The Role of Phe-92 in the Ca-induced Conformational Transition in the C-terminal Domain of Calmodulin

Recent studies have shown that substitution of Ala for one or more Phe residues in calmodulin (CaM) imparts a temperature-sensitive phenotype to yeast (Ohya, Y., and Botstein, D. (1994) Science 263, 963-966). The Phe residue immediately preceding the first Ca ligand in site III of CaM (Phe-92) was found to be of particular importance because the mutation at this position alone was sufficient to induce this phenotype. In the present work we have studied the functional and structural consequences of the Phe-92 Ala mutation in human liver calmodulin. We found that the mutant (CaMF92A) is incapable of activating phosphodiesterase, and the maximal activation of calcineurin is reduced by 40% as compared with the wild type CaM. Impaired regulatory properties of CaMF92A are accompanied by an increase in affinity for Ca at the C-terminal domain. To investigate the structural consequences of the F92A mutation, we constructed four recombinant C-terminal domain fragments (C-CaM) of calmodulin (residues 78-148): 1) wild type (C-CaMW); 2) Ala substituted for Phe-92 (C-CaMF92A); 3) cysteine residues introduced at position 85 and 112 to lock the domain with a disulfide bond in the Ca -free (closed) conformation (C-CaM85/112); and 4) mutations 2 and 3 combined (C-CaM85/112F92A). The Cys-containing mutants readily form intramolecular disulfide bonds regardless whether Phe or Ala is present at position 92. The F92A mutation causes a decrease in stability of the domain in the absence of Ca as indicated by an 11.8°C shift in the far UV circular dichroism thermal unfolding curve. This effect is reversed by the disulfide bond in the C-CaM85/112F92A mutant. The C-CaMW peptide shows a characteristic Ca -dependent increase in solvent-exposed hydrophobic surface which was monitored by an increase in the fluorescence of the hydrophobic probe 1,1′-bis(4-anilino)naphthalene-5,5′-disulfonic acid. The fluorescence increase induced by C-CaMF92A is 45% lower than that induced by C-CaMW suggesting that the F92A mutation causes a decrease in the accessibility of several hydrophobic side chains in the C-terminal domain of CaM in the presence of Ca . The Cys-85-Cys-112 disulfide bond causes a 10- or 5.9-fold decrease in Ca affinity depending on whether Phe or Ala is present at position 92, respectively, suggesting that coupling between Ca binding and the conformational transition is weaker in the absence of the phenyl ring at position 92. Our results indicate that Phe-92 makes an importan