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

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Abstract
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.