BLOCKING THE CA2-INDUCED CONFORMATIONAL TRANSITIONS IN CALMODULIN WITH DISULFIDE BONDS()

Calcium dependent regulation of intracellular processes is mediated by proteins that on binding Ca2+ assume a new conformation, which enable s them to bind to their specific target proteins and to modulate their function. Calmodulin (CaM) and troponin C, the two best characterized Ca2+-regulatory proteins, are members of the family of Ca2+-binding p roteins utilizing the helix-loop-helix structural motif (EF-hand), Her zberg, Moult, and James (Herzberg, O., Moult, J., and James, M. N. G. (1986) J. Biol. Chem. 261, 2638-2644) proposed that the Ca2+-induced c onformational transition in troponin C involves opening of the interfa ce between the a helical segments in the N-terminal domain of this pro tein. Here we have tested the hypothesis that a similar transition is the key Ca2+-induced regulatory event in calmodulin. Using site-direct ed mutagenesis we have substituted cysteine residues for Gln(41) and L ys(75) (CaM41/ 75) or Ile(85) and Leu(112) (CaM85/112) in the N-termin al and C-terminal domains, respectively, of human liver calmodulin. Ba sed on molecular modeling, cysteines at these positions were expected to form intramolecular disulfide bonds in the Ca2+-free conformation o f the protein, thus blocking the putative Ca2+-induced transition. We found that intramolecular disulfide bonds are readily formed in both m utants causing a decrease in affinity for Ca2+ and the loss of ability to activate target enzymes, phosphodiesterase and calcineurin, The re gulatory activity is fully recovered in CaM41/75 and partially recover ed in CaM85/112 upon reduction of the disulfide bonds with dithiotreit ol and blocking the Cys residues by carboxyamidomethylation or cyanyla tion. These results indicate that the Ca2+-induced opening of the inte rfaces between helical segments in both domains of CaM is critical for its regulatory properties consistent with the Herzberg-Moult-James mo del.