Structural comparison of alanine-substituted analogues of the calcitonin gene-related peptide 8-37. Importance of the C-terminal segment for antagonistic activity.

Replacement of specific residues of the antagonistic fragment human calcitonin gene-related peptide 8-37 (hCGRP 8-37) by alanine residues produces good antagonists to CGRP1 receptors when the replacement is made 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 1H NMR spectroscopy and molecular modeling. Following the complete assignment 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 [Ala17]-hCGRP 8-37 and [Ala20]-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 [Ala21]-hCGRP 8-37, differences were observed all along the chain. Molecular modeling calculations were performed by distance geometry, simulated annealing and energy minimization using NOE distance constraints. Molecular models showed a structural homology between [Ala17]-hCGRP 8-37, [Ala20]-hCGRP 8-37 and hCGRP 8-37 in the C-terminal segment Asn31-Phe37 as well as hydrogen bonding between Val28 and Asn31. These structural similarities are not observed with [Ala21]-hCGRP 8-37. Therefore, the structure of the C-terminal segment of hCGRP 8-37 appears to be critical for antagonistic activity at CGRP1 receptors.