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.