Crystal structure of the D94S/G98E variant of rat α-parvalbumin. An explanation for the reduced divalent ion affinity

Simultaneous replacement of Asp-94 with serine and Gly-98 with glutamate in rat α-parvalbumin creates a CD-site ligand array in the context of the EF-site binding loop. Previous work has shown that, relative to the wild-type CD site, this engineered site has markedly reduced Ca2+ affinity. Seeking an explanation for this phenomenon, we have obtained the crystal structure of the α D94S/G98E variant. The Ca2+ coordination within the engineered EF site of the 94/98E variant is nearly identical to that within the CD site, suggesting that the attenuated affinity of the EF site in 94/98E is not a consequence of suboptimal coordination geometry. We have also examined the divalent ion binding properties of the α 94/98E variant in both Na+- and K+-containing buffers. Although the Ca2+ and Mg2+ affinities are higher in K+ solution, the increases are comparable to those observed for wild-type α. Consistent with that finding, the apparent Na+ stoichiometry, estimated from stability studies conducted as a function of ...