Strontium Binding to α-Parvalbumin, a Canonical Calcium-Binding Protein of the “EF-Hand” Family

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