title: 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 CRF(1) 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 CRF(1) 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 (cCRF(30-41)), was calculated while bound to the recombinant ECD using transferred nuclear Overhauser effect spectroscopy. Although the peptide is unstructured in solution, it adopts an alpha-helical conformation when bound to the ECD. Residues of cCRF(30-41) comprising the binding interface with the ECD were mapped using saturation transfer difference NMR. Two hydrophobic residues (Met(38) and Ile(41)) as well as two amide groups (Asn(34) 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.
authors.affiliation: Department of Medicinal Chemistry, Neurocrine Biosciences, Inc., San Diego, California 92130, USA.
authors.forename: Michael F
authors.initials: MF
authors.lastname: Mesleh
authors.forename: William A
authors.initials: WA
authors.lastname: Shirley
authors.forename: Christopher E
authors.initials: CE
authors.lastname: Heise
authors.forename: Nicholas
authors.initials: N
authors.lastname: Ling
authors.forename: Richard A
authors.initials: RA
authors.lastname: Maki
authors.forename: Richard P
authors.initials: RP
authors.lastname: Laura
mesh_terms: D000596:Amino Acids; D001665:Binding Sites; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D010446:Peptide Fragments / Q000494:pharmacology; D010455:Peptides / Q000737:chemistry / Q000494:pharmacology*; D011485:Protein Binding; D011487:Protein Conformation; D018019:Receptors, Corticotropin-Releasing Hormone / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D000099052:CRF Receptor, Type 1
chemical_list: D000596:Amino Acids; D010446:Peptide Fragments; D010455:Peptides; D018019:Receptors, Corticotropin-Releasing Hormone; D000099052:CRF Receptor, Type 1