Escherichia coli K-12 substr. MG1655 Polypeptide: copper / silver efflux system - periplasmic binding protein and metallochaperone

Gene: cusF Accession Numbers: G6321 (EcoCyc), b0573, ECK0565

Synonyms: agrF, cusX, ylcC, ORF110

Regulation Summary Diagram: ?

Regulation summary diagram for cusF

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].

Comment: [Franz08]

Citations: [Bagchi13]

Gene Citations: [Franke01]

Locations: periplasmic space

Map Position: [596,354 -> 596,686] (12.85 centisomes, 46°)
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

Relationship Links: InterPro:IN-FAMILY:IPR021647 , PDB:Structure:1zeq , PDB:Structure:2qcp , PDB:Structure:2vb2 , PDB:Structure:2vb3 , PDB:Structure:3E6Z , Pfam:IN-FAMILY:PF11604

Gene-Reaction Schematic: ?

Gene-Reaction Schematic

Genetic Regulation Schematic: ?

Genetic regulation schematic for cusF

GO Terms:

Biological Process: GO:0006878 - cellular copper ion homeostasis Inferred from experiment [Franke03]
GO:0010043 - response to zinc ion Inferred from experiment [Lee05b]
GO:0010272 - response to silver ion Inferred from experiment [Franke01, Lok08]
GO:0010273 - detoxification of copper ion Inferred from experiment [Franke03]
GO:0046688 - response to copper ion Inferred from experiment [Franke01, Yamamoto05a]
Molecular Function: GO:0005507 - copper ion binding Inferred from experiment [Franke03, Astashkin05, Loftin05, Kittleson06, Loftin07, Xue08]
GO:0005515 - protein binding Inferred from experiment [Franke03]
GO:0016530 - metallochaperone activity Inferred from experiment [Bagai08]
GO:0016531 - copper chaperone activity Inferred from experiment [Bagai08]
GO:0046914 - transition metal ion binding Inferred from experiment [Bagai08, Kittleson06, Loftin07]
GO:0046872 - metal ion binding Inferred by computational analysis [UniProtGOA11a]
Cellular Component: GO:0030288 - outer membrane-bounded periplasmic space Inferred from experiment Inferred by computational analysis [DiazMejia09, Franke03]
GO:0042597 - periplasmic space Inferred from experiment Inferred by computational analysis [UniProtGOA11, UniProtGOA11a, Franke03]

MultiFun Terms: cell processes adaptations

Essentiality data for cusF knockouts: ?

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]

Last-Curated ? 12-Nov-2008 by Johnson A , JCVI

Subunit of: copper / silver efflux system

Synonyms: CusCFBA

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 (extended 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].

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]. 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, consistent with the 'funnel' model of efflux. Alternatively CusB may act as a switch whereby metal binding to CusB promotes opening of a CusA channel and docking of substrate-loaded CusF (reviewed in [Kim11b]). Using selenomethionine labeling of active site methionine residues [Chacon14] provide in vitro evidence that loaded CusB activates CusA to accept copper from CusF. Back-transfer of copper from CusB to CusF was also observed and this may function as a regulatory mechanism

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, Gupta01]. 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 [Lee05b]. 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].

Reviews: [Rensing03, Nies03, Silver03, Su11a, Long12, Mealman12, Delmar13]
Comment: [Outten13]

Citations: [Franz08, Bleuel05, Outten01]

Relationship Links: PDB:Structure:3NE5

GO Terms:

Molecular Function: GO:0005375 - copper ion transmembrane transporter activity Inferred from experiment [Franke03]
GO:0015080 - silver ion transmembrane transporter activity Inferred from experiment [Lok08]
Cellular Component: GO:0030313 - cell envelope Inferred by computational analysis Inferred from experiment [Su11]

Last-Curated ? 02-Feb-2014 by Mackie A , Macquarie University

Enzymatic reaction of: export of Cu+ (copper / silver efflux system)

Transport reaction diagram for export of Cu+

Enzymatic reaction of: Export of Ag+ (copper / silver efflux system)

Transport reaction diagram for Export of Ag+

Sequence Features

Protein sequence of copper / silver efflux system - periplasmic binding protein and metallochaperone with features indicated

Feature Class Location Common Name Citations Comment
Signal-Sequence 1 -> 21 CusF signal sequence
Chain 22 -> 110  
UniProt: Cation efflux system protein cusF;
Mutagenesis-Variant 25 -> 27  
[Franke03, UniProt11]
UniProt: No change in copper resistance.
Mutagenesis-Variant 48  
[Franke03, UniProt11]
UniProt: No change in copper resistance.
Mutagenesis-Variant 57 -> 58  
[Franke03, UniProt15]
UniProt: Slight decrease in copper resistance.
Metal-Binding-Site 58, 69, 71 CusF metal binding site
Mutagenesis-Variant 69  
[Franke03, UniProt11]
UniProt: Loss of copper resistance and strong decrease in copper binding; when associated with I-71.
Mutagenesis-Variant 71  
[Franke03, UniProt11]
UniProt: Loss of copper resistance and strong decrease in copper binding; when associated with I-69.
Mutagenesis-Variant 73  
[Franke03, UniProt11]
UniProt: No change in copper resistance.

Gene Local Context (not to scale): ?

Gene local context diagram

Transcription Units:

Transcription-unit diagram

Transcription-unit diagram


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

Bagai08: Bagai I, Rensing C, Blackburn NJ, McEvoy MM (2008). "Direct metal transfer between periplasmic proteins identifies a bacterial copper chaperone." Biochemistry 47(44);11408-14. PMID: 18847219

Bagchi13: Bagchi P, Morgan MT, Bacsa J, Fahrni CJ (2013). "Robust affinity standards for Cu(I) biochemistry." J Am Chem Soc 135(49);18549-59. PMID: 24298878

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

Chacon14: Chacon KN, Mealman TD, McEvoy MM, Blackburn NJ (2014). "Tracking metal ions through a Cu/Ag efflux pump assigns the functional roles of the periplasmic proteins." Proc Natl Acad Sci U S A 111(43);15373-8. PMID: 25313055

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

Delmar13: Delmar JA, Su CC, Yu EW (2013). "Structural mechanisms of heavy-metal extrusion by the Cus efflux system." Biometals. PMID: 23657864

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

Franke03: Franke S, Grass G, Rensing C, Nies DH (2003). "Molecular analysis of the copper-transporting efflux system CusCFBA of Escherichia coli." J Bacteriol 185(13);3804-12. PMID: 12813074

Franz08: Franz KJ (2008). "Copper shares a piece of the pi." Nat Chem Biol 4(2);85-6. PMID: 18202673

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

Grass01: Grass G, Rensing C (2001). "Genes involved in copper homeostasis in Escherichia coli." J Bacteriol 183(6);2145-7. PMID: 11222619

Gupta01: Gupta A, Phung LT, Taylor DE, Silver S (2001). "Diversity of silver resistance genes in IncH incompatibility group plasmids." Microbiology 147(Pt 12);3393-402. PMID: 11739772

Hirakawa05: Hirakawa H, Inazumi Y, Masaki T, Hirata T, Yamaguchi A (2005). "Indole induces the expression of multidrug exporter genes in Escherichia coli." Mol Microbiol 55(4);1113-26. PMID: 15686558

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

Kim11b: Kim EH, Nies DH, McEvoy MM, Rensing C (2011). "Switch or funnel: how RND-type transport systems control periplasmic metal homeostasis." J Bacteriol 193(10);2381-7. PMID: 21398536

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

Lee05b: Lee LJ, Barrett JA, Poole RK (2005). "Genome-wide transcriptional response of chemostat-cultured Escherichia coli to zinc." J Bacteriol 187(3);1124-34. PMID: 15659689

Li97: Li XZ, Nikaido H, Williams KE (1997). "Silver-resistant mutants of Escherichia coli display active efflux of Ag+ and are deficient in porins." J Bacteriol 179(19);6127-32. PMID: 9324262

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

Mealman12: Mealman TD, Blackburn NJ, McEvoy MM (2012). "Metal export by CusCFBA, the periplasmic Cu(I)/Ag(I) transport system of Escherichia coli." Curr Top Membr 69;163-96. PMID: 23046651

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

Nies03: Nies DH (2003). "Efflux-mediated heavy metal resistance in prokaryotes." FEMS Microbiol Rev 27(2-3);313-39. PMID: 12829273

Nishino01: Nishino K, Yamaguchi A (2001). "Analysis of a complete library of putative drug transporter genes in Escherichia coli." J Bacteriol 2001;183(20);5803-12. PMID: 11566977

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

Outten13: Outten FW, Munson GP (2013). "Lability and liability of endogenous copper pools." J Bacteriol 195(20);4553-5. PMID: 23913325

Rensing03: Rensing C, Grass G (2003). "Escherichia coli mechanisms of copper homeostasis in a changing environment." FEMS Microbiol Rev 27(2-3);197-213. PMID: 12829268

Serres01: Serres MH, Gopal S, Nahum LA, Liang P, Gaasterland T, Riley M (2001). "A functional update of the Escherichia coli K-12 genome." Genome Biol 2(9);RESEARCH0035. PMID: 11574054

Silver03: Silver S (2003). "Bacterial silver resistance: molecular biology and uses and misuses of silver compounds." FEMS Microbiol Rev 27(2-3);341-53. PMID: 12829274

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

Su11: Su CC, Long F, Zimmermann MT, Rajashankar KR, Jernigan RL, Yu EW (2011). "Crystal structure of the CusBA heavy-metal efflux complex of Escherichia coli." Nature 470(7335);558-62. PMID: 21350490

Su11a: Su CC, Long F, Yu EW (2011). "The Cus efflux system removes toxic ions via a methionine shuttle." Protein Sci 20(1);6-18. PMID: 20981744

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

UniProt09: UniProt Consortium (2009). "UniProt version 15.8 released on 2009-10-01 00:00:00." Database.

UniProt11: UniProt Consortium (2011). "UniProt version 2011-06 released on 2011-06-30 00:00:00." Database.

UniProt15: UniProt Consortium (2015). "UniProt version 2015-01 released on 2015-01-16 00:00:00." Database.

UniProtGOA11: UniProt-GOA (2011). "Gene Ontology annotation based on the manual assignment of UniProtKB Subcellular Location terms in UniProtKB/Swiss-Prot entries."

UniProtGOA11a: UniProt-GOA (2011). "Gene Ontology annotation based on manual assignment of UniProtKB keywords in UniProtKB/Swiss-Prot entries."

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

Yamamoto05a: Yamamoto K, Ishihama A (2005). "Transcriptional response of Escherichia coli to external copper." Mol Microbiol 56(1);215-27. PMID: 15773991

Other References Related to Gene Regulation

Urano15: Urano H, Umezawa Y, Yamamoto K, Ishihama A, Ogasawara H (2015). "Cooperative regulation of the common target genes between H2O2-sensing YedVW and Cu2+-sensing CusSR in Escherichia coli." Microbiology 161(Pt 4);729-38. PMID: 25568260

Yang12a: 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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Please cite the following article in publications resulting from the use of EcoCyc: Nucleic Acids Research 41:D605-12 2013
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