Ligand-induced structural alterations in human iron regulatory protein-1 revealed by protein footprinting

Human iron regulatory protein-1 (IRP-1) is a bifunctional protein that regu lates iron metabolism by binding to mRNAs encoding proteins involved in iro n uptake, storage, and utilization. Intracellular iron accumulation regulat es IRP-1 function by promoting the assembly of an iron-sulfur cluster, conf erring aconitase activity to IRP-1, and hindering RNA binding. Using protei n footprinting, we have studied the structure of the two functional forms o f IRP-1 and have mapped the surface of the iron-responsive element (IRE) bi nding site. Binding of the ferritin IRE or of the minimal regulatory region of transferrin receptor mRNA induced strong protections against proteolysi s in the region spanning amino acids 80 to 187, which are located in the pu tative cleft thought to be involved in RNA binding. In addition, IRE-induce d protections were also found in the C-terminal domain at Arg-721 and Arg-7 28. These data implicate a bipartite IRE binding site located in the putati ve cleft of IRP-1. The aconitase form of IRP-1 adopts a more compact struct ure because strong reductions of cleavage were detected in two defined area s encompassing residues 149 to 187 and 721 to 735. Thus both ligands of apo -IRP-1, the IRE and the 4Fe-4S cluster, induce distinct but overlapping alt erations in protease accessibility. These data provide evidences for struct ural changes in IRP-1 upon cluster formation that affect the accessibility of residues constituting the RNA binding site.