Escherichia coli K-12 substr. MG1655 Enzyme: chaperone for [Fe-S] cluster biosynthesis

Gene: hscA Accession Numbers: EG12130 (EcoCyc), b2526, ECK2523

Synonyms: hsc, Hsc66, chaperone, member of Hsp70 protein family

Regulation Summary Diagram: ?

Regulation summary diagram for hscA

HscA together with HscB comprises a chaperone/cochaperone system similar to DnaK/DnaJ [Vickery97, Silberg98]. HscA is required for the assembly of iron-sulfur clusters [Takahashi99, Tokumoto01]. Models for chaperone-facilitated Fe-S cluster transfer have been proposed. One model involves an exchange of Fe-S cluster ligands in the iron-sulfur cluster scaffold protein IscU upon chaperone binding that facilitates cluster transfer [Bonomi11]; another involves chaperone-mediated disordering of IscU that promotes the transfer of the Fe-S cluster to an acceptor protein [Kim12].

HscA consists of two domains, a nucleotide-binding domain (NBD) that binds and hydrolyzes ATP [Vickery97] and a substrate-binding domain that binds IscU [Silberg01]. The inter-domain linker peptide is able to stimulate the rate of ATP hydrolysis of the isolated NBD [Alderson14].

Under steady-state conditions, ATP hydrolysis rather than ADP/ATP nucleotide exchange is the rate-limiting step in the HscA reaction cycle [Silberg00]; the dissociation rates for ATP and ADP are comparatively fast [Brehmer01]. IscU is a substrate for HscA [Silberg01]; in the presence of HscB, IscU stimulates the ATPase activity of HscA up to 480-fold [Hoff00, Hoff03]. The in vivo turnover rate of the chaperone cycle may be determined by the availability of the IscU-HscB complex [Silberg04]. In the presence of HscA and HscB, conformational changes in the [2Fe-2S] cluster of IscU appear to be coupled to ATP hydrolysis by HscA [Bonomi08]. Interaction of HscA with IscU occurs via the LPPVK sequence motif in IscU, is highly directional [Hoff02, Hoff03, Tapley04], and occurs in both the ATP- and ADP-bound state, while interaction with HscB occurs primarily in the ATP-bound state [Silberg04, Kim14]. HscA alone or in the ADP-bound state preferentially binds to and stabilizes the dynamically disordered state of IscU. Binding of ATP leads to release of IscU [Kim12].

HscA interacts with the apo form of biotin synthase and improves the efficiency of the assembly of the [2Fe-2S] cluster on the enzyme, which is thought to provide the sulfur atom in the biotin synthase reaction. The HscA-BioB complex also binds IscU [Reyda09].

A crystal structure of the HscA substrate binding domain in complex with the IscU recognition peptide has been reported [CuppVickery04]. The sequence requirements for recognition of IscU by HscA have been explored by site-directed mutagenesis [Tapley06].

Expression of hscA is induced by cold shock, but not by heat shock [Lelivelt95]. An hscA mutant does not have a general protein folding defect, and overproduction of HscA does not complement a ΔdnaK52 mutant [Hesterkamp98]. An hscA mutant has increased doubling time in rich media and has lower activity of the Fe-S enzymes GltS and SDH than wild type [Tokumoto01].

HscA: "heat shock cognate protein A" [Kawula94, Seaton94]

Reviews: [Markley13, Roche13, Genevaux07, Vickery07, Dougan02]

Citations: [Aoto05]

Gene Citations: [Tokumoto02]

Locations: cytosol

Map Position: [2,655,107 <- 2,656,957] (57.23 centisomes, 206°)
Length: 1851 bp / 616 aa

Molecular Weight of Polypeptide: 65.652 kD (from nucleotide sequence), 62.0 kD (experimental) [Vickery97 ]

Unification Links: ASAP:ABE-0008315 , CGSC:32977 , DIP:DIP-47348N , EchoBASE:EB2051 , EcoGene:EG12130 , EcoliWiki:b2526 , ModBase:P0A6Z1 , OU-Microarray:b2526 , PortEco:hscA , PR:PRO_000022925 , Pride:P0A6Z1 , Protein Model Portal:P0A6Z1 , RefSeq:NP_417021 , RegulonDB:EG12130 , SMR:P0A6Z1 , String:511145.b2526 , Swiss-Model:P0A6Z1 , UniProt:P0A6Z1

Relationship Links: InterPro:IN-FAMILY:IPR010236 , InterPro:IN-FAMILY:IPR013126 , InterPro:IN-FAMILY:IPR018181 , InterPro:IN-FAMILY:IPR029047 , InterPro:IN-FAMILY:IPR029048 , PDB:Structure:1U00 , Pfam:IN-FAMILY:PF00012 , Prints:IN-FAMILY:PR00301 , Prosite:IN-FAMILY:PS00297 , Prosite:IN-FAMILY:PS00329 , Prosite:IN-FAMILY:PS01036

In Paralogous Gene Group: 5 (5 members)

Gene-Reaction Schematic: ?

Gene-Reaction Schematic

GO Terms:

Biological Process: GO:0070417 - cellular response to cold Inferred from experiment [Lelivelt95]
GO:0097428 - protein maturation by iron-sulfur cluster transfer Inferred from experiment [Bonomi08, Takahashi99]
GO:0006457 - protein folding Inferred by computational analysis [GOA06, GOA01]
GO:0016226 - iron-sulfur cluster assembly Inferred by computational analysis [GOA01]
Molecular Function: GO:0005515 - protein binding Inferred from experiment [Reyda09, Tokumoto02, Silberg04, Hoff03]
GO:0005524 - ATP binding Inferred from experiment Inferred by computational analysis [UniProtGOA11, GOA01, Brehmer01, Silberg00]
GO:0016887 - ATPase activity Inferred from experiment Inferred by computational analysis [GOA06, GOA01, Silberg00]
GO:0043531 - ADP binding Inferred from experiment [Brehmer01]
GO:0000166 - nucleotide binding Inferred by computational analysis [UniProtGOA11]
GO:0051082 - unfolded protein binding Inferred by computational analysis [GOA01]
Cellular Component: GO:0005829 - cytosol Inferred from experiment Inferred by computational analysis [DiazMejia09, Ishihama08]
GO:1990230 - iron-sulfur cluster transfer complex Inferred from experiment [Bonomi08]

MultiFun Terms: information transfer protein related chaperoning, repair (refolding)

Essentiality data for hscA knockouts: ?

Growth Medium Growth? T (°C) O2 pH Osm/L Growth Observations
LB Lennox Yes 37 Aerobic 7   Yes [Baba06, Comment 1]
M9 medium with 1% glycerol Yes 37 Aerobic 7.2 0.35 Yes [Joyce06, Comment 2]
MOPS medium with 0.4% glucose Yes 37 Aerobic 7.2 0.22 Yes [Baba06, Comment 1]

Last-Curated ? 12-Nov-2014 by Keseler I , SRI International

Enzymatic reaction of: non-chaperonin molecular chaperone ATPase (chaperone for [Fe-S] cluster biosynthesis)

EC Number: 3.6.4.-

a [chaperone-ADP]-[disordered-form scaffold protein] complex + ATP <=> an [Fe-S cluster biosynthesis chaperone]-ATP + a [disordered-form [Fe-S] cluster scaffold protein] + ADP

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the direction of enzyme catalysis.

The reaction is physiologically favored in the direction shown. [Silberg00]

Kinetic Parameters:

Km (μM)

Sequence Features

Protein sequence of chaperone for [Fe-S] cluster biosynthesis with features indicated

Feature Class Location Citations Comment
Cleavage-of-Initial-Methionine 1
Mutagenesis-Variant 212
A T212V mutant lacks ATPase activity [Bonomi08].
Extrinsic-Sequence-Variant 216
UniProt: In hsca1..
Sequence-Conflict 456 -> 465
[Kawula94, UniProt10a]
UniProt: (in Ref. 1; AAA18300);
Sequence-Conflict 516
[Kawula94, UniProt10a]
UniProt: (in Ref. 1; AAA18300);
Sequence-Conflict 557
[Kawula94, UniProt10a]
UniProt: (in Ref. 1; AAA18300);
Sequence-Conflict 583
[Seaton94, UniProt10a]
UniProt: (in Ref. 2; AAD13473);

Gene Local Context (not to scale): ?

Gene local context diagram

Transcription Unit:

Transcription-unit diagram


10/20/97 Gene b2526 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG12130; confirmed by SwissProt match.


Alderson14: Alderson TR, Kim JH, Cai K, Frederick RO, Tonelli M, Markley JL (2014). "Specialized Hsp70 (HscA) interdomain linker binds to its nucleotide-binding domain and stimulates ATP hydrolysis in both cis and trans configurations." Biochemistry. PMID: 25372495

Aoto05: Aoto PC, Ta DT, Cupp-Vickery JR, Vickery LE (2005). "X-ray diffraction analysis of a crystal of HscA from Escherichia coli." Acta Crystallogr Sect F Struct Biol Cryst Commun 61(Pt 7);715-7. PMID: 16511138

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

Bonomi08: Bonomi F, Iametti S, Morleo A, Ta D, Vickery LE (2008). "Studies on the mechanism of catalysis of iron-sulfur cluster transfer from IscU[2Fe2S] by HscA/HscB chaperones." Biochemistry 47(48);12795-801. PMID: 18986169

Bonomi11: Bonomi F, Iametti S, Morleo A, Ta D, Vickery LE (2011). "Facilitated transfer of IscU-[2Fe2S] clusters by chaperone-mediated ligand exchange." Biochemistry 50(44);9641-50. PMID: 21977977

Brehmer01: Brehmer D, Rudiger S, Gassler CS, Klostermeier D, Packschies L, Reinstein J, Mayer MP, Bukau B (2001). "Tuning of chaperone activity of Hsp70 proteins by modulation of nucleotide exchange." Nat Struct Biol 8(5);427-32. PMID: 11323718

CuppVickery04: Cupp-Vickery JR, Peterson JC, Ta DT, Vickery LE (2004). "Crystal structure of the molecular chaperone HscA substrate binding domain complexed with the IscU recognition peptide ELPPVKIHC." J Mol Biol 342(4);1265-78. PMID: 15351650

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

Dougan02: Dougan DA, Mogk A, Bukau B (2002). "Protein folding and degradation in bacteria: to degrade or not to degrade? That is the question." Cell Mol Life Sci 59(10);1607-16. PMID: 12475170

Genevaux07: Genevaux P, Georgopoulos C, Kelley WL (2007). "The Hsp70 chaperone machines of Escherichia coli: a paradigm for the repartition of chaperone functions." Mol Microbiol 66(4);840-57. PMID: 17919282

GOA01: GOA, DDB, FB, MGI, ZFIN (2001). "Gene Ontology annotation through association of InterPro records with GO terms."

GOA06: GOA, SIB (2006). "Electronic Gene Ontology annotations created by transferring manual GO annotations between orthologous microbial proteins."

Hesterkamp98: Hesterkamp T, Bukau B (1998). "Role of the DnaK and HscA homologs of Hsp70 chaperones in protein folding in E.coli." EMBO J 17(16);4818-28. PMID: 9707441

Hoff00: Hoff KG, Silberg JJ, Vickery LE (2000). "Interaction of the iron-sulfur cluster assembly protein IscU with the Hsc66/Hsc20 molecular chaperone system of Escherichia coli." Proc Natl Acad Sci U S A 97(14);7790-5. PMID: 10869428

Hoff02: Hoff KG, Ta DT, Tapley TL, Silberg JJ, Vickery LE (2002). "Hsc66 substrate specificity is directed toward a discrete region of the iron-sulfur cluster template protein IscU." J Biol Chem 277(30);27353-9. PMID: 11994302

Hoff03: Hoff KG, Cupp-Vickery JR, Vickery LE (2003). "Contributions of the LPPVK motif of the iron-sulfur template protein IscU to interactions with the Hsc66-Hsc20 chaperone system." J Biol Chem 278(39):37582-9. PMID: 12871959

Ishihama08: Ishihama Y, Schmidt T, Rappsilber J, Mann M, Hartl FU, Kerner MJ, Frishman D (2008). "Protein abundance profiling of the Escherichia coli cytosol." BMC Genomics 9;102. PMID: 18304323

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

Kawula94: Kawula TH, Lelivelt MJ (1994). "Mutations in a gene encoding a new Hsp70 suppress rapid DNA inversion and bgl activation, but not proU derepression, in hns-1 mutant Escherichia coli." J Bacteriol 176(3);610-9. PMID: 8300516

Kim12: Kim JH, Tonelli M, Frederick RO, Chow DC, Markley JL (2012). "Specialized Hsp70 chaperone (HscA) binds preferentially to the disordered form, whereas J-protein (HscB) binds preferentially to the structured form of the iron-sulfur cluster scaffold protein (IscU)." J Biol Chem 287(37);31406-13. PMID: 22782893

Kim14: Kim JH, Alderson TR, Frederick RO, Markley JL (2014). "Nucleotide-dependent interactions within a specialized Hsp70/Hsp40 complex involved in Fe-S cluster biogenesis." J Am Chem Soc 136(33);11586-9. PMID: 25080945

Lelivelt95: Lelivelt MJ, Kawula TH (1995). "Hsc66, an Hsp70 homolog in Escherichia coli, is induced by cold shock but not by heat shock." J Bacteriol 1995;177(17);4900-7. PMID: 7665466

Markley13: Markley JL, Kim JH, Dai Z, Bothe JR, Cai K, Frederick RO, Tonelli M (2013). "Metamorphic protein IscU alternates conformations in the course of its role as the scaffold protein for iron-sulfur cluster biosynthesis and delivery." FEBS Lett. PMID: 23333622

Reyda09: Reyda MR, Fugate CJ, Jarrett JT (2009). "A complex between biotin synthase and the iron-sulfur cluster assembly chaperone HscA that enhances in vivo cluster assembly." Biochemistry 48(45);10782-92. PMID: 19821612

Roche13: Roche B, Aussel L, Ezraty B, Mandin P, Py B, Barras F (2013). "Reprint of: Iron/sulfur proteins biogenesis in prokaryotes: formation, regulation and diversity." Biochim Biophys Acta 1827(8-9);923-37. PMID: 23660107

Seaton94: Seaton BL, Vickery LE (1994). "A gene encoding a DnaK/hsp70 homolog in Escherichia coli." Proc Natl Acad Sci U S A 1994;91(6);2066-70. PMID: 8134349

Silberg00: Silberg JJ, Vickery LE (2000). "Kinetic characterization of the ATPase cycle of the molecular chaperone Hsc66 from Escherichia coli." J Biol Chem 275(11);7779-86. PMID: 10713091

Silberg01: Silberg JJ, Hoff KG, Tapley TL, Vickery LE (2001). "The Fe/S assembly protein IscU behaves as a substrate for the molecular chaperone Hsc66 from Escherichia coli." J Biol Chem 276(3);1696-700. PMID: 11053447

Silberg04: Silberg JJ, Tapley TL, Hoff KG, Vickery LE (2004). "Regulation of the HscA ATPase reaction cycle by the co-chaperone HscB and the iron-sulfur cluster assembly protein IscU." J Biol Chem 279(52):53924-31. PMID: 15485839

Silberg98: Silberg JJ, Hoff KG, Vickery LE (1998). "The Hsc66-Hsc20 chaperone system in Escherichia coli: chaperone activity and interactions with the DnaK-DnaJ-grpE system." J Bacteriol 180(24);6617-24. PMID: 9852006

Ta92: Ta DT, Vickery LE (1992). "Cloning, sequencing, and overexpression of a [2Fe-2S] ferredoxin gene from Escherichia coli." J Biol Chem 1992;267(16);11120-5. PMID: 1317854

Takahashi99: Takahashi Y, Nakamura M (1999). "Functional assignment of the ORF2-iscS-iscU-iscA-hscB-hscA-fdx-ORF3 gene cluster involved in the assembly of Fe-S clusters in Escherichia coli." J Biochem (Tokyo) 1999;126(5);917-26. PMID: 10544286

Tapley04: Tapley TL, Vickery LE (2004). "Preferential substrate binding orientation by the molecular chaperone HscA." J Biol Chem 279(27);28435-42. PMID: 15100228

Tapley06: Tapley TL, Cupp-Vickery JR, Vickery LE (2006). "Structural determinants of HscA peptide-binding specificity." Biochemistry 45(26);8058-66. PMID: 16800630

Tokumoto01: Tokumoto U, Takahashi Y (2001). "Genetic analysis of the isc operon in Escherichia coli involved in the biogenesis of cellular iron-sulfur proteins." J Biochem (Tokyo) 130(1);63-71. PMID: 11432781

Tokumoto02: Tokumoto U, Nomura S, Minami Y, Mihara H, Kato S, Kurihara T, Esaki N, Kanazawa H, Matsubara H, Takahashi Y (2002). "Network of protein-protein interactions among iron-sulfur cluster assembly proteins in Escherichia coli." J Biochem (Tokyo) 131(5);713-9. PMID: 11983079

UniProt10a: UniProt Consortium (2010). "UniProt version 2010-11 released on 2010-11-02 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 manual assignment of UniProtKB keywords in UniProtKB/Swiss-Prot entries."

Vickery07: Vickery LE, Cupp-Vickery JR (2007). "Molecular chaperones HscA/Ssq1 and HscB/Jac1 and their roles in iron-sulfur protein maturation." Crit Rev Biochem Mol Biol 42(2);95-111. PMID: 17453917

Vickery97: Vickery LE, Silberg JJ, Ta DT (1997). "Hsc66 and Hsc20, a new heat shock cognate molecular chaperone system from Escherichia coli." Protein Sci 6(5);1047-56. PMID: 9144776

Other References Related to Gene Regulation

Giel06: Giel JL, Rodionov D, Liu M, Blattner FR, Kiley PJ (2006). "IscR-dependent gene expression links iron-sulphur cluster assembly to the control of O-regulated genes in Escherichia coli." Mol Microbiol 60(4);1058-75. PMID: 16677314

Kaleta10: Kaleta C, Gohler A, Schuster S, Jahreis K, Guthke R, Nikolajewa S (2010). "Integrative inference of gene-regulatory networks in Escherichia coli using information theoretic concepts and sequence analysis." BMC Syst Biol 4;116. PMID: 20718955

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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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