IF1, the natural inhibitor protein of F(O)F(1)ATP synthase able to regulate the ATP hydrolytic activity of both mitochondria] and cell surface enzyme, exists in two oligomeric states depending on pH: an inactive, highly helical, tetrameric form above pH 6.7 and an active, inhibitory, dimeric form below pH 6.7 [Cabezon, E., Butler, P. J., Runswick, M. J., and Walker, J. E. (2000) J. Biol. Chem. 275, 25460-25464]. IF1 is known to interact in vitro with the archetypal EF-hand calcium sensor calmodulin (CaM), as well to colocalize with CaM on the plasma membrane of cultured cells. Low resolution structural data were herein obtained in order to get insights into the molecular interaction between IF1 and CaM. A combined structural proteomicstrategy was used which integrates limited proteolysis and chemical cross-linking with mass spectrometric analysis. Specifically, chemical cross-linking data clearly indicate that the C-terminal lobe of CaM molecule contacts IF1 within the inhibitory, flexible N-terminal region that is not involved in the dimeric interface in Nevertheless, native mass spectrometry analysis demonstrated that in the micromolar range the stoichiometry of the IF1-CaM complex is 1:1, thereby indicating that binding to CaM promotes IF1 dimer dissociation without directly interfering with the intersubunit contacts of the IF1 dimer. The relevance of the finding that only the C-terminal lobe of CaM is involved in the interaction is two fold: (i) the IF1-CaM complex can be included in the category of noncanonical structures of CaM complexes; (ii) it can be inferred that the N-terminal region of CaM might have the opportunity to bind to a second target.
