Regulation of the HscA ATPase Reaction Cycle by the Co-chaperone HscB and the Iron-Sulfur Cluster Assembly Protein IscU

Silberg, Jonathan J.; Tapley, Tim L.; Hoff, Kevin G.; Vickery, Larry E.

doi:10.1074/jbc.m410117200

Cited by 85 publications

(100 citation statements)

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“…Taken together with the evidence for high-affinity binding of IscU to the R-state HscA/ADP complex (17) and the observation that ADP binding to the R-state HscA/IscU complex does result in a modest (∼2-fold) increase in the rate of the cluster transfer, leads us to propose two possibilities for the interaction with apo-Fdx and the mechanism of ATPdependent cluster transfer, see Possible mechanistic schemes for how the HscA/HscB/IscU chaperone cycle stimulates cluster transfer from [2Fe-2S]IscU to apo-IscFdx. The HscA chaperone cycle proposed by Silberg et al (17) has been modified to incorporate the cluster transfer results reported in this work.…”

Section: Discussionmentioning

confidence: 54%

“…This body of work has recently led to a detailed kinetic analysis of the regulation of the HscA ATPase reaction cycle by HscB and apo IscU (17). ATP binding to HscA leads to a tense (T) state with decreased substrate-binding affinity.…”

Section: Discussionmentioning

confidence: 99%

“…HscA and HscB function together as a nucleotide-dependent molecular chaperone system that is specific for IscU (15)(16)(17). HscA is a specialized member of the ubiquitous 70-kDa heat shock protein (Hsp70 or DnaK) family of molecular chaperones that facilitate protein folding, (dis)assembly and transport via nucleotide-dependent binding to unfolded, misfolded or unstable polypeptides in order to prevent non-specific aggregation processes (18).…”

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HscA and HscB Stimulate [2Fe-2S] Cluster Transfer from IscU to Apoferredoxin in an ATP-Dependent Reaction

2006

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The role of the Azotobacter vinelandii HscA/HscB co-chaperone system in ISC-mediated iron-sulfur cluster biogenesis has been investigated in vitro by using CD and EPR spectrometry to monitor the effect of HscA, HscB, MgATP, and MgADP on the time course of cluster transfer from [2Fe-2S] IscU to apo-Isc ferredoxin. CD spectra indicate that both HscB and HscA interact with [2Fe-2S] IscU and the rate of cluster transfer was stimulated more than 20-fold in the presence stoichiometric HscA and HscB and excess MgATP. No stimulation was observed in the absence of either HscB or MgATP and cluster transfer was found to be an ATP-dependent reaction based on concomitant phosphate production and the enhanced rates of cluster transfer in the presence of KCl which is known to stimulated HscA ATPase activity. The results demonstrate a role of the ISC HscA/HscB cochaperone system in facilitating efficient [2Fe-2S] cluster transfer from the IscU scaffold protein to acceptor proteins and that [2Fe-2S] cluster transfer from IscU is an ATP-dependent process. The data are consistent with the proposed regulation of the HscA ATPase cycle by HscB and IscU (Silberg, J. J., Tapley, T. L., Hoff, K. G., and Vickery, L. E. (2004) J. Biol. Chem. 279,[53924][53925][53926][53927][53928][53929][53930][53931], and mechanistic proposals for coupling of the HscA ATPase cycle with cluster transfer from [2Fe-2S] IscU to apo-IscFdx are discussed.Iron-sulfur cluster biogenesis proteins in bacteria are commonly encoded by a highly conserved isc (iron-sulfur cluster) gene cluster, iscRSUA-hscBA-fdx (1). Moreover, with the exception of the regulatory protein IscR, homologs of each of these proteins have been shown to be involved in Fe-S cluster biogenesis in eukaryotic organisms (2). Extensive biochemical and genetic studies have established well-defined roles for IscS and IscU, but specific roles for the IscA, HscA, HscB and Fdx proteins remain elusive. IscS is a cysteine desulfurase that catalyzes the conversion of cysteine to alanine (3;4) and thereby provides the inorganic sulfur for assembly or [4Fe-4S] clusters on the IscU scaffold protein (5). In vitro studies have shown thatIscA has the ability to function as either an alternative scaffold for IscS-mediated cluster assembly of or [4Fe-4S] clusters (6;7) or as a specific Fe donor for cluster assembly on IscU (8), but the physiologically relevant role has yet to be determined. Likewise, the specific redox role of the Isc [2Fe-2S] 2+,+ Fdx (ferredoxin) in IscS-mediated cluster assembly has yet to be identified, with reduction of S 0 to S 2− , oxidation of Fe 2+ to Fe 3+ in the assembly of [2Fe-2S] 2+ clusters, and reductive coupling of two [2Fe-2S] 2+ clusters on IscU to yield one [4Fe-4S] 2+ cluster as the most likely candidates (5;9).In vivo gene knockout studies have demonstrated that both HscA (heat shock cognate 66-kDa; Hsc66) and HscB (heat shock cognate 20-kDa; Hsc20), and their eukaryotic homologs, Ssq1 and Jac1, have crucial roles in the maturation of Fe-S proteins in both prokary...

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Section: Discussionmentioning

confidence: 54%

Section: Discussionmentioning

confidence: 99%

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HscA and HscB Stimulate [2Fe-2S] Cluster Transfer from IscU to Apoferredoxin in an ATP-Dependent Reaction

2006

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“…RecA and RecN have important roles in the repair of DNA damage which have been extensively characterized in UV damage experiments in many organisms, including E. coli and H. influenzae (43,50,51,77,82). Further repair mechanisms are potentially afforded by the gene products of NTHI0493 and NTHI0495, which encode homologues of the cochaperone proteins HscA and HscB, respectively, and that have roles in iron-sulfur cluster assembly (2,31,78). NTHI1588 is a homologue of a gene that encodes ImpA, an error-prone DNA polymerase originally identified in the incompatibility group plasmid TP110.…”

Section: Resultsmentioning

confidence: 99%

The OxyR Regulon in Nontypeable Haemophilus influenzae

et al. 2007

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Nontypeable Haemophilus influenzae (NTHi) is a gram-negative bacterium and a common commensal organism of the upper respiratory tract in humans. NTHi causes a number of diseases, including otitis media, sinusitis, conjunctivitis, exacerbations of chronic obstructive pulmonary disease, and bronchitis. During the course of colonization and infection, NTHi must withstand oxidative stress generated by insult due to multiple reactive oxygen species produced endogenously by other copathogens and by host cells. Using an NTHi-specific microarray containing oligonucleotides representing the 1821 open reading frames of the recently sequenced NTHi isolate 86-028NP, we have identified 40 genes in strain 86-028NP that are upregulated after induction of oxidative stress due to hydrogen peroxide. Further comparisons between the parent and an isogenic oxyR mutant identified a subset of 11 genes that were transcriptionally regulated by OxyR, a global regulator of oxidative stress. Interestingly, hydrogen peroxide induced the OxyR-independent upregulation of expression of the genes encoding components of multiple iron utilization systems. This finding suggested that careful balancing of levels of intracellular iron was important for minimizing the effects of oxidative stress during NTHi colonization and infection and that there are additional regulatory pathways involved in iron utilization.In the evolution of life, a delicate physiological balance has arisen to compensate for both an organism's need for oxygen and the attendant lethal consequences of oxygen's toxicity. For example, in the electron transport chain, electrons can be inadvertently transferred from redox-active proteins to oxygen, producing reactive oxygen species (ROS) such as superoxide and hydrogen peroxide (H 2 O 2 ). The effects of ROS on cells can then be exacerbated by the presence of free iron which, via the Fenton reaction, can react with H 2 O 2 to produce more highly reactive hydroxyl radicals (34, 40, 52). Within a host, this problem is compounded by the release of extracellular ROS by phagocytes, which again, can have deleterious effects on invading pathogens (13). Thus, bacteria have evolved an array of increasingly well-defined mechanisms to abrogate the effects of oxidative stress. Proteins involved in such processes include catalase, which decomposes H 2 O 2 , and members of both the AhpCF/TsaA family of alkylhydroperoxidases and the organic hydroperoxide resistance proteins (Ohr) that decompose organic peroxides. Also, DNA-binding ferritin-like proteins (Dps) sequester free iron and bind to DNA, thus protecting DNA from damage (27,33,45,53,59,60,67). In addition, many bacterial species possess genes encoding OxyR, a global regulator of antioxidant defenses (24, 66).Protection against oxidative stress is especially important to nontypeable Haemophilus influenzae (NTHi). NTHi is one of three bacterial commensal organisms, the others being Streptococcus pneumoniae and Moraxella catarrhalis, which can ascend a virus-compromised eustachian tube and caus...

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“…In T. potens, there are ORFs coding for homologs of the transcription factor IscR (TherJR_1914, 37% identical to the E. coli protein) (7), the cysteine desulfurase IscS (TherJR_1913) (4,20), and the scaffold IscU (TherJR_1912) (21) from the ISC pathway. An additional suf-like operon in T. potens comprises homologs of sufC (TherJR_0923), sufB, and sufD (TherJR_0924) from the E. coli SUF pathway, and the chaperones hcsA (TherJR_0925) and hcsB (TherJR_0926) from the E. coli isc operon (22)(23)(24).…”

Section: Resultsmentioning

confidence: 99%

The unique regulation of iron-sulfur cluster biogenesis in a Gram-positive bacterium

Santos

Alonso-García

Macedo-Ribeiro

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Iron-sulfur clusters function as cofactors of a wide range of proteins, with diverse molecular roles in both prokaryotic and eukaryotic cells. Dedicated machineries assemble the clusters and deliver them to the final acceptor molecules in a tightly regulated process. In the prototypical Gram-negative bacterium Escherichia coli, the two existing iron-sulfur cluster assembly systems, iron-sulfur cluster (ISC) and sulfur assimilation (SUF) pathways, are closely interconnected. The ISC pathway regulator, IscR, is a transcription factor of the helix-turn-helix type that can coordinate a [2Fe-2S] cluster. Redox conditions and iron or sulfur availability modulate the ligation status of the labile IscR cluster, which in turn determines a switch in DNA sequence specificity of the regulator: cluster-containing IscR can bind to a family of gene promoters (type-1) whereas the clusterless form recognizes only a second group of sequences (type-2). However, iron-sulfur cluster biogenesis in Gram-positive bacteria is not so well characterized, and most organisms of this group display only one of the iron-sulfur cluster assembly systems. A notable exception is the unique Gram-positive dissimilatory metal reducing bacterium Thermincola potens, where genes from both systems could be identified, albeit with a diverging organization from that of Gram-negative bacteria. We demonstrated that one of these genes encodes a functional IscR homolog and is likely involved in the regulation of iron-sulfur cluster biogenesis in T. potens. Structural and biochemical characterization of T. potens and E. coli IscR revealed a strikingly similar architecture and unveiled an unforeseen conservation of the unique mechanism of sequence discrimination characteristic of this distinctive group of transcription regulators.Rrf2-like regulator | transcription regulation | helix-turn-helix motif | DNA recognition | specificity modulation I ron-sulfur (Fe/S) proteins play crucial roles for the functioning of both prokaryotic and eukaryotic cells, being required for biological functions ranging from electron transport to redox and nonredox catalysis, and from DNA synthesis and repair to sensing in regulatory processes (1). The main role of the Fe/S cluster assembly machineries is to mobilize iron and sulfur atoms from their storage sources, assemble the two components into an Fe/S cluster, and then transfer the newly formed cluster to the final protein acceptors (2). In Escherichia coli, there are two of these Fe/S cluster ''factories,'' the ISC (iron-sulfur cluster) and SUF (sulfur assimilation) systems, whose corresponding genes are organized in two operons, iscSUA-hscBA-fdx and sufABCDSE, respectively (2, 3). Deletion mutants of the ISC system display a variety of growth defects due to loss of Fe/S cluster-containing enzyme activity and disruption of sulfur metabolism whereas failure of both the ISC and SUF systems leads to synthetic lethality (4, 5).In E. coli, the ISC machinery is considered the housekeeping system responsible for the maturation of a lar...

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Regulation of the HscA ATPase Reaction Cycle by the Co-chaperone HscB and the Iron-Sulfur Cluster Assembly Protein IscU

Cited by 85 publications

References 57 publications

HscA and HscB Stimulate [2Fe-2S] Cluster Transfer from IscU to Apoferredoxin in an ATP-Dependent Reaction

HscA and HscB Stimulate [2Fe-2S] Cluster Transfer from IscU to Apoferredoxin in an ATP-Dependent Reaction

The OxyR Regulon in Nontypeable Haemophilus influenzae

The unique regulation of iron-sulfur cluster biogenesis in a Gram-positive bacterium

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