72. PMID: 11283292
Fung13: Fung DK, Lau WY, Chan WT, Yan A (2013). "Copper efflux is induced during anaerobic amino acid limitation in Escherichia coli to protect iron-sulfur cluster enzymes and biogenesis." J Bacteriol 195(20);4556-68. PMID: 23893112
Gerdes03: Gerdes SY, Scholle MD, Campbell JW, Balazsi G, Ravasz E, Daugherty MD, Somera AL, Kyrpides NC, Anderson I, Gelfand MS, Bhattacharya A, Kapatral V, D'Souza M, Baev MV, Grechkin Y, Mseeh F, Fonstein MY, Overbeek R, Barabasi AL, Oltvai ZN, Osterman AL (2003). "Experimental determination and system level analysis of essential genes in Escherichia coli MG1655." J Bacteriol 185(19);5673-84. PMID: 13129938
Joyce06: Joyce AR, Reed JL, White A, Edwards R, Osterman A, Baba T, Mori H, Lesely SA, Palsson BO, Agarwalla S (2006). "Experimental and computational assessment of conditionally essential genes in Escherichia coli." J Bacteriol 188(23);8259-71. PMID: 17012394
Kittleson06: Kittleson JT, Loftin IR, Hausrath AC, Engelhardt KP, Rensing C, McEvoy MM (2006). "Periplasmic metal-resistance protein CusF exhibits high affinity and specificity for both CuI and AgI." Biochemistry 45(37);11096-102. PMID: 16964970
Loftin05: Loftin IR, Franke S, Roberts SA, Weichsel A, Heroux A, Montfort WR, Rensing C, McEvoy MM (2005). "A novel copper-binding fold for the periplasmic copper resistance protein CusF." Biochemistry 44(31);10533-40. PMID: 16060662
Loftin07: Loftin IR, Franke S, Blackburn NJ, McEvoy MM (2007). "Unusual Cu(I)/Ag(I) coordination of Escherichia coli CusF as revealed by atomic resolution crystallography and X-ray absorption spectroscopy." Protein Sci 16(10);2287-93. PMID: 17893365
Loftin09: Loftin IR, Blackburn NJ, McEvoy MM (2009). "Tryptophan Cu(I)-pi interaction fine-tunes the metal binding properties of the bacterial metallochaperone CusF." J Biol Inorg Chem 14(6);905-12. PMID: 19381697
Lok08: Lok CN, Ho CM, Chen R, Tam PK, Chiu JF, Che CM (2008). "Proteomic identification of the Cus system as a major determinant of constitutive Escherichia coli silver resistance of chromosomal origin." J Proteome Res 7(6);2351-6. PMID: 18419149
Long12: Long F, Su CC, Lei HT, Bolla JR, Do SV, Yu EW (2012). "Structure and mechanism of the tripartite CusCBA heavy-metal efflux complex." Philos Trans R Soc Lond B Biol Sci 367(1592);1047-58. PMID: 22411977
Munson00: Munson GP, Lam DL, Outten FW, O'Halloran TV (2000). "Identification of a copper-responsive two-component system on the chromosome of Escherichia coli K-12." J Bacteriol 182(20);5864-71. PMID: 11004187
Outten01: Outten FW, Huffman DL, Hale JA, O'Halloran TV (2001). "The independent cue and cus systems confer copper tolerance during aerobic and anaerobic growth in Escherichia coli." J Biol Chem 276(33);30670-7. PMID: 11399769
Stroebel07: Stroebel D, Sendra V, Cannella D, Helbig K, Nies DH, Coves J (2007). "Oligomeric behavior of the RND transporters CusA and AcrB in micellar solution of detergent." Biochim Biophys Acta 1768(6);1567-73. PMID: 17467658
Su12: Su CC, Long F, Lei HT, Bolla JR, Do SV, Rajashankar KR, Yu EW (2012). "Charged amino acids (R83, E567, D617, E625, R669, and K678) of CusA are required for metal ion transport in the Cus efflux system." J Mol Biol 422(3);429-41. PMID: 22683351
Xue08: Xue Y, Davis AV, Balakrishnan G, Stasser JP, Staehlin BM, Focia P, Spiro TG, Penner-Hahn JE, O'Halloran TV (2008). "Cu(I) recognition via cation-pi and methionine interactions in CusF." Nat Chem Biol 4(2);107-9. PMID: 18157124
Yamada06: Yamada S, Awano N, Inubushi K, Maeda E, Nakamori S, Nishino K, Yamaguchi A, Takagi H (2006). "Effect of drug transporter genes on cysteine export and overproduction in Escherichia coli." Appl Environ Microbiol 72(7);4735-42. PMID: 16820466
Yang12: Yang C, Huang TW, Wen SY, Chang CY, Tsai SF, Wu WF, Chang CH (2012). "Genome-wide PhoB binding and gene expression profiles reveal the hierarchical gene regulatory network of phosphate starvation in Escherichia coli." PLoS One 7(10);e47314. PMID: 23071782
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