Intrinsically disordered proteins drive enamel formation via an evolutionarily conserved self-assembly motif

Abstract

The formation of mineralized tissues is governed by extracellular matrix proteins that assemble into a 3D organic matrix directing the deposition of hydroxyapatite. Although the formation of bones and dentin depends on the self-assembly of type I collagen via the Gly-X-Y motif, the molecular mechanism by which enamel matrix proteins (EMPs) assemble into the organic matrix remains poorly understood. Here we identified a Y/F-x-x-Y/L/F-x-Y/F motif, evolutionarily conserved from the first tetrapods to man, that is crucial for higher order structure self-assembly of the key intrinsically disordered EMPs, ameloblastin and amelogenin. Using targeted mutations in mice and high-resolution imaging, we show that impairment of ameloblastin self-assembly causes disorganization of the enamel organic matrix and yields enamel with disordered hydroxyapatite crystallites. These findings define a paradigm for the molecular mechanism by which the EMPs self-assemble into supramolecular structures and demonstrate that this process is crucial for organization of the organic matrix and formation of properly structured enamel.

Co-Immunoprecipitation Reveals Interactions Between Amelogenin and Ameloblastin via Their Self-Assembly Domains

“…AB2 was further divided into N-and C-terminal peptides peptide derived from N-terminus of AB2 (AB2N) and peptide derived from C-terminus of AB2 (AB2C; Chempeptide Ltd., China). A mutant of AB2N was designed by replacing the key amino acids in the YSRLGF motif with glycine, making it GSRGGG (Wald et al, 2017), which we refer to as peptide AB2N with mutation Abbreviations: AB2, Ambn-exon 5 derived peptide; AB2C, Peptide derived from C-terminus of AB2; AB2N, Peptide derived from N-terminus of AB2; AB2N-GGG, Peptide AB2N with mutation p.Y67G_L70G_F72G; AB4, Ambn-exon 6 derived peptide; AI, Amelogenesis imperfecta; Ambn/rAmbn, Ameloblastin/recombinant ameloblastin; AmbnΔ5, Ambn p.Y67_Q103del; AmbnΔ6, Ambn p.P104_V168del; Amel/rAmel, Amelogenin/recombinant amelogenin; Co-IP, Co-immunoprecipitation; EMP, Enamel matrix protein; MCC, Manders' colocalization coefficient; SDS-PAGE, Sodium dodecyl sulfate polyacrylamide gel electrophoresis; TRAP, Tyrosine rich amelogenin polypeptide. p.Y67G_L70G_F72G (AB2N-GGG; Biomertech, United States; Supplementary Figures 3, 4; Supplementary Table 1).…”

Xenohybrid Bone Graft Containing Intrinsically Disordered Proteins Shows Enhanced In Vitro Bone Formation

“…This study uses a new and rapidly popular protein family called "intrinsically disordered proteins" (IDPs). 23 IDPs have been shown to be involved in biomineralization, particularly through signaling and regulation of the direction and extend of mineral crystal growth. 24 It possesses high-specificity/lowaffinity interactions which play a crucial role and its unique structural feature enables the IDPs to participate in both oneto-many and many-to-one signaling.…”

Proteins in Calcium Phosphates Biomineralization

“…Full-length amelogenin undergoes hierarchical stepwise assembly, first forming oligomers, which in turn assemble into higher order structures and stabilize mineral prenucleation clusters, and organize them into parallel arrays of linear chains, yielding the formation of crystallite bundles [102]. There is a hypothesis that the higher order structures of the self-assembled ameloblastin (or most likely its N-terminal moiety) contribute to the oriented growth of the linear chains of amelogenin in the 3D space [104].…”