title: Blocking the Ca2+-induced conformational transitions in calmodulin with disulfide bonds.
abstract: Calcium-dependent regulation of intracellular processes is mediated by proteins that on binding Ca2+ assume a new conformation, which enables 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 proteins utilizing the helix-loop-helix structural motif (EF-hand). Herzberg, Moult, and James (Herzberg, O., Moult, J., and James, M.N.G. (1986) J. Biol. Chem. 261, 2638-2644) proposed that the Ca2+-induced conformational transition in troponin C involves opening of the interface between the alpha-helical segments in the N-terminal domain of this protein. Here we have tested the hypothesis that a similar transition is the key Ca2+-induced regulatory event in calmodulin. Using site-directed mutagenesis we have substituted cysteine residues for Gln41 and Lys75 (CaM41/75) or Ile85 and Leu112 (CaM85/112) in the N-terminal and C-terminal domains, respectively, of human liver calmodulin. Based on molecular modeling, cysteines at these positions were expected to form intramolecular disulfide bonds in the Ca2+-free conformation of the protein, thus blocking the putative Ca2+-induced transition. We found that intramolecular disulfide bonds are readily formed in both mutants causing a decrease in affinity for Ca2+ and the loss of ability to activate target enzymes, phosphodiesterase and calcineurin. The regulatory activity is fully recovered in CaM41/75 and partially recovered in CaM85/112 upon reduction of the disulfide bonds with dithiothreitol and blocking the Cys residues by carboxyamidomethylation or cyanylation. These results indicate that the Ca2+-induced opening of the interfaces between helical segments in both domains of CaM is critical for its regulatory properties consistent with the Herzberg-Moult-James model.
authors.affiliation: Muscle Research Group, Boston Biomedical Research Institute, Massachusetts 02114, USA.
authors.forename: R Y
authors.initials: RY
authors.lastname: Tan
authors.forename: Y
authors.initials: Y
authors.lastname: Mabuchi
authors.forename: Z
authors.initials: Z
authors.lastname: Grabarek
mesh_terms: D001665:Binding Sites; D019703:Calcineurin; D002118:Calcium / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D002147:Calmodulin / Q000737:chemistry* / Q000187:drug effects / Q000302:isolation & purification; D002148:Calmodulin-Binding Proteins / Q000378:metabolism; D003001:Cloning, Molecular; D003545:Cysteine; D004220:Disulfides; D004789:Enzyme Activation; D005973:Glutamine; D018257:Helix-Loop-Helix Motifs; D006801:Humans; D007532:Isoleucine; D007700:Kinetics; D007930:Leucine; D008099:Liver / Q000378:metabolism; D008239:Lysine; D008958:Models, Molecular; D008961:Models, Structural; D016297:Mutagenesis, Site-Directed; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D011487:Protein Conformation / Q000187:drug effects; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000187:drug effects / Q000302:isolation & purification; D014336:Troponin / Q000737:chemistry*; D019209:Troponin C
chemical_list: D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D004220:Disulfides; D011994:Recombinant Proteins; D014336:Troponin; D019209:Troponin C; D007532:Isoleucine; D005973:Glutamine; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases; D010727:Phosphoric Diester Hydrolases; D007930:Leucine; D008239:Lysine; D003545:Cysteine; D002118:Calcium