NMR characterization of monomeric and oligomeric conformations of human calcitonin and its interaction with EGCG.
Calcitonin is a 32-residue peptide hormone known for its hypocalcemic effect and its inhibition of bone resorption. While calcitonin has been used in therapy for osteoporosis and Paget's disease for decades, human calcitonin (hCT) forms fibrils in aqueous solution that limit its therapeutic application. The molecular mechanism of fiber formation by calcitonin is not well understood. Here, high-resolution structures of hCT at concentrations of 0.3 mM and 1 mM have been investigated using NMR spectroscopy. Comparing the structures of hCT at different concentrations, we discovered that the peptide undergoes a conformational transition from an extended to a β-hairpin structure in the process of molecular association. This conformational transition locates the aromatic side chains of Tyr12 and Phe16 in a favorable way for intermolecular π-π stacking, which is proposed to be a crucial interaction for peptide association and fibrillation. One-dimensional (1)H NMR experiments confirm that oligomerization of hCT accompanies the conformational transition at 1 mM concentration. The effect of the polyphenol epigallocatechin 3-gallate (EGCG) on hCT fibrillation was also investigated by NMR and electron microscopy, which show that EGCG efficiently inhibits fibril formation of hCT by preventing the initial association of hCT before fiber formation. The NMR experiments also indicate that the interaction between aromatic rings of EGCG and the aromatic side chains of the peptide may play an important role in inhibiting fibril formation of hCT.
Biophysics and Department of Chemistry, University of Michigan, Ann Arbor, MI 48109-1055, USA.
Rui
R
Huang
Subramanian
S
Vivekanandan
Jeffrey R
JR
Brender
Yuki
Y
Abe
Akira
A
Naito
Ayyalusamy
A
Ramamoorthy
D000595:Amino Acid Sequence; D002116:Calcitonin / Q000737:chemistry* / Q000378:metabolism*; D002392:Catechin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008854:Microscopy, Electron / Q000379:methods; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D011485:Protein Binding; D017433:Protein Structure, Secondary
D002392:Catechin; D002116:Calcitonin; C045651:epigallocatechin gallate