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 Ca2+ binding to proteins of the EF-hand family, we studied Sr2+/Ca2+ interaction with a canonical EF-hand protein, alpha-parvalbumin (alpha-PA). Evaluation of the equilibrium metal association constants for the active Ca2+ binding sites of recombinant human alpha-PA ('CD' and 'EF' sites) from fluorimetric titration experiments and isothermal titration calorimetry data gave 4 x 10(9) M-1 and 4 x 10(9) M-1 for Ca2+, and 2 x 10(7) M-1 and 2 x 10(6) M-1 for Sr2+. Inactivation of the EF site by homologous substitution of the Ca2+-coordinating Glu in position 12 of the EF-loop by Gln decreased Ca2+/Sr2+ affinity of the protein by an order of magnitude, whereas the analogous inactivation of the CD site induced much deeper suppression of the Ca2+/Sr2+ affinity. These results suggest that Sr2+ and Ca2+ bind to CD/EF sites of alpha-PA and the Ca2+/Sr2+ binding are sequential processes with the CD site being occupied first. Spectrofluorimetric Sr2+ titration of the Ca2+-loaded alpha-PA revealed presence of secondary Sr2+ binding site(s) with an apparent equilibrium association constant of 4 x 10(5) M-1. Fourier-transform infrared spectroscopy data evidence that Ca2+/Sr2+-loaded forms of alpha-PA exhibit similar states of their COO - groups. Near-UV circular dichroism (CD) data show that Ca2+/Sr2+ binding to alpha-PA induce similar changes in symmetry of microenvironment of its Phe residues. Far-UV CD experiments reveal that Ca2+/Sr2+ binding are accompanied by nearly identical changes in secondary structure of alpha-PA. Meanwhile, scanning calorimetry measurements show markedly lower Sr2+-induced increase in stability of tertiary structure of alpha-PA, compared to the Ca 2 +-induced effect. Theoretical modeling using Density Functional Theory computations with Polarizable Continuum Model calculations confirms that Ca2+-binding sites of alpha-PA are well protected against exchange of Ca2+ for Sr2+ regardless of coordination number of Sr2+, solvent exposure or rigidity of sites. The latter appears to be a key determinant of the Ca2+/Sr2+ selectivity. Overall, despite lowered affinity of alpha-PA to Sr2+, the latter competes with Ca2+ for the same EF-hands and induces similar structural rearrangements. The presence of a secondary Sr2+ binding site(s) could be a factor contributing to Sr2+ impact on the functional activity of proteins.