STRUCTURAL COMPARISON OF ALANINE-SUBSTITUTED ANALOGS OF THE CALCITONIN-GENE-RELATED PEPTIDE-8-37

Replacement of specific residues of the antagonistic fragment human ca lcitonin gene-related peptide 8-37 (hCGRP 8-37) by alanine residues pr oduces good antagonists to CGRP1 receptors when the replacement is mad e at positions 17 and 20 but a poor antagonist when the replacement is made at position 21. The solution structures of hCGRP 8-37 and of the three alanine analogues have been determined by two-dimensional H-1 N MR spectroscopy and molecular modeling. Following the complete assignm ent of the NMR spectra, a comparison of the chemical shifts and of the temperature dependence of the amide chemical shifts showed that these parameters differed for [Ala(17)]-hCGRP 8-37 and [Ala(20)]-hCGRP 8-37 relative to hCGRP 8-37 in the N-terminal and central segments but not in the C-terminal segment (residues 31-37). In the case of [Ala(21)]- hCGRP 8-37, differences were observed all along the chain. Molecular m odeling calculations were performed by distance geometry, simulated an nealing and energy minimization using NOE distance constraints. Molecu lar models showed a structural homology between [Ala(17)]-hCGRP 8-37, [Ala(20)]-hCGRP 8-37 and hCGRP 8-37 in the C-terminal segment Asn(31)- Phe(37) as well as hydrogen. bonding between Val(28) and Asn(31). Thes e structural similarities are not observed with [Ala(21)]-hCGRP 8-37. Therefore, the structure of the C-terminal segment of hCGRP 8-37 appea rs to be critical for antagonistic activity at CGRP1 receptors.