Identification of a conserved calmodulin-binding motif in the sequence of F0F1 ATPsynthase inhibitor protein.
The 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 clarified.
Contessi
Stefania
S
Department of Biomedical Sciences and Technologies, MATI Centre of Excellence, CIME Centre, University of Udine, Udine, Italy.
Haraux
Francis
F
Mavelli
Irene
I
Lippe
Giovanna
G
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002147:Calmodulin / Q000032:analysis / Q000737:chemistry*; D002417:Cattle; D017124:Conserved Sequence; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000737:chemistry; D025261:Mitochondrial Proton-Translocating ATPases / Q000037:antagonists & inhibitors*; D008969:Molecular Sequence Data; D011485:Protein Binding; D011506:Proteins / Q000032:analysis / Q000737:chemistry*; D016415:Sequence Alignment; D000097795:ATPase Inhibitory Protein
D002147:Calmodulin; D004791:Enzyme Inhibitors; D025261:Mitochondrial Proton-Translocating ATPases; D011506:Proteins; D000097795:ATPase Inhibitory Protein