Hierarchical self-assembly of amelogenin and the regulation of biomineralization at the nanoscale

Abstract

Enamel is a highly organized hierarchical nanocomposite, which consists of parallel arrays of elongated apatitic crystallites forming an intricate three-dimensional microstructure. Amelogenin, the major extracellular matrix protein of dental enamel, regulates the formation of these crystalline arrays via cooperative interactions with forming mineral phase. Using cryoelectron microscopy, we demonstrate that amelogenin undergoes stepwise hierarchical self-assembly. Furthermore, our results indicate that interactions between amelogenin hydrophilic C-terminal telopeptides are essential for oligomer formation and for subsequent steps of hierarchical self-assembly. We further show that amelogenin assemblies stabilize mineral prenucleation clusters and guide their arrangement into linear chains that organize as parallel arrays. The prenucleation clusters subsequently fuse together to form needle-shaped mineral particles, leading to the formation of bundles of crystallites, the hallmark structural organization of the forming enamel at the nanoscale. These findings provide unique insight into the regulation of biological mineralization by specialized macromolecules and an inspiration for bottom-up strategies for the materials design.

Fig. 1.

Cryo-EM micrographs of rM179 after 1 (A) and 10 min (B) in PBS at pH 8.0. Different classes of rM179 assemblies found in the sample after 1 min of incubation: (C) monomers, (D–F) oligomers of different sizes, (G) pairs of oligomers. After 10 min, (H) nanospheres

Fig. 2.

Single-particle reconstruction models of rM179 monomers (A), rM166 monomers (B), and rM179 dodecamers (C). Asterisks indicate the extension (C telopeptide) in monomers and dodecamers. Green shading roughly outlines the contour of one monomer in the dodecamer. (D) Models of rM179 self-assembly and rM166 aggregation into nanospheres.

Fig. 3.

Cryo-EM micrographs of calcium phosphate mineralization in the presence of full-length amelogenin rM179. (A, D, and E) 10 min in the reaction; (B, F, and G) 30 min in the reaction; and (C, H, and I) 120 min in the reaction. (C, Inset) Isolated mineral prenucleation clusters. Arrowheads in D–I point to individual prenucleation clusters.

Fig. 4.

A scheme depicting the proposed mechanism by which the amelogenin assemblies manipulate the arrangement of prenucleation clusters into organized mesostructures. (Phase 1) Mixing of mineral ions (calcium, green; phosphate, red) and the full-length amelogenin monomers. (Phase 2) At physiological pH values, amelogenin oligomers and mineral prenucleation clusters are formed. (Phase 3) Amelogenin assemblies and prenucleation clusters then assemble into the proposed composite structures. (Phase 4) Protein-mineral nanoparticles assemble into linear chains. This latter process will require some rearrangement of the amelogenin assemblies. (Phase 5) The linear protein-mineral nanoparticle chains organize into parallel arrays. (Phase 6) Prenucleation clusters fuse, leading to the formation of bundles of elongated crystallites of secretory enamel.