Turn Structures in CGRP C-Terminal Analogues Promote Stable Arrangements of Key Residue Side Chains

• Published in: Biochemistry (Easton) 2001-07, Vol.40 (28), p.8317-8325
• Main Authors: Carpenter, Katharine A, Schmidt, Ralf, von Mentzer, Bengt, Haglund, Ulla, Roberts, Edward, Walpole, Chris
• Format: Article
• Language: English
• Subjects: Alanine - metabolism; Amides; Amino Acid Sequence; Amino Acid Substitution; Amino Acids - chemistry; Amino Acids - metabolism; Calcitonin Gene-Related Peptide - chemistry; Calcitonin Gene-Related Peptide - metabolism; Humans; MEDICIN; MEDICINE; Models, Molecular; Molecular Sequence Data; Nuclear Magnetic Resonance, Biomolecular - methods; Peptide Fragments - chemical synthesis; Peptide Fragments - chemistry; Peptide Fragments - metabolism; Protein Binding; Protein Conformation; Protein Structure, Secondary; Protons; Receptors, Calcitonin Gene-Related Peptide - metabolism; Spectroscopy, Fourier Transform Infrared; Structure-Activity Relationship; Temperature; Tumor Cells, Cultured
• ISSN: 0006-2960; 1520-4995; 1520-4995
• DOI: 10.1021/bi0102860

The 37-amino acid calcitonin gene-related peptide (CGRP) is a potent endogenous vasodilator thought to be implicated in the genesis of migraine attack. CGRP antagonists may thus have therapeutic value for the treatment of migraine. The CGRP C-terminally derived peptide [D31,P34,F35]CGRP(27−37)-NH2 was recently identified as a high-affinity hCGRP1 receptor selective antagonist. Reasonable CGRP1 affinity has also been demonstrated for several related analogues, including [D31,A34,F35]CGRP(27−37)-NH2. In the study presented here, conformational and structural features in CGRP(27−37)-NH2 analogues that are important for hCGRP1 receptor binding were explored. Structure−activity studies carried out on [D31,P34,F35]CGRP(27−37)-NH2 resulted in [D31,P34,F35]CGRP(30−37)-NH2, the shortest reported CGRP C-terminal peptide analogue exhibiting reasonable hCGRP1 receptor affinity (K i = 29.6 nM). Further removal of T30 from the peptide's N-terminus greatly reduced receptor affinity from the nanomolar to micromolar range. Additional residues deemed critical for hCGRP1 receptor binding were identified from an alanine scan of [A34,F35]CGRP(28−37)-NH2 and included V32 and F37. Replacement of the C-terminal amide in this same peptide with a carboxyl, furthermore, resulted in a greater than 50-fold reduction in hCGRP1 affinity, thus suggesting a direct role for the amide moiety in receptor binding. The conformational properties of two classes of CGRP(27−37)-NH2 peptides, [D31,X34,F35]CGRP(27−37)-NH2 (X is A or P), were examined by NMR spectroscopy and molecular modeling. A β-turn centered on P29 was a notable feature consistently observed among active peptides in both series. This turn led to exposure of the critical T30 residue to the surrounding environment. Peptides in the A34 series were additionally characterized by a stable C-terminal helical turn that resulted in the three important residues (T30, V32, and F37) adopting consistent interspatial positions with respect to one another. Peptides in the P34 series were comparatively more flexible at the C-terminus, although a large proportion of the [D31,P34,F35]CGRP(27−37)-NH2 calculated conformers contained a γ-turn centered on P34. These results collectively suggest that turn structures at both the C-terminus and N-terminus of CGRP(27−37)-NH2 analogues may help to appropriately orient critical residues (T30, V32, and F37) for hCGRP1 receptor binding.