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Mutational analysis of the [4Fe‐4S]‐cluster converting iron regulatory factor from its RNA‐binding form to cytoplasmic aconitase.
Author(s) -
Hirling H.,
Henderson B.R.,
Kühn L.C.
Publication year - 1994
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1002/j.1460-2075.1994.tb06280.x
Subject(s) - aconitase , biology , biochemistry , rna binding protein , rna , cysteine , binding site , cytoplasm , messenger rna , microbiology and biotechnology , gene , mitochondrion , enzyme
The control of cellular iron homeostasis involves the coordinate post‐transcriptional regulation of ferritin mRNA translation and transferring receptor mRNA stability. These regulatory events are mediated by a soluble cytoplasmic protein, iron regulatory factor (IRF), which binds specifically to mRNA hairpin structures, termed iron‐responsive elements (IREs), in the respective mRNAs. IRF is modulated by variations of cellular iron levels and exists as either an apo‐protein or a [4Fe‐4S]‐cluster protein. The two conformations show distinct, mutually exclusive functions. High‐affinity IRE binding is observed with the apo‐form induced by iron deprivation, but is lost under high iron conditions when IRF is converted to the [4Fe‐4S]‐cluster form which shows cytoplasmic aconitase activity. Moreover, IRE binding is inactivated by the sulfhydryl‐oxidizing agent diamide and fully activated in vitro by 2% 2‐mercapto‐ethanol, whereas alkylation of IRF inhibits IRE binding. In the present study, we analyzed each of the above features using site‐directed mutants of recombinant human IRF. The results support the bifunctional nature of IRF. We conclude that cysteines 437, 503 and 506 anchor the [4Fe‐4S]‐cluster, and are essential to the aconitase activity. Mutagenesis changing any of the cysteines to serine leads to constitutive RNA binding in 0.02% 2‐mercaptoethanol. Cysteine 437 is particularly critical to the RNA‐protein interaction. The spontaneous or diamide‐induced formation of disulfide bonds between cysteines 437 and 503 or 437 and 506, in apo‐IRF, as well as its alkylation by N‐ethylmaleimide, inhibit binding to the IRE.(ABSTRACT TRUNCATED AT 250 WORDS)