| Summary: | Strontium salts are used for treatment of osteoporosis and bone cancer, but their impact on calcium-mediated physiological processes remains obscure. To explore Sr<sup>2+</sup> interference with Ca<sup>2+</sup> binding to proteins of the EF-hand family, we studied Sr<sup>2+</sup>/Ca<sup>2+</sup> interaction with a canonical EF-hand protein, α-parvalbumin (α-PA). Evaluation of the equilibrium metal association constants for the active Ca<sup>2+</sup> binding sites of recombinant human α-PA (‘CD’ and ‘EF’ sites) from fluorimetric titration experiments and isothermal titration calorimetry data gave 4 × 10<sup>9</sup> M<sup>−1</sup> and 4 × 10<sup>9</sup> M<sup>−1</sup> for Ca<sup>2+</sup>, and 2 × 10<sup>7</sup> M<sup>−1</sup> and 2 × 10<sup>6</sup> M<sup>−1</sup> for Sr<sup>2+</sup>. Inactivation of the EF site by homologous substitution of the Ca<sup>2+</sup>-coordinating Glu in position 12 of the EF-loop by Gln decreased Ca<sup>2+</sup>/Sr<sup>2+</sup> affinity of the protein by an order of magnitude, whereas the analogous inactivation of the CD site induced much deeper suppression of the Ca<sup>2+</sup>/Sr<sup>2+</sup> affinity. These results suggest that Sr<sup>2+</sup> and Ca<sup>2+</sup> bind to CD/EF sites of α-PA and the Ca<sup>2+</sup>/Sr<sup>2+</sup> binding are sequential processes with the CD site being occupied first. Spectrofluorimetric Sr<sup>2+</sup> titration of the Ca<sup>2+</sup>-loaded α-PA revealed presence of secondary Sr<sup>2+</sup> binding site(s) with an apparent equilibrium association constant of 4 × 10<sup>5</sup> M<sup>−1</sup>. Fourier-transform infrared spectroscopy data evidence that Ca<sup>2+</sup>/Sr<sup>2+</sup>-loaded forms of α-PA exhibit similar states of their COO<sup>−</sup> groups. Near-UV circular dichroism (CD) data show that Ca<sup>2+</sup>/Sr<sup>2+</sup> binding to α-PA induce similar changes in symmetry of microenvironment of its Phe residues. Far-UV CD experiments reveal that Ca<sup>2+</sup>/Sr<sup>2+</sup> binding are accompanied by nearly identical changes in secondary structure of α-PA. Meanwhile, scanning calorimetry measurements show markedly lower Sr<sup>2+</sup>-induced increase in stability of tertiary structure of α-PA, compared to the Ca<sup>2+</sup>-induced effect. Theoretical modeling using Density Functional Theory computations with Polarizable Continuum Model calculations confirms that Ca<sup>2+</sup>-binding sites of α-PA are well protected against exchange of Ca<sup>2+</sup> for Sr<sup>2+</sup> regardless of coordination number of Sr<sup>2+</sup>, solvent exposure or rigidity of sites. The latter appears to be a key determinant of the Ca<sup>2+</sup>/Sr<sup>2+</sup> selectivity. Overall, despite lowered affinity of α-PA to Sr<sup>2+</sup>, the latter competes with Ca<sup>2+</sup> for the same EF-hands and induces similar structural rearrangements. The presence of a secondary Sr<sup>2+</sup> binding site(s) could be a factor contributing to Sr<sup>2+</sup> impact on the functional activity of proteins.
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