Essence of Small Molecule-Mediated Control of Hydroxyapatite Growth: Free Energy Calculations of Amino Acid Side Chain Analogues

Much effort has been exerted to unravel the bone biomineralization mechanism by which the growth of plate-shaped crystals of hydroxyapatite (HAP) is regulated within a fibrillar collagen matrix. Acidic noncollagenous acidic proteins (NCPs) bearing a large number of carboxylate and phosphorylate amino acid residues are effective regulators of HAP crystal morphology. To reveal the energetic and structural essence of the growth-regulation mechanism, we performed here advanced molecular dynamics simulations to obtain adsorption free energies of side chain analogues (SCAs) of acidic and uncharged polar amino acids as well as the corresponding zwitterionic backbone (BB) fragment and compared the results to those for the entire amino acid molecule. We observed that negatively charged SCAs (phosphoserine in two protonation states, aspartate and glutamate) and the zwitterionic BB preferentially bind to the HAP (100) surface compared to the (001) face, consistent with [100] being the preferred growth direction for plate-shaped HAP crystals. Charged SCAs and zwitterionic BB bind via the formation of salt bridges between the −COO– group and Ca²⁺ ions on the surface, or hydrogen bonds between the −NH₃⁺ and surface PO₄³– ions, and adsorption of uncharged, polar serine SCA was thermodynamically unfavorable. Intriguingly, however, binding free energies depend on the number of charged groups rather than simply on net charge. Thus, zwitterionic (net neutral) BB adsorbed more strongly than monovalent Asp and Glu or uncharged Ser SCAs. Second, it was more difficult for larger molecules to adsorb presumably because of less favorable enthalpic and entropic contributions required to penetrate the tightly bound surface water layer. Accordingly, Asp adsorbed more strongly than homoionic Glu, and the sum of the SCA and BB binding affinities was greater than that of the entire amino acid molecule. The present results shed light on some previously unidentified subtle effects that the number of charged motifs rather than net charge and molecule size control a delicate balance of molecule–surface and water–surface interactions. These results should help understand the mechanisms of NCP-mediated HAP crystal growth in bone biomineralization and have broad implications for the design of new peptides and small molecules for biomimetic crystal synthesis.