STRUCTURE CALCIUM AFFINITY RELATIONSHIPS OF SITE-III OF CALMODULIN - TESTING THE ACID PAIR HYPOTHESIS USING CALMODULIN MUTANTS

Calmodulin mutants in which the calcium binding affinity of site IV wa s greatly reduced by a D133E mutation were prepared using site-specifi c, cassette-mediated mutagenesis as a multisite calcium binding protei n model to examine structure/calcium affinity relationships in site II I of calmodulin, Tryptophan was introduced in position 92 of the calmo dulin mutants as a fluorescent label to monitor the calcium-induced st ructural changes in the C-terminal domain of calmodulin, The five calm odulin mutants, 3xCaM, 3zCaM, 4xCaM, 4zCaM, and 4xzCaM, were designed so that there were three or four acidic amino acid residues in chelati ng positions of site III with acid pairs on either the X and/or Z coor dinating axes, The calcium dissociation constant of site III, K-III, o f the five calmodulin mutants changes in a descending order from 3xCaM (237 mu M), 3zCaM (140 mu M), 4xCaM (5.8 mu M), 4zCaM (3 mu M), to 4x zCaM (2 mu M), and these K-III values are significantly lower than tha t of F92W/D133E calmodulin (335 mu M) in which three acidic residues w ith no acid pairs were present in site III [Wu, X., & Reid, R. E. (199 7) Biochemistry 36, 3608-3616]. These results indicate that the calciu m affinity of site III increases when the number of the acidic chelati ng residues increases from three to four, when the number of acid pair s increases from zero to one and further to two, and when the location of the acid pair is changed from the X axis to the Z axis. This study provides the first evidence that the acid pair hypothesis which corre lates the nature of the chelating residues with the calcium affinity o f the hlh motif is applicable to a multisite calcium binding protein m odel. The Hill coefficients indicate that reversal of the sequence of filling of the calcium binding sites in the C-terminal domain from IV --> III to III --> IV also changes the site cooperativity from positiv e to negative. The cooperativity returns to positive when the proteins are titrated in the presence of a calmodulin-binding peptide. Data fr om the present study also demonstrate that calmodulin mutants with a d ecreased calcium affinity have a reduced efficiency in phosphodiestera se regulation at low calcium concentrations (50 mu M). However, high c alcium concentrations (15 mM)restore the phosphodiesterase regulatory activity of the calmodulin mutants to a level obtained with F92W calmo dulin, indicating that the mutations alter calcium regulation of calmo dulin-mediated phosphodiesterase activity without affecting the intera ction between calmodulin and the enzyme.