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).
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.
From the Center for Eukaryotic Structural Genomics and.
Kai
K
Cai
Ronnie O
RO
Frederick
Jin Hae
JH
Kim
Nichole M
NM
Reinen
Marco
M
Tonelli
John L
JL
Markley
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D001681:Biological Assay; D013437:Carbon-Sulfur Lyases / Q000378:metabolism*; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D050957:HSP110 Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D035925:Iron-Regulatory Proteins / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008928:Mitochondria / Q000378:metabolism*; D008954:Models, Biological; D018832:Molecular Chaperones / Q000378:metabolism*; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D016415:Sequence Alignment
C490651:HSCB protein, human; D050957:HSP110 Heat-Shock Proteins; C121993:HSPA4 protein, human; C506160:ISCU protein, human; D035925:Iron-Regulatory Proteins; D007506:Iron-Sulfur Proteins; C544445:LYRM4 protein, human; D018832:Molecular Chaperones; D050505:Mutant Proteins; D000251:Adenosine Triphosphatases; D013437:Carbon-Sulfur Lyases; C513492:NFS1 protein, human
ATPases; Chaperone Chaperonin; Enzyme Catalysis; Mitochondria; NMR; Protein Conformation; Protein-Protein Interactions; Scaffold Proteins; Spectroscopy