Structure of the N-terminal domain of a type B1 G protein-coupled receptor in complex with a peptide ligand

doi:10.1073/pnas.0700682104

Structure of the N-terminal domain of a type B1 G protein-coupled receptor in complex with a peptide ligand

*Structural Biology Laboratory and
^†The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037

Contributed by Wylie W. Vale, January 29, 2007 (received for review January 5, 2007)

Abstract

The corticotropin releasing factor (CRF) family of ligands and their receptors coordinate endocrine, behavioral, autonomic, and metabolic responses to stress and play additional roles within the cardiovascular, gastrointestinal, and other systems. The actions of CRF and the related urocortins are mediated by activation of two receptors, CRF-R1 and CRF-R2, belonging to the B1 family of G protein-coupled receptors. The short-consensus-repeat fold (SCR) within the first extracellular domain (ECD1) of the CRF receptor(s) comprises the major ligand binding site and serves to dock a peptide ligand via its C-terminal segment, thus positioning the N-terminal segment to interact with the receptor's juxtamembrane domains to activate the receptor. Here we present the 3D NMR structure of ECD1 of CRF-R2β in complex with astressin, a peptide antagonist. In the structure of the complex the C-terminal segment of astressin forms an amphipathic helix, whose entire hydrophobic face interacts with the short-consensus-repeat motif, covering a large intermolecular interface. In addition, the complex is characterized by intermolecular hydrogen bonds and a salt bridge. These interactions are quantitatively weighted by an analysis of the effects on the full-length receptor affinities using an Ala scan of CRF. These structural studies identify the major determinants for CRF ligand specificity and selectivity and support a two-step model for receptor activation. Furthermore, because of a proposed conservation of the fold for both the ECD1s and ligands, this structure can serve as a model for ligand recognition for the entire B1 receptor family.

Footnotes

^‡To whom correspondence may be addressed. E-mail: vale{at}salk.edu or riek{at}salk.edu
Author contributions: C.R.R.G., M.H.P., M.R.D., J.P.C., and R.R. performed research; J.G. contributed new reagents/analytic tools; C.R.R.G., M.H.P., and R.R. analyzed data; and C.R.R.G., M.H.P., J.E.R., W.W.V., and R.R. wrote the paper.
Conflict of interest statement: W.W.V. is a cofounder, consultant, equity holder, and member of the Board of Directors of Neurocrine Biosciences and Acceleron Pharma. The following have been licensed by The Salk Institute for Biological Studies and/or The Clayton Foundation: CRF to Ferring Pharmaceuticals, CRF1 receptor and Ucn 2 to Neurocrine Biosciences, and Ucn 3 to Johnson & Johnson.
Data deposition: The 3D NMR structures of free ECD1–CRF-R2β and ECD1–CRF-R2β in complex with astressin have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 2JNC and 2JND, respectively).
This article contains supporting information online at www.pnas.org/cgi/content/full/0700682104/DC1.
Abbreviations:

CRF,

corticotropin releasing factor;

oCRF,

ovine CRF;

CRF-Rn,

CRF receptor n;

ECD,

extracellular domain;

GPCR,

G protein-coupled receptor;

Ucn,

urocortin;

SCR,

short-consensus-repeat.