Site-specific replacement of amino acid residues in the CD site of rat parvalbumin changes the metal specificity of this Ca2+/Mg(2+)-mixed site toward a Ca(2+)-specific site.

Abstract

Rat parvalbumin (PV) and oncomodulin (OM) display considerable sequence similarity and structural similarity, but differ in the affinity and selectivity of metal binding to their CD site, a Ca2+/Mg(2+)-mixed site in PV and a Ca(2+)-specific site in OM. In an attempt to identify the structural basis for these differences, mutations were introduced in the previously generated [W102]PV mutant, which contains a unique tryptophan as a conformational-sensitive fluorescent probe inside the hydrophobic core. In the present report, we substituted selected amino acid residues in the CD site of PV by those present at identical positions in OM. One mutant protein, named [F66, W102]PV, has one new substitution in which isoleucine at position 66 was exchanged by phenylalanine. The second mutant protein, [I46, I50, L58, F66, W102]PV, has four new substitutions, namely V46-->I, L50-->I, I58-->L and I66-->F. Tryptophan fluorescence and difference spectrophotometry indicated that the mutations do not alter significantly the hydrophobic core. Both mutant proteins display two metal-binding sites of identical affinities with intrinsic affinity constants K'Ca2+ of 2.9 x 10(7) M-1 for [F66, W102]PV and 1.7 x 10(7) M-1 for [I46, I50, L58, F66, W102]PV and K'Mg2+ of 3.1 x 10(4) M-1 for [F66, W102]PV and 1.9 x 10(4) M-1 for [I46, I50, L58, F66, W102]PV. Thus, the five-residue substitution, but not the two-residue one, leads to a small decrease of affinity compared to [W102]PV (K'Ca2+ = 2.7 x 10(7) M-1, K'Mg2+ = 4.4 x 10(4) M-1). Despite these similarities, the Mg2+ effect on Ca2+ binding is different for the two mutant parvalbumins: the Ca(2+)-binding isotherms of [F66, W102]PV undergo a parallel shift upon increasing Mg2+ concentrations, which indicates that the Mg2+ effect on the two Ca(2+)-binding sites is the same and quantitatively very similar to that described for [W102]PV. In [I46, I50, L58, F66, W102]PV, Mg2+ antagonizes the binding of the second Ca2+ (likely at the EF site) much more than that of the first Ca2+ (likely the CD site). According to the competition equation, the two sites display KMg2+.compet values of 390 M-1 and 3.9 x 10(3) M-1, respectively. These data indicate that (a) the single I66-->F mutation does not modify the cation binding parameters. (b) Multiple modifications in the hydrophobic core still do not change the affinity for Ca2+ and Mg2+, but strongly affect the Mg2+ antagonism and probably the selectivity of the CD site.