Identification of RNA-binding surfaces in iron regulatory protein-1

Post-transcriptional regulation of mRNA translation and stability in iron m etabolism involves the interaction between the trans-acting cytoplasmic iro n regulatory proteins (IRP-1 and IRP-2) and cis-acting iron-responsive elem ents (IREs) in mRNA 5'- or 3'-untranslated regions. IRP-1 can adopt two con formations: one with a [4Fe-4S]-cluster, unable to bind IREs, which functio ns as a cytoplasmic aconitase; one lacking this cluster, which accumulates in iron-deprived cells and binds mRNA firmly, We investigated which surface s of IRP-1 interact with IREs, Surface areas were predicted on the basis of the crystallized porcine mitochondrial aconitase structure, We selected ni ne sequences absent or different in mitochondrial and Escherichia coil acon itases, both being devoid of RNA-binding properties, Mutations in two regio ns of domain 4 of IRP-1 lowered the affinity for a wild-type IRE up to 7-fo ld in vitro, whereas the aconitase activity, a control for structural integ rity, was not affected. Scatchard plot analysis with mutant IREs indicated that domain 4 is involved in the binding specificity, This conclusion was c onfirmed with hybrid proteins in which IRP-1 surface loops were grafted int o IRP-2, The results indicate that arginines 728 and 732 contact the IRE bu lge, whereas region 685-689 is necessary for recognition of the IRE loop.