title: Modification of a free Fe-S cluster cysteine residue in the active iron-responsive element-binding protein prevents RNA binding.
abstract: The iron-responsive element-binding protein (IRE-BP) binds to specific RNA stem-loop structures called iron-responsive elements (IREs), which mediate the post-transcriptional regulation of a variety of genes involved in iron metabolism. The IRE-BP is cytosolic aconitase, and a [4Fe-4S] cubane cluster is required for aconitase activity but is associated with loss of IRE binding affinity. Chemical modification of the IRE-BP can abrogate RNA binding and the 3 cysteines predicted to coordinate the Fe-S cluster in the IRE-BP could be targets for modification. We report the expression of recombinant IRE-BP in which the three putative cluster cysteines (Cys-437, Cys-503, and Cys-506) have been mutated to serine residues. Replacement of any or all of these cysteine residues results in a complete loss of aconitase activity. While all of the mutants bind RNA, substitution of Cys-437 specifically renders the IRE-BP resistant to inactivation by low concentrations of N-ethylmaleimide or diamide. These results identify Cys-437 as the target of in vitro regulation of RNA binding in the IRE-BP and suggest that, in the RNA-binding form of the protein, Cys-437 is free and therefore available for modifications that inhibit RNA binding.
authors.affiliation: Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: C C
authors.initials: CC
authors.lastname: Philpott
authors.forename: D
authors.initials: D
authors.lastname: Haile
authors.forename: T A
authors.initials: TA
authors.lastname: Rouault
authors.forename: R D
authors.initials: RD
authors.lastname: Klausner
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D003545:Cysteine; D003600:Cytosol / Q000378:metabolism; D003676:Deferoxamine / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D006427:Hemin / Q000494:pharmacology; D035925:Iron-Regulatory Proteins; D007506:Iron-Sulfur Proteins / Q000378:metabolism*; D051379:Mice; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012694:Serine
chemical_list: D035925:Iron-Regulatory Proteins; D007506:Iron-Sulfur Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D012694:Serine; D006427:Hemin; D003676:Deferoxamine; D003545:Cysteine; D005033:Ethylmaleimide