Amelogenin proteolysis orchestrates functional amyloid pathways in enamel development

Amelogenin, the most abundant protein in developing enamel, self-assembles into supramolecular structures that serve as templates for apatite growth. Recent studies revealed that amelogenin nanoribbons exhibit hallmark features of functional amyloids, yet the molecular mechanisms governing their formation remain incompletely understood. Here, we combine atomic force microscopy, transmission electron microscopy, and spectroscopic analyses to define the assembly pathways of full-length amelogenin (rH174) alongside its major proteolytic products generated by metalloproteinase-20 (MMP20). We demonstrate that both rH174 and the C-terminally truncated rH146 follow a nucleated conformational conversion mechanism, progressing from spherical oligomers through proto-ribbons to ordered β-sheet-rich nanoribbons. rH174 assembly progresses slowly, displaying an extended lag phase and delayed maturation, whereas rH146 nucleates rapidly, completing these stages within a shorter timeframe. Cross-seeding of rH146 into rH174 monomers (1:10) eliminates the delay in rH174 assembly, rapidly driving the system into elongation and leading to an earlier stabilization of the assembly system. C-terminus-driven interactions in rH174 trigger secondary nucleation that evolves into bundled nanoribbons resembling enamel organization, a process largely absent in rH146. Cross-seeding, therefore, exemplifies the in vivo mechanism whereby nascent amelogenin is immediately added to existing nanoribbon scaffolds, a cooperative strategy that generates a heterogeneous matrix, coupling the ability of rapid nucleation and spatial organization. Unexpectedly, the MMP20 cleavage product - TRAP, which comprises the cross-beta assembly domain, does not form nanoribbons and diverts from the assembly pathway full-length amelogenin takes when hydrolyzed at the C-terminal. Hence, a MMP20-driven mechanism exists that could contribute to an enamel matrix that acts as a spacer and prevents early crystal fusion during the secretory stage of amelogenesis. These findings offer insights into a proteolysis-triggered assembly pathway that may reconcile long-standing supramolecular models of amelogenin and establish amelogenin as a vertebrate example of a functional amyloid that can be tuned to enable ordered enamel biomineralization.