Role of aromatic residues in amyloid fibril formation of human calcitonin by solid-state 13C NMR and molecular dynamics simulation

Abstract

Calcitonin (CT) is an amyloid fibril forming peptide. Since salmon calcitonin (sCT), having Leu residues (Leu12, Leu16 or Leu19) instead of Tyr12, Phe16 or Phe19 for human calcitonin (hCT), is known to form the fibrils much slower than hCT, hCTs mutated to Leu residues at the position of 16 (F16L-hCT), 19 (F19L-hCT), and 12, 16 and 19 (TL-hCT) were examined to reveal the role of aromatic side-chains on amyloid fibrillation using solid-state (13)C NMR. The detailed kinetics were analyzed using a two-step reaction mechanism such as nucleation and fibril elongation with the rate constants of k1 and k2, respectively. The k2 values of hCT mutants were significantly slower than that of hCT at a neutral pH, although they were almost the same at an acidic pH. The (13)C chemical shifts of the labeled sites showed that the conformations of monomeric hCT mutants take α-helices as viewed from the Gly10 moiety. The hCT mutants formed fibrils and during the fibril formation, the α-helix around Gly10-Phe22 changed to the β-sheet, and the major structures around Ala26-Ala31 were random coil in the fibrils. Molecular dynamics simulation was performed for the β-sheet system of hCT9-23 and its mutants F16L-hCT9-23, F19L-hCT9-23 and TL-hCT9-23. In one of the stable fibril structures, Phe16 of hCT interacts with Phe19 of the next strand alternatively. In the hCT mutants, lack of Phe16 and Phe19 interaction causes significant instability as compared with the hCT fibril, leading to the reduction of k2 values, as observed experimentally in the hCT mutants at a neutral pH.

Nanostructure and stability of calcitonin amyloids

“…The β-sheet content stabilizes to 40.5 ± 1.8% for the parallel octamer, to 46.0 ± 2.4% for the antiparallel 1 model, and to 40.0 ± 1.9 for the antiparallel 2 model. The antiparallel 1 model thus presents the highest content of amino acid forming β-sheets, suggesting that this conformation is slightly more stable than the two other proposed models, in accordance with previous NMR results (13). To test the stability of each system we performed 100-ns long simulations at increasing temperatures (from 350 to 450 K), showing a decrease of the β-sheet content for all models (Fig.…”

“…On the basis of NMR studies, Naito and colleagues (13) proposed two antiparallel packing conformations that differ for displacement along the polypeptide axis. In addition to these two packing conformations, we tested the stability of the parallel arrangement, based on previous works (14), suggesting that DFNKF peptides assemble in parallel conformation (Table 1, models 1, 2, 3).…”

“…A similar distance has been used in the past to model assembly of DFNKF peptides (16). In the case of antiparallel octamers, every second monomer is rotated by 180° around the y axis and displaced according to NMR studies (13) (see also Table 1). We then minimize the structure while applying a position restrain to the backbone of residues from 12 to 32, to allow the relaxation of the disulfide bond between Cys 1 and Cys 7 .…”

“…The fact that DFNKF is strongly amyloidogenic (but its mutations are not) can explain the lower amyloidogenic properties of salmon calcitonin (sCT), the hormone used for the treatment of hypercalcemia or osteoporosis, which presents two leucine residues (Leu 16 and Leu 19 ) instead of Phe 16 and Phe 19 in hCT (Fig. 1) (13). …”

Synchrotron-Infrared Microscopy Analysis of Amyloid Fibrils Irradiated by Mid-Infrared Free-Electron Laser

“…In the case of the short peptide, however, FTIR spectroscopy could not determine the conformational changes in detail because the peptide has several conformations and a flexible structure in solution [9]. X-ray crystallography and nuclear magnetic resonance (NMR) are usually employed to determine the structure of peptides [15]- [17], but while these analytical methods are excellent for three-dimensional structural determination at the atomic level, they are also time consuming, and once analyzed, samples cannot be re-used for other analytical methods. In contrast, IR absorption spectral measurements are comparatively simple, and the spectra are sensitive to the secondary structures of the peptides [18].…”