Phosphorylated/Nonphosphorylated Motifs in Amelotin Turn Off/On the Acidic Amorphous Calcium Phosphate-to-Apatite...

Amelotin (AMTN) as a matrix protein exerts a direct effect on biomineralization by modulating apatite (HAP) formation during the dental enamel maturation stage through the specific interaction of a potentially phosphorylated Ser-Ser-Glu-Glu-Leu (SSEEL) peptide fragment with calcium phosphate (Ca-P) surfaces. However, the roles of (non)phosphorylation of this evolutionarily conserved subdomain within AMTN remain poorly understood. Here, we show, by time-resolved atomic force microscopy (AFM) imaging of in situ HAP crystallization via the HPO₄^2--rich amorphous calcium phosphate (acidic ACP), the on/off switching of the phase transformation process through a nonphosphorylation-to-phosphorylation transition of the SSEEL motif. Using high-resolution transmission electron microscopy (HRTEM), we observed that the acidic ACP phase is stabilized by the phosphorylated SSEEL motif, delaying its transformation to HAP, whereas the nonphosphorylated counterpart promotes HAP formation by accelerating the dissolution-recrystallization of the acidic ACP substrate. Dynamic force spectroscopy measurements demonstrate greater binding energies of nonphosphorylated SSEEL to the acidic ACP substrate by the formation of molecular peptide-ACP bonding, explaining the enhanced dissolution of the acidic ACP substrate by stronger complexion with surface Ca²⁺ ions. Our findings demonstrate direct evidence for the switching role of (non)phosphorylation of an evolutionarily conserved subdomain within AMTN in controlling the phase transition of growing enamel and designing tissue regeneration biomaterials.