NMR Structural Characterization of a Minimal Peptide Antagonist Bound to the Extracellular Domain of the Corticotropin-releasing Factor1 Receptor

Abstract Natural peptide agonists of corticotrophin-releasing factor (CRF) receptors bind to the receptor by a two-site mechanism as follows: the carboxyl end of the ligand binds the N-terminal extracellular domain (ECD) of the receptor and the amino portion of the ligand binds the extracellular face of the seven transmembrane region. Recently, peptide antagonists homologous to the 12 C-terminal residues of CRF have been derived, which bind the CRF1 receptor through an interaction with the ECD. Here we characterized the binding of a minimal 12-residue peptide antagonist while bound to the isolated ECD of the CRF1 receptor. We have expressed and purified soluble and properly folded ECD independent from the seven-transmembrane region as a thioredoxin fusion protein in Escherichia coli. A model of the peptide antagonist, cyclic corticotrophin-releasing factor residues 30–41 (cCRF30–41), was calculated while bound to the recombinant ECD using transferred nuclear Overhauser effect spectroscopy. Although the peptide is unstructured in solution, it adopts an α-helical conformation when bound to the ECD. Residues of cCRF30–41 comprising the binding interface with the ECD were mapped using saturation transfer difference NMR. Two hydrophobic residues (Met38 and Ile41) as well as two amide groups (Asn34 and the C-terminal amide) on one face of the helix defined the binding epitope of the antagonist. This epitope may be used as a starting point for development of non-peptide antagonists targeting the ECD of this receptor.