RT Journal Article
SR Electronic
T1 Zinc(II) binding on human wild-type ISCU and Met140 variants modulates Fe-S complex activity
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 262477
DO 10.1101/262477
A1 Nicholas G. Fox
A1 Alain Martelli
A1 Joseph F. Nabhan
A1 Jay Janz
A1 Oktawia Borkowska
A1 Christine Bulawa
A1 Wyatt W. Yue
YR 2018
UL http://biorxiv.org/content/early/2018/02/10/262477.abstract
AB The human de novo iron-sulfur (Fe-S) assembly complex consists of the cysteine desulfurase NFS1, accessory protein ISD11, scaffold protein ISCU, and allosteric activator frataxin (FXN). FXN has been shown to bind the NFS1-ISD11-ISCU complex (SDU), to activate the desulfurase activity and thus Fe-S cluster biosynthesis. Conversely, in the absence of FXN, the NFS1-ISD11 (SD) complex was reported to be inhibited by the binding of recombinant ISCU. Here, we show that recombinant ISCU binds zinc(II) ion, and that the presence of zinc in as-isolated ISCU has impacts on the SDU desulfurase activity as measured by sulfide production. Indeed, the removal of this zinc(II) ion from ISCU causes a moderate but significant increase in activity compared to SD alone, and FXN can activate both zinc-depleted and zinc-bound forms of ISCU complexed to SD. Recent yeast studies have reported a substitution on the yeast ISCU orthologue Isu, at position Met141 (Met140 in human numbering of precursor protein) to Ile, Leu, Val, or Cys that could bypass the requirement of FXN for Fe-S cluster assembly and cell viability. Using recombinant human proteins, we report no significant differences in the biochemical and biophysical properties observed between wild-type and variants M140I, M140 L, and M140 V of ISCU. Importantly, in the absence of FXN, ISCU variants behaved like wild-type and did not stimulate the desulfurase activity of the SD complex. This study therefore identifies an important regulatory role for ISCU-bound zinc in modulation of the human Fe-S assembly system in vitro but no ‘FXN bypass’ effect on mutations at position Met140 in human ISCU.ACPacyl carrier transfer proteinBLIbiolayer interferometryBSAbovine serum albuminCDcircular dichroismDMPDNN-dimethyl-p-phenylenediamineDSFdifferential scanning fluorimetryDTTdithiothreitol; EDTA, ethylenediaminetetracetic acidFe-Siron sulfurFRDAFriedreich’s ataxiaFXNfrataxinHEPES4-(2-hydroxyethyl)-1-piperazineethanesulfonic acidIPTGisopropyl β-D-1-thiogalactopyranosidePLPpyridoxal 5′-phosphateSDprotein complex composed of NFS 1 and ISD11SDUprotein complex composed of NFS 1, ISD11ISCUSDUF, protein complex composed of NFS 1, ISD11, ISCU, and frataxinTCAtrichloroacetic acidTCEPtris(2-carboxyethyl) phosphineTristris(hydroxymethyl)aminomethane