Interaction of iron regulatory protein-1 (IRP-1) with ATP/ADP maintains a non-IRE-binding state.

In its aconitase-inactive form, IRP-1 (iron regulatory protein-1)/cytosolic aconitase binds to the IRE (iron-responsive element) of several mRNAs to effect post-transcriptional regulation. We have shown previously that IRP-1 has ATPase activity and that binding of ATP suppresses the IRP-1/IRE interaction. In the present study, we characterize the binding activity further. Binding is observed with both [alpha-32P]ATP and [alpha-32P]ADP, but not with [gamma-32P]ATP. Recombinant IRP-1 binds approximately two molecules of ATP, and positive co-operativity is observed with a Hill coefficient of 1.67+/-0.36 (EC50=44 microM) commencing at 1 microM ATP. Similar characteristics are observed with both apoprotein and the aconitase form. On binding, ATP is hydrolysed to ADP, and similar binding parameters and co-operativity are seen with ADP, suggesting that ATP hydrolysis is not rate limiting in product formation. The non-hydrolysable analogue AMP-PNP (adenosine 5'-[beta,gamma-imido]triphosphate) does not induce co-operativity. Upon incubation of IRP-1 with increasing concentrations of ATP or ADP, the protein migrates more slowly on agarose gel electrophoresis, and there is a shift in the CD spectrum. In this new state, adenosine nucleotide binding is competed for by other nucleotides (CTP, GTP and AMP-PNP), although ATP and ADP, but not the other nucleotides, partially stabilize the protein against spontaneous loss of aconitase activity when incubated at 37 degrees C. A mutant IRP-1(C437S) lacking aconitase activity shows only one ATP-binding site and lacks co-operativity. It has increased IRE-binding capacity and lower ATPase activity (Km=75+/-17 nmol/min per mg of protein) compared with the wild-type protein (Km=147+/-48 nmol/min per mg of protein). Under normal cellular conditions, it is predicted that ATP/ADP will maintain IRP-1 in a non-IRE-binding state.