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Human mitochondrial chaperone (mtHSP70) and cysteine desulfurase (NFS1) bind preferentially to the disordered conformation, whereas co-chaperone (HSC20) binds to the structured conformation of the iron-sulfur cluster scaffold protein (ISCU).

ABSTRACT: Human ISCU is the scaffold protein for mitochondrial iron-sulfur (Fe-S) cluster biogenesis and transfer. NMR spectra have revealed that ISCU populates two conformational states; that is, a more structured state (S) and a partially disordered state (D). We identified two single amino acid substitutions (D39V and N90A) that stabilize the S-state and two (D39A and H105A) that stabilize the D-state. We isolated the two constituent proteins of the human cysteine desulfurase complex (NFS1 and ISD11) separately and used NMR spectroscopy to investigate their interaction with ISCU. We found that ISD11 does not interact directly with ISCU. By contrast, NFS1 binds preferentially to the D-state of ISCU as does the NFS1-ISD11 complex. An in vitro Fe-S cluster assembly assay showed that [2Fe-2S] and [4Fe-4S] clusters are assembled on ISCU when catalyzed by NFS1 alone and at a higher rate when catalyzed by the NFS1-ISD11 complex. The DnaK-type chaperone (mtHSP70) and DnaJ-type co-chaperone (HSC20) are involved in the transfer of clusters bound to ISCU to acceptor proteins in an ATP-dependent reaction. We found that the ATPase activity of mtHSP70 is accelerated by HSC20 and further accelerated by HSC20 plus ISCU. NMR studies have shown that mtHSP70 binds preferentially to the D-state of ISCU and that HSC20 binds preferentially to the S-state of ISCU.

SUBMITTER: Cai K

PROVIDER: S-EPMC3789972 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature

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Human mitochondrial chaperone (mtHSP70) and cysteine desulfurase (NFS1) bind preferentially to the disordered conformation, whereas co-chaperone (HSC20) binds to the structured conformation of the iron-sulfur cluster scaffold protein (ISCU).

Cai Kai K Frederick Ronnie O RO Kim Jin Hae JH Reinen Nichole M NM Tonelli Marco M Markley John L JL

The Journal of biological chemistry 20130812 40

Human ISCU is the scaffold protein for mitochondrial iron-sulfur (Fe-S) cluster biogenesis and transfer. NMR spectra have revealed that ISCU populates two conformational states; that is, a more structured state (S) and a partially disordered state (D). We identified two single amino acid substitutions (D39V and N90A) that stabilize the S-state and two (D39A and H105A) that stabilize the D-state. We isolated the two constituent proteins of the human cysteine desulfurase complex (NFS1 and ISD11) s ...[more]

PMID: 23940031

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Project description:Human de novo iron-sulfur (Fe-S) assembly complex consists of cysteine desulfurase NFS1, accessory protein ISD11, acyl carrier protein ACP, scaffold protein ISCU, and allosteric activator frataxin (FXN). FXN binds the NFS1-ISD11-ACP-ISCU complex (SDAU), to activate the desulfurase activity and Fe-S cluster biosynthesis. In the absence of FXN, the NFS1-ISD11-ACP (SDA) complex was reportedly inhibited by binding of recombinant ISCU. Recent studies also reported a substitution at position Met141 on the yeast ISCU orthologue Isu, to Ile, Leu, Val, or Cys, could bypass the requirement of FXN for Fe-S cluster biosynthesis and cell viability. Here, we show that recombinant human ISCU binds zinc(II) ion, as previously demonstrated with the E. coli orthologue IscU. Surprisingly, the relative proportion between zinc-bound and zinc-depleted forms varies among purification batches. Importantly the presence of zinc in ISCU impacts SDAU desulfurase activity. Indeed, removal of zinc(II) ion from ISCU causes a moderate but significant increase in activity compared to SDA alone, and FXN can activate both zinc-depleted and zinc-bound forms of ISCU complexed to SDA. Taking into consideration the inhibition of desulfurase activity by zinc-bound ISCU, we characterized wild type ISCU and the M140I, M140L, and M140V variants under both zinc-bound and zinc-depleted conditions, and did not observe significant differences in the biochemical and biophysical properties between wild-type and variants. Importantly, in the absence of FXN, ISCU variants behaved like wild-type and did not stimulate the desulfurase activity of the SDA complex. This study therefore identifies an important regulatory role for zinc-bound ISCU in modulation of the human Fe-S assembly system in vitro and reports no 'FXN bypass' effect on mutations at position Met140 in human ISCU. Furthermore, this study also calls for caution in interpreting studies involving recombinant ISCU by taking into consideration the influence of the bound zinc(II) ion on SDAU complex activity.

Dataset Information

Human mitochondrial chaperone (mtHSP70) and cysteine desulfurase (NFS1) bind preferentially to the disordered conformation, whereas co-chaperone (HSC20) binds to the structured conformation of the iron-sulfur cluster scaffold protein (ISCU).

Publications

Human mitochondrial chaperone (mtHSP70) and cysteine desulfurase (NFS1) bind preferentially to the disordered conformation, whereas co-chaperone (HSC20) binds to the structured conformation of the iron-sulfur cluster scaffold protein (ISCU).

Similar Datasets

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