Synthetic fragments of calmodulin calcium-binding site III. A test of the acid pair hypothesis

The acid pair hypothesis describing the interaction of calcium with the helix-loop-helix conformation of EF hands in calmodulin and related proteins predicts that these calcium-binding sites will have increased affinity for calcium if the anionic amino acid dentates in the loop region which interact directly with the cation are paired on the axial vertices of the resulting octahedral arrangement of chelating residues about the cation. As a test of this hypothesis, synthetic 33 residue analogs of bovine brain calmodulin calcium-binding site III have been prepared by the solid-phase method and analyzed for calcium affinity. The native sequence has a Kd of 735 microM for calcium and contains three anionic ligands which assume the +x, +y, and -z coordinates of the octahedral arrangement about the cation, thus precluding any pairing of the anionic ligands. This dissociation constant is 26 times weaker than that obtained from a synthetic analog of the sequentially homologous calcium-binding site III of rabbit skeletal TnC (Kd = 28 microM) which has four anionic ligands paired on the x and z axes. An analog of calmodulin site III with substitutions in the chelating residues at positions 1, 3, 5, 7, 9, and 12 of the 12-residue loop region to make these positions identical to those of rabbit skeletal troponin C site III decreased the calcium dissociation constant of the calmodulin peptide to 19 microM, similar to the troponin C peptide. Two synthetic analogs of calmodulin site III which contain three anionic ligands with two ligands paired on the x axis and two on the z axis have a Kd for calcium of 524 and 59 microM, respectively. This study provides strong support for and a better definition of the acid pair hypothesis and further demonstrates the usefulness of synthetic calcium-binding fragments in delineating the mechanism of calcium regulation of calmodulin and related proteins.