<p>Amelogenin self-assembly is a critical step in enamel matrix organization, supporting the spatial confinement of transient ACP and orchestrating hydroxyapatite nucleation and ordered growth. Studies have demonstrated that amelogenin self-assembles into nanoribbons by forming an amyloid-like structure that matches the cross-β pattern observed in the developing enamel matrix. However, molecular determinants remain unclear. In this work, we demonstrate that Ser16 phosphorylation (pS16) and Mmp20-mediated C-terminal truncation exert complementary control over amelogenin self-assembly and templated mineralization. Using advanced microscopy and spectroscopic methods, we show that C-terminal truncation lowers the energetic threshold for the disordered-to-ordered β-sheet phase transformation, producing rapid nanoribbon growth, whereas pS16 prolongs this transformation barrier and channels assembly into a gradual, more orderly templating process. Remarkably, pS16 alone is sufficient to drive nanoribbon formation under ion-free conditions, facilitating ion-mediated charge compensation at the self-assembly N-terminal domain that otherwise requires calcium and phosphate ions. These ions play complementary roles: phosphate appears to promote longitudinal elongation through interactions with protonated His-rich motifs, whereas calcium strengthens lateral cohesion and bundling by binding acidic C-terminal residues and engaging with the pS16 site, which could also foster ACP accumulation along the nanoribbon central zone. In a revised structural model, this central zone likely represents a longitudinal interface between two strands of beta-sheets, forming a steric-zipper by interactions of isoleucine and phenylalanine. Together, these results establish a framework in which pS16, C-terminal truncation, and ion-specific interactions cooperate to fine-tune amelogenin self-assembly and direct ribbon-like mineral formation in enamel.</p>