Inhibition of an iron‐responsive element/iron regulatory protein‐1 complex by ATP binding and hydrolysis

• Published in: The FEBS journal 2007-06, Vol.274 (12), p.3108-3119
• Main Authors: Popovic, Zvezdana, Templeton, Douglas M.
• Format: Article
• Language: English
• Subjects: Adenosine Diphosphate - pharmacology; Adenosine Triphosphate - metabolism; Adenylyl Imidodiphosphate - pharmacology; ATP binding; ATP hydrolysis; Binding sites; Biochemistry; Cell Line; Cross-Linking Reagents - chemistry; energy metabolism; Humans; Hydrolysis; Iron; Iron - metabolism; Iron Regulatory Protein 1 - metabolism; iron regulatory proteins; iron‐responsive element; Metabolism; Molecular biology; Oxidative Phosphorylation; Protein Binding; Proteins; Recombinant Proteins - metabolism; Response Elements; Ribonucleic acid; RNA; RNA, Messenger - metabolism
• ISSN: 1742-464X; 1742-4658
• DOI: 10.1111/j.1742-4658.2007.05843.x

Iron regulatory protein‐1 binding to the iron‐responsive element of mRNA is sensitive to iron, oxidative stress, NO, and hypoxia. Each of these agents changes the level of intracellular ATP, suggesting a link between iron levels and cellular energy metabolism. Furthermore, restoration of iron regulatory protein‐1 aconitase activity after NO removal has been shown to require mitochondrial ATP. We demonstrate here that the iron‐responsive element‐binding activity of iron regulatory protein is ATP‐dependent in HepG2 cells. Iron cannot decrease iron regulatory protein binding activity in cell extracts if they are simultaneously treated with an uncoupler of oxidative phosphorylation. Physiologic concentrations of ATP inhibit iron‐responsive element/iron regulatory protein binding in cell extracts and binding of iron‐responsive element to recombinant iron regulatory protein‐1. ADP has the same effect, in contrast to the nonhydrolyzable analog adenosine 5′‐(β,γ‐imido)triphosphate, indicating that in order to inhibit iron regulatory protein‐1 binding activity, ATP must be hydrolyzed. Indeed, recombinant iron regulatory protein‐1 binds ATP with a Kd of 86 ± 17 µm in a filter‐binding assay, and can be photo‐crosslinked to azido‐ATP. Upon binding, ATP is hydrolyzed. The kinetic parameters [Km = 5.3 µm, Vmax = 3.4 nmol·min−1·(mg protein)−1] are consistent with those of a number of other ATP‐hydrolyzing proteins, including the RNA‐binding helicases. Although the iron‐responsive element does not itself hydrolyze ATP, its presence enhances iron regulatory protein‐1's ATPase activity, and ATP hydrolysis results in loss of the complex in gel shift assays.