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 Hodges [(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 Ca(2+)-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 sites for Ca2+. The variants resulting from replacement of serine-55 by aspartate (S55D), glycine-98 by aspartate (G98D), and the combined mutations (55/98) have been examined in Ca(2+)- and Mg(2+)-binding studies, titration calorimetry, and differential scanning calorimetry. The KCa for the CD site is reduced from 800 to 67 nM by the S55D mutation, while KCa for the EF site is reduced from 45 to 4 nM by the G98D mutation. Both mutations destabilize the apo form of the protein and increase the thermal stability on the Ca(2+)-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 microM. This increase in affinity is reflected in a substantially increased thermal stability of the Mg(2+)-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 mutations 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 apo-protein by the additional carboxylate.
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Department of Biochemistry, University of Missouri, Columbia 65211, USA.
Hapak
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Goodpasture
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D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002152:Calorimetry, Differential Scanning; D002264:Carboxylic Acids / Q000378:metabolism; D004563:Electrochemistry; D066298:In Vitro Techniques; D007700:Kinetics; D008024:Ligands; D008274:Magnesium / Q000378:metabolism; D008956:Models, Chemical; D008969:Molecular Sequence Data; D017354:Point Mutation; D017433:Protein Structure, Secondary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics
D002135:Calcium-Binding Proteins; D002264:Carboxylic Acids; D008024:Ligands; D011994:Recombinant Proteins; C034733:oncomodulin; D008274:Magnesium; D002118:Calcium