Introduction of a fifth carboxylate ligand heightens the affinity of the oncomodulin CD and EF sites for Ca2+

The acid-pair hypothesis, proposed by Reid and Hedges [(1980) J. Theor. Biol. 84, 401-444], suggests that the affinity of an EF-hand motif for Ca2+ will be maximal with four acidic ligands, paired along the +x, -x and +z-z axes. Addition of a fifth anionic ligand is predicted to reduce Ca2+-binding affinity, as a consequence of increased electrostatic repulsion. Interestingly, for oncomodulin, we observe that introduction of a fifth carboxylate residue at the +z position in the CD coordination sphere or at the -x position in the EF coordination sphere significantly increases the affinity of those aspartate (G98D), and the combined mutations (55/98) have been examined in Ca2+- and Mg2+-binding studies, titration calorimetry, and differential scanning calorimetry. The K-Ca for the CD site is reduced from 800 to 67 nM by the S55D mutation, while K-Ca for the EF site is reduced from 45 to 4 nM by the G98D mutation. Both mutations destabilize the ape form of the protein and increase the thermal stability on the Ca2+-bound state. Interestingly, the S55D mutation also increases the affinity of the oncomodulin CD site for Mg2+, decreasing the dissociation constant from >1 mM to approximately 30 mu M. This increase in affinity is reflected in a substantially increased thermal stability of the Mg2+-bound form of the protein. In 0.15 M NaCl, 0.025 M Hepes (pH 7.4), and 0.01 M Mg2+, the wild-type protein denatures at 68.5 degrees C. By contrast, under identical conditions, the S55D mutation denatures at 79.0 degrees C. The increased metal ion-binding affinity displayed by the variant proteins may result in part from preferential destabilization of the ape-protein by the additional carboxylate.