Experimental NOE, Chemical Shift, and Proline Isomerization Data Provide Detailed Insights into Amelotin Oligomerization

Amelotin
is an intrinsically disordered protein (IDP)
rich in Pro
residues and is involved in hydroxyapatite mineralization. It rapidly
oligomerizes under physiological conditions of pH and pressure but
reverts to its monomeric IDP state at elevated pressure. We identified
a 105-residue segment of the protein that becomes ordered upon oligomerization,
and we used pressure-jump NMR spectroscopy to measure long-range NOE
contacts that exist exclusively in the oligomeric NMR-invisible state.
The kinetics of oligomerization and dissociation were probed at the
residue-specific level, revealing that the oligomerization process
is initiated in the C-terminal half of the segment. Using pressure-jump
NMR, the degree of order in the oligomer at the sites of Pro residues
was probed by monitoring changes in cis/trans equilibria relative to the IDP state after long-term equilibration
under oligomerizing conditions. Whereas most Pro residues revert to trans in the oligomeric state, Pro-49 favors a cis configuration and three Pro residues retain an unchanged cis fraction, pointing to their local lack of order in the
oligomeric state. NOE contacts and secondary 13C chemical
shifts in the oligomeric state indicate the presence of an 11-residue
α-helix, preceded by a small intramolecular antiparallel β-sheet,
with slower formation of long-range intermolecular interactions to
N-terminal residues. Although none of the models generated by AlphaFold2
for the amelotin monomer was consistent with experimental data, subunits
of a hexamer generated by AlphaFold-Multimer satisfied intramolecular
NOE and chemical shift data and may provide a starting point for developing
atomic models for the oligomeric state.