Mineral-binding region predicted and demonstrated within exon 6ABC of Amelogenin

Objectives:

To recapitulate enamel in regenerative medicine, we must understand the mechanisms of X-chromosomal Amelogenin (AMELX), the most abundant protein in enamel during all stages of the mineral development. We implement contemporary computational biology approaches, piece together existing in vitro and in vivo evidence to formulate a comprehensive model of AMELX structure and function, and dissect AMELX into functional regions.

Methods:

To ascertain quantitative importance of every amino acid to AMELX function, we assess evolutionary conservation and distinction using evolutionary trees built with multiple sequence alignments for each residue. We predict hydroxyapatite-binding regions using sequence similarity scoring matrices previously derived from a set of hydroxyapatite-binding peptides generated by combinatorial biology techniques (phage display). To determine calcium and phosphate ion binding regions we apply spatial and electrostatic assessments to putative AMELX protein structures predicted by us. We decipher structural importance for each amino acid by applying knowledge-based scoring functions to virtual mutant structures.

We performed hydroxyapatite biomineralization experiments in the presence of peptide constructs comprised by two regions predicted for hydroxyapatite-binding and an intervening stabilization region (ADP7), a previously designed peptide optimized for hydroxyapatite-binding (HABP1), no peptide control, and other AMELX-derived constructs including Exon-4 (ADP5) and the C-terminal region previously thought to control mineralization (ADP3).

Results:

Novel functional regions emerge from predictive and reviewed data, including two hydroxyapatite-binding regions, one novel AMELX-AMELX interaction region, and two regions effecting structural stabilization, each interspersed between mineralization and interaction regions, respectively, creating specific spatial approximation for pairs of regions with similar function.

ADP7 mineralization dynamics and final crystal morphology closely mimicked those of HABP1. ADP5 induced rapid dynamics and crystal seeding. Other peptides including ADP3 mimicked no peptide control.

Conclusions:

Our predictions led directly to demonstration of AMELX region responsible for hydroxyapatite-binding, and serendipitously, crystal seeding.

Funded by NIDCR/NIH F30DE017522 and NSF-MRSEC UW-GEMSEC.