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.

Figure 1.

Conformational dynamics of each monomeric calcitonin peptide. Time evolution of the secondary structure of each residue from hCT (a), TL-hCT (b), phCT (c), and sCT (d) peptide in the monomeric peptide simulation is shown on the upper panel. Transient ordered conformations formed along the simulation (the time-stamped blew) are also presented. For each system, one 500 ns DMD trajectory is randomly selected out of thirty independent simulations. The N-terminal Cα atom is highlighted by a sphere for clarity.

Figure 2.

Secondary structure analysis for each calcitonin monomer. (a) Average secondary structure propensity in the monomers of each calcitonin. (b) Probability of each residue adopting different secondary structures. The mutation sites in TL-hCT and phCT are labeled in orange and purple. (c) The frequency of intra-molecular residue-pairwise contact formed between atoms from main-chain (upper diagonal) and side-chain (lower diagonal). The representative structured contact patterns and their corresponding structures (selected according to the contact frequency) are labeled and presented as 1–5. (d) The residue-pairwise contact frequency differences of TL-hCT, phCT, and sCT with respect to the wild-type hCT are calculated by subtracting each corresponding residue-pairwise contact propensity of hCT from the hCT derivatives. Only the last 200 ns data from 30 independent 500 ns DMD simulations are used for the above analysis. The error bars of secondary structure propensities correspond to the standard deviations of means from 30 independent simulations.

Figure 3.

Oligomerization dynamics and conformational changes of each calcitonin variant. The time evolution of secondary structure per residue (a, d, g, j), and the oligomer size into which a peptide aggregated (b, e, h, k) are shown for representative trajectories. Four snapshots with time stamps below (c, f, i, l) are also shown to illustrate transient conformations. For each system, the trajectory is randomly selected out of 30 independent DMD simulations.

Figure 4.

Secondary structure analysis for calcitonin oligomers. (a) The secondary structure contents in oligomers formed by hCT, TL-hCT, phCT, and sCT. (b) Probability of each residue adopting different secondary structures. The mutation sites in TL-hCT and phCT are labeled in orange and purple. The averaging was calculated over all 30 independent simulations during the last 300 ns. The error bars of secondary structure propensities correspond to the standard deviations of means from 30 independent simulations. (c) Simulation trajectories are sorted according to the β-sheet content from high to low, and the corresponding trajectories’ helical content is shown below. (d) Snapshot structures with high β-sheet contents from the top ranked trajectories are also shown.

Figure 5.

The population of β-barrel oligomers. (a) Probability of β-barrel oligomers observed in the 30 independent DMD trajectories for each calcitonin. (b) One representative β-barrel structure formed by hCT and TL-hCT is also presented in two different views (side and top).

Figure 6.

Inter-residue interactions driving the self-assembly of calcitonin variants. The intra-chain (lower diagonal) and inter-chain (upper diagonal) residue-wise contact frequencies of main-chain (left column) and side-chain (right column) atoms for the aggregates of (a) hCT, (b) TL-hCT, (c) phCT, and (d) sCT. The average number of inter-chain (topper) and intra-chain (bottom) contacts per residue formed by atoms from the main-chain (left panel) and side-chain (right panel) are also computed for each molecular system. For each molecular system, two representative aggregate structures selected according to contact frequency are also presented with N-terminal Cα atoms highlighted by spheres. For clarity, residues 1–11, 12–22, and 22–32 are colored from light, to moderate and deep, respectively.

Figure 7.

Conformational free energy landscape analysis. The conformational free energy landscape as a function of the helix and β-sheet contents for the self-assemblies aggregated by hCT (a), TL-hCT (b), phCT (c), and sCT (d) peptides. For each molecular system, four representative structures with the helix and β-sheet contents featuring low free energy (labelled as 1–4 on the free energy landscape surface) are also presented. The N-terminal Cα atoms are highlighted as a sphere for clarity. The probability distribution as a function of average helix content (e) and the β-sheet ratio of the whole peptide (f), as well as the β-sheet ratio around the central helical region (residues 12–22) (g) and C-terminus (residues 23–32) (h) in each calcitonin variants oligomer are also present.

Figure 8.

Proposed mechanism of hCT forming β-sheet rich aggregates. (a) The hCT monomers are dynamic and mainly adopted unstructured conformations with partial helix and transient β-sheet structures. Oligomers of hCT are abundant in diverse β-sheets formed by the central region and C-terminus of hCT. The interactions among aromatic and hydrophobic residues from the central region and C-terminus stabilize β-sheets and promoted the helix-to-sheet transition. (b) The Y12L, F16L, and F19L substitutions enhance the helix propensity and decrease the helix-to-sheet conformational conversion, because the intrinsic helical tendency of leucine amino acid is stronger than aromatic tyrosine and phenylalanine. Oligomeric β-sheets of TL-hCT are mainly formed by C-terminal residues. (c) The Y12L, N17H, A26N, I27T, and A31T replacements disrupt the interaction between the central region and C-terminus that stabilize transient β-sheet in monomers and protect the central helix of phCT monomer forming conformational convert. The β-sheet formations of phCT oligomers mainly span central region stabilized by interactions among aromatic residues. The C-terminal tail of phCT mostly adopts unstructured conformation.