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J. Biol. Chem., Vol. 282, Issue 9, 6338-6346, March 2, 2007

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

Michael F. Mesleh, William A. Shirley, Christopher E. Heise, Nicholas Ling^¶, Richard A. Maki^||1, and Richard P. Laura^||

From the Departments of Medicinal Chemistry, Pharmacology, ^¶Biological Chemistry, and ^||Discovery Biology, Neurocrine Biosciences, Inc., San Diego, California 92130

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 (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 -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 (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.

Received for publication, October 18, 2006 , and in revised form, December 6, 2006.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains a detailed description of the protocol used for the purification of the trx-ECD protein, the ¹³C-edited NMR data used for assignment and epitope mapping, an alignment of the amino acid sequences of the larger family of CRF-related peptides, supplemental Figs. 1–3, and additional Refs 1–5.

¹ To whom correspondence should be addressed: Dept. of Discovery Biology, Neurocrine Biosciences, 12790 El Camino Real, San Diego, CA 92130. Tel.: 858-617-7681; Fax: 858-617-7696; E-mail: rmaki{at}neurocrine.com.

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