EF-hand motifs of α, β and γ isoforms of diacylglycerol kinase bind calcium with different affinities and conformational changes

The three diacylglycerol kinase isoenzymes (DGKα, DGKβ and DGKγ) cloned so far contain in common a tandem repeat of EF-hand motifs. However, the Ca²⁺ dependences of the DGK activities are known to be variable between isoenzymes, and the Ca²⁺-binding activities of these motifs have not been tested except for those present in DGKα. We therefore attempted to define the intrinsic properties of EF-hands occurring in the DGK isoenzymes. For this purpose we bacterially expressed and purified the EF-hand motifs (termed DKE forms) of the three DGKs. Equilibrium dialysis with the purified DKE forms showed that all of the expressed proteins could bind approx. 2 mol of Ca²⁺ per mol. However, the apparent dissociation constant (K_d) for calcium binding to α-DKE (9.9 µM) was an order of magnitude greater than those estimated for β-DKE (0.89 µM) and γ-DKE (0.40 µM). Experiments with 2-p-toluidinylnaphthalene 6-sulphonate, a probe for hydrophobic regions of proteins, showed that the binding of Ca²⁺ to β-DKE resulted in the exposure of hydrophobic amino acids, whereas hydrophobic regions of α-DKE and γ-DKE were masked by the addition of Ca²⁺. Taken together, these results indicate that DGKα, DGKβ and DGKγ possess EF-hand structures with intrinsic properties different from each other with respect to affinities for Ca²⁺ and Ca²⁺-induced conformational changes.