Article: article from journal or magazin.
Identification of RNA-binding surfaces in iron regulatory protein-1.
Publication types: Journal Article
Post-transcriptional regulation of mRNA translation and stability in iron metabolism involves the interaction between the trans-acting cytoplasmic iron regulatory proteins (IRP-1 and IRP-2) and cis-acting iron-responsive elements (IREs) in mRNA 5'- or 3'-untranslated regions. IRP-1 can adopt two conformations: one with a [4Fe-4S]-cluster, unable to bind IREs, which functions as a cytoplasmic aconitase; one lacking this cluster, which accumulates in iron-deprived cells and binds mRNA firmly. We investigated which surfaces of IRP-1 interact with IREs. Surface areas were predicted on the basis of the crystallized porcine mitochondrial aconitase structure. We selected nine sequences absent or different in mitochondrial and Escherichia coli aconitases, both being devoid of RNA-binding properties. Mutations in two regions of domain 4 of IRP-1 lowered the affinity for a wild-type IRE up to 7-fold in vitro, whereas the aconitase activity, a control for structural integrity, was not affected. Scatchard plot analysis with mutant IREs indicated that domain 4 is involved in the binding specificity. This conclusion was confirmed with hybrid proteins in which IRP-1 surface loops were grafted into IRP-2. The results indicate that arginines 728 and 732 contact the IRE bulge, whereas region 685-689 is necessary for recognition of the IRE loop.
Aconitate Hydratase/chemistry, Amino Acid Sequence, Animals, Binding Sites, Escherichia coli/enzymology, Ferritins/genetics, Humans, Iron Regulatory Protein 1, Iron Regulatory Protein 2, Iron-Regulatory Proteins, Iron-Sulfur Proteins/chemistry, Iron-Sulfur Proteins/genetics, Mitochondria/enzymology, Models, Molecular, Molecular Sequence Data, Mutagenesis, Mutation, Protein Binding, Protein Conformation, Proto-Oncogene Proteins/genetics, RNA, Messenger/metabolism, RNA-Binding Proteins/chemistry, RNA-Binding Proteins/genetics, Recombinant Fusion Proteins, Sequence Homology, Amino Acid, Swine, Wnt2 Protein
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