Chimeras of parvalbumin and oncomodulin involving exchange of the complete CD site show that the Ca2+/Mg2+ specificity is an intrinsic property of the site

Rat parvalbumin (PV) and oncomodulin (OM) differ in the affinity and selectivity of metal binding to their CD site, which is a high-affinity Ca2+/Mg2+-mixed site in PV and a low-affinity Ca2+-specific site in OM. To assess to what degree the Ca2+/Mg2+ specificity and affinity of an EF-hand motif in a protein is intrinsically determined by its sequence, the complete CD sites were exchanged, yielding two chimeras, [S41-Q71]PV and [D41-S71]OM. The optical characteristics of a Trp102, inserted in the hydrophobic core of PV, OM and the two chimeras, are very similar in all four proteins, which suggests that the hydrophobic core is qualitatively similar in the chimeras as in the parent proteins. Direct Ca2+ and Mg2+ binding monitored by flow dialysis and gel filtration revealed that [S41-Q71]PV binds only one Mg2+ with an intrinsic affinity K-Mg2+' of 3.0 x 10(4) M(-1) and two Ca2+ with an identical K-Ca2+' of 4.4 x 10(6) M(-1), whereas [D41-S71]OM binds two Mg2+ with a mean K-Mg2+' of 2 x 10(4) M(-1) and two Ca2+ with a K-Ca2+' of 1.3 x 10(7) M(-1). K-Ca2+' of the CD site of [S41-Q71]PV was 2.5-fold higher than of the CD site in [W102]OM, but 5-6-fold lower than that of the CD site in [W102]PV. In [D41-S71]OM, K-Ca2+' of the CD site was twofold lower than in [W102]PV, but eightfold higher than in [W102]OM. These results indicate that the sequence of the CD site determines its Ca2+Mg2+-specificity, whereas its affinity for Ca2+ influenced by the protein into which the CD site is inserted. The inserted CD site in turn influences the affinity of the EF site to which it is paired in the host protein and the paired sites display an equalized affinity for Ca2+. Mg2+ decreases the affinity of the chimeras for Ca2+, but not according to a simple competition model. The Mg2+ antagonism is much more pronounced in [D41-S71]OM than in [S41-Q71]PV, but in each chimera the CD and EF site are quantitatively affected in the same manner. Thus, [S41-Q71]PV which can only bind a single Mg2+ ion, displays a Ca2+/Mg2+-antagonism for both sites with a K-Mg . compet of 2.3 x 10(2) M(-1). These results confirm the 'equalizer' principle in the cation-binding parameters of [S41-Q71]PV: both sites display the same Ca2+ affinity and Mg2+ antagonism. In [D41-S71]OM with its two Ca2+/Mg2+ sites the antagonism shows qualitatively the same complexity as in wild-type PV, although it is somewhat weaker in amplitude.