Structural Perturbation of Monomers Determines the Amyloid Aggregation Propensity of Calcitonin Variants.

Abstract

Human calcitonin (hCT) is a polypeptide hormone that participates in calcium-phosphorus metabolism. Irreversible aggregation of 32-amino acid hCT into β-sheet-rich amyloid fibrils impairs physiological activity and increases the risk of medullary carcinoma of the thyroid. Amyloid-resistant hCT derivatives substituting critical amyloidogenic residues are of particular interest for clinical applications as therapeutic drugs against bone-related diseases. Uncovering the aggregation mechanism of hCT at the molecular level, therefore, is important for the design of amyloid-resistant hCT analogues. Here, we investigated the aggregation dynamics of hCT, non-amyloidogenic salmon calcitonin (sCT), and two hCT analogues with reduced aggregation tendency─TL-hCT and phCT─using long timescale discrete molecular dynamics simulations. Our results showed that hCT monomers mainly adopted unstructured conformations with dynamically formed helices around the central region. hCT self-assembled into helix-rich oligomers first, followed by a conformational conversion into β-sheet-rich oligomers with β-sheets formed by residues 10-30 and stabilized by aromatic and hydrophobic interactions. Our simulations confirmed that TL-hCT and phCT oligomers featured more helices and fewer β-sheets than hCT. Substitution of central aromatic residues with leucine in TL-hCT and replacing C-terminal hydrophobic residue with hydrophilic amino acid in phCT only locally suppressed β-sheet propensities in the central region and C-terminus, respectively. Having mutations in both central and C-terminal regions, sCT monomers and dynamically formed oligomers predominantly adopted helices, confirming that both central aromatic and C-terminal hydrophobic residues played important roles in the fibrillization of hCT. We also observed the formation of β-barrel intermediates, postulated as the toxic oligomers in amyloidosis, for hCT but not for sCT. Our computational study depicts a complete picture of the aggregation dynamics of hCT and the effects of mutations. The design of next-generation amyloid-resistant hCT analogues should consider the impact on both amyloidogenic regions and also take into account the amplification of transient β-sheet population in monomers upon aggregation.