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

ABSTRACT: Strontium salts are used for treatment of osteoporosis and bone cancer, but their impact on calcium-mediated physiological processes remains obscure. To explore Sr²⁺ interference with Ca²⁺ binding to proteins of the EF-hand family, we studied Sr²⁺/Ca²⁺ interaction with a canonical EF-hand protein, α-parvalbumin (α-PA). Evaluation of the equilibrium metal association constants for the active Ca²⁺ binding sites of recombinant human α-PA ('CD' and 'EF' sites) from fluorimetric titration experiments and isothermal titration calorimetry data gave 4 × 10⁹ M^-1 and 4 × 10⁹ M^-1 for Ca²⁺, and 2 × 10⁷ M^-1 and 2 × 10⁶ 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 α-PA and the Ca²⁺/Sr²⁺ binding are sequential processes with the CD site being occupied first. Spectrofluorimetric Sr²⁺ titration of the Ca²⁺-loaded α-PA revealed presence of secondary Sr²⁺ binding site(s) with an apparent equilibrium association constant of 4 × 10⁵ M^-1. Fourier-transform infrared spectroscopy data evidence that Ca²⁺/Sr²⁺-loaded forms of α-PA exhibit similar states of their COO^- groups. Near-UV circular dichroism (CD) data show that Ca²⁺/Sr²⁺ binding to α-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 α-PA. Meanwhile, scanning calorimetry measurements show markedly lower Sr²⁺-induced increase in stability of tertiary structure of α-PA, compared to the Ca²⁺-induced effect. Theoretical modeling using Density Functional Theory computations with Polarizable Continuum Model calculations confirms that Ca²⁺-binding sites of α-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 α-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.

SUBMITTER: Vologzhannikova AA 

PROVIDER: S-EPMC8392015 | biostudies-literature |

REPOSITORIES: biostudies-literature