Energetic Basis for Inhibition of Calcium Phosphate Biomineralization by Osteopontin

Calcium oxalate kidney stones form attached to Randall's plaques (RP), calcium phosphate (Ca-P) deposits on the renal papillary surface. Osteopontin (OPN) suppresses crystal growth in the complex process of urinary stone formation, but the inhibitory role of active domains of OPN involved in the initial formation of the RPs attached to epithelial cells has yet to be clarified. Here we demonstrate the thermodynamic basis for how OPN sequences regulate the onset of Ca-P mineral formation on lipid rafts as a model membrane. We first quantify the kinetics of hydroxyapatite (HAP) nucleation on membrane substrates having liquid-condensed (LC) and liquid-expanded (LE) phases using in situ atomic force microscopy (AFM). We find that rates are sequence-dependent, and the thermodynamic barrier to nucleation is reduced by minimizing the interfacial free energy γ. Combined with single-molecule determination of the binding energy (ΔG_B) of the OPN peptide segments adsorbed to the HAP (100) face, we show a linear relationship of γ and ΔG_B, suggesting that the increase in the nucleation barriers correlates with strong peptide-crystal nuclei binding. These findings reveal fundamental energetic clues for inhibition of membrane-mediated nucleation by sequence motifs and subdomains within the OPN protein through spatial location of charged moieties and provide insight connecting peripheral cell membranes to pathological mineralization.