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

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 ß-sheet, and the major structures around Ala26-Ala31 were random coil in the fibrils. Molecular dynamics simulation was performed for the ß-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.

Structural diversity of amyloid fibril formed in human calcitonin as revealed by site-directed 13C solid-state NMR spectroscopy.

Fibril formation in human calcitonin (hCT) from aqueous solution at pH 4.1 was examined and compared with those at pH 3.3 and 7.5 corresponding to three different net charges by means of site-directed (13)C solid-state NMR spectroscopy. Notably, the observed (13)C chemical shifts and lineshapes of the (13)C CP/MAS spectra differed substantially among fibrils prepared at different pHs. It was found that antiparallel beta-sheet structures were formed at pH 7.5 and 4.1 in the central core regions. In the C-terminal region, random coils were formed at both pH 7.5 and 4.1, although the random coil region at pH 4.1 was larger than that at pH 7.5. Fibrillation kinetics analyzed by a two-step autocatalytic reaction mechanism showed that the rate constants k(1) and k(2) for nucleation and maturation reactions of the fibril formation, respectively, were separately determined and the values correlated well with the net positive charges of Lys(18) and His(20) rather than the existence of a negative charge of Asp(15). Further, an attempt was made to assess interatomic distances between amide nitrogen and carbonyl carbon of neighboring chains of (13)C, (15)N-labeled hCT and a model pentapeptide by (13)C REDOR measurements by taking into account its dipolar interaction analyzed by the 3 spin system proposed previously. A unique chain packing of the antiparallel beta-sheets was proposed as a dominant fibril structure, although the possibility of a contribution of chain packing consisting of sliding one or two residues perpendicular to the fibril direction cannot be ruled out. In addition, it appears that the phenyl rings of Phe(16) are aligned on the same side of the beta-sheet and make the beta-sheet stable by forming pi-pi interactions between the beta-strands.