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The inhibitory region of mitochondrial F0F1ATPsynthase inhibitor protein IF1 contains a calmodulin-binding motif
No full textTopology of the Calmodulin Complex with ATPsynthase Inhibitor Protein IF1: a Structural Proteomic analysis
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Diazoxide affects the IF1 inhibitor protein binding to F1 sector of beef heart F0F1ATPsynthase
No full textDiazoxide, a selective opener of the mitochondrial ATP-sensitive K+ channel (mitoK(ATP)), has been reported to enhance F(0)F(1)ATP-synthase inhibition during ischemia, but the underlying mechanisms are still unclear. Here, we demonstrate that diazoxide directly interacts with the F-1 sector of beef heart F(0)F(1)ATPsynthase markedly promoting the binding of the inhibitor protein (IF1) to beta subunit. More specifically, the treatment of soluble F-1 with one equivalent of diazoxide was sufficient to decrease the K-d of IF1-F-1 complex at low pH. Such effect was revealed only on the cycling enzyme, while no effect was observed in the absence of Mg-ATP. However, diazoxide binding occurred independently from the catalysis, as shown by the structural changes induced by the drug in not catalytically active F-1 and revealed by CD spectra. In addition, kinetic analysis of ATP hydrolysis demonstrated that diazoxide exerts a stabilising role on Mg-ADP bound in the catalytic site of the beta subunit adopting the tight conformation (beta(DP)). In accordance, a stabilising effect of Mg-ADP at the nucleotide binding domain (NBD) has been reported also for K-ATP channel. These results suggest that diazoxide binds to beta subunit at NBD, which is highly conserved in the ATP-binding cassette protein family, thus inducing nucleotide stabilisation and favouring F-1 conformation suitable for IF, binding. Finally, diazoxide also increased IF1 binding to membrane bound F1, while it did not influence the energisation-dependent IF1 release. As IF1 binding mediates the F(0)F(1)ATPsynthase inhibition, we suggest that such mechanism may contribute to cardioprotection during ischemia
Iron binding to additional sites of bacterial and mammalian F1ATPases: structure stabilisation vs. pro-oxidant side-effects
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Identification of a conserved calmodulin-binding motif in the sequence of F0F1ATPsynthase inhibitor protein
No full textThe natural inhibitor proteins IF1 regulate mitochondrial F0F1 ATPsynthase in a wide range of species. We characterized the interaction of CaM with purified bovine IF1, two bovine IF1 synthetic peptides, as well as two homologous proteins from yeast, namely IF1 and STF1. Fluorometric analyses showed that bovine and yeast inhibitors bind CaM with a 1:1 stoichiometry in the pH range between 5 and 8 and that CaM-IF1 interaction is Ca2+-dependent. Bovine and yeast IF1 have intermediate binding affinity for CaM, while the Kd (dissociation constant) of the STF1-CaM interaction is slightly higher. Binding studies of CaM with bovine IF1 synthetic peptides allowed us to identify bovine IF1 sequence 33-42 as the putative CaM-binding region. Sequence alignment revealed that this region contains a hydrophobic motif for CaM binding, highly conserved in both yeast IF1 and STF1 sequences. In addition, the same region in bovine IF1 has an IQ motif for CaM binding, conserved as an IQ-like motif in yeast IF1 but not in STF1. Based on the pH and Ca2+ dependence of IF1 interaction with CaM, we suggest that the complex can be formed outside mitochondria, where CaM could regulate IF1 trafficking or additional IF1 roles not yet clarifie
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