Residue-Specific Insights into the Intermolecular Protein-Protein Interfaces Driving Amelogenin Self-Assembly in Solution

Amelogenin, the dominant organic component (>90%) in the early stages of amelogenesis, orchestrates the mineralization of apatite crystals into enamel. The self-association properties of amelogenin as a function of pH and protein concentration have been suggested to play a central role in this process; however, the large molecular weight of the self-assembled quaternary structures has largely prevented structural studies of the protein in solution under physiological conditions using conven-tional approaches. Here, using perdeuterated murine amelogenin (0.25 mM, 5 mg/mL) and TROSY-based NMR experiments to improve spectral resolution, we assigned the 1H -15N spectra of murine amelogenin over a pH range (5.5 to 8.0) where amelogenin is reported to exist as oligomers (pH <=similar to 6.8) and nanospheres (pH >=similar to 7.2). The disappearance or intensity reduction of amide resonances in the 1H -15N HSQC spectra was interpreted to reflect changes in dynamics (intermediate millisecond-to-microsecond motion) and/or heterogenous interfaces of amide nuclei at protein- protein interfaces. The intermolecular interfaces were concentrated toward the N-terminus of amelogenin (L3-G8, V19-G38, L46 -Q49, and Q57-L70) at pH 6.6 (oligomers) and at pH 7.2 (nanospheres) including the entire N-terminus up to Q76 and regions distributed through the central hydrophobic region (Q82-Q101, S125-Q139, and F151-Q154). At all pH levels, the C-terminus appeared disordered, highly mobile, and not involved in self-assembly, suggesting nanosphere structures with solvent-exposed C -termini. These findings present unique, residue-specific insights into the intermolecular protein-protein interfaces driving amelogenin quaternary structure formation and suggest that nanospheres in solution predominantly contain disordered, solvent-exposed C-termini.

Automated summary

This study investigated the structure of Amelogenin under different pH conditions using NMR experiments, revealing that its self-assembly form changes with pH variation, with the N-terminus playing a key role in intermolecular interactions.