Conformational changes induced by binding of divalent cations to calregulin

Scatchard analysis of equilibrium dialysis studies have revealed that in the presence of 3.0 mM MgCl₂ and 150 mM KCl, calregulin has a single binding site for Ca²⁺ with an apparent dissociation constant (apparent K(d)) of 0.05 μM and 14 binding sites for Zn²⁺ with apparent K(d)(Zn²⁺) of 310 μM. Ca²⁺ binding to calregulin induces a 5% increase in the intensity of intrinsic fluorescence and a 2-3-nm blue shift in emission maximum. Zn²⁺ binding to calregulin causes a dose-dependent increase of about 250% in its intrinsic fluorescence intensity and a red shift in the emission maximum of about 11 nm. Half-maximal wavelength shift occurs at 0.4 mol of Zn²⁺/mol of calregulin, and 100% of the wavelength shift is complete at 2 mol of Zn²⁺/mol of calregulin. In the presence of Zn²⁺ and calregulin the fluorescence intensity of the hydrophobic fluorescent probe 8-anilino-1-naphthalenesulfonate (ANS) was enhanced 300-400% with a shift in emission maximum from 500 to 480 nm. Half-maximal Zn²⁺-induced shift in ANS emission maximum occurred at 1.2 mol of Zn²⁺/mol of calregulin, and 100% of this shift occurred at 6 mol of Zn²⁺/mol of calregulin. Of 12 cations tested, only Zn²⁺ and Ca²⁺ produced changes in calregulin intrinsic fluorescence, and none of these metal ions could inhibit the Zn²⁺-induced red shift in intrinsic fluorescence emission maximum. Furthermore, none of these cations could inhibit or mimic the Zn²⁺-induced blue shift in ANS emission maximum. These results suggest that calregulin contains distinct and specific ligand-binding sites for Ca²⁺ and Zn²⁺. While Ca²⁺ binding results in the movement of tryptophan away from the solvent, Zn²⁺ causes a movement of tryptophan into the solvent and the exposure of a domain with considerable hydrophobic character.