THE ROLE OF PHE-92 IN THE CA2-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 phenotyp e to yeast (Ohya, Y., and Botstein, D. (1994) Science 263, 963-966). T he Phe residue immediately preceding the first Ca2+ 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 phenotyp e. In the present work we have studied the functional and structural c onsequences of the Phe-92 --> Ala mutation in human liver calmodulin. We found that the mutant (CaMF92A) is incapable of activating phosphod iesterase, 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 Ca2+ at the C- terminal domain. To investigate the structural consequences of the F92 A mutation, we constructed four recombinant C-terminal domain fragment s (C-CaM) of calmodulin (residues 78-148): 1) wild type (C-CaMW); 2) A la substituted for Phe-92 (C-CaMF92A); 3) cysteine residues introduced at position 85 and 112 to lock the domain with a disulfide bond in th e Ca2+-free (closed) conformation (C-CaM85/112); and 4) mutations 2 an d 3 combined (C-CaM85/112F92A). The Cys-containing mutants readily for m intramolecular disulfide bonds regardless whether Phe or Ala is pres ent at position 92. The F92A mutation causes a decrease in stability o f the domain in the absence of Ca2+ as indicated by an 11.8 degrees C shift in the far UV circular dichroism thermal unfolding curve. This e ffect is reversed by the disulfide bond in the C-CaM85/112F92A mutant. The C-CaMW peptide shows a characteristic Ca2+-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)-naph thalene-5,5'-disulfonic acid. The fluorescence increase induced by C-C aMF92A is similar to 45% lower than that induced by C-CaMW suggesting that the F92A mutation causes a decrease in the accessibility of sever al hydrophobic side chains in the C-terminal domain of CaM in the pres ence of Ca2+. The Cys-85-Cys-112 disulfide bond causes a 10- or 5.9-fo ld decrease in Ca2+ affinity depending on whether Phe or Ala is presen t at position 92, respectively, suggesting that coupling between Ca2binding and the conformational transition is weaker in the absence of the phenyl ring at position 92. Our results indicate that Phe-92 makes an important contribution to the Ca2+-induced transition in the C-ter minal domain of CaM. This is most likely the reason for the severely i mpaired regulatory properties of the CaM mutants having Ala substitute d for Phe-92.