Molecular Recognition of Corticotropin-releasing Factor by Its G-protein-coupled Receptor CRFR1

Abstract

The bimolecular interaction between corticotropin-releasing factor (CRF), a neuropeptide, and its type 1 receptor (CRFR1), a class B G-protein-coupled receptor (GPCR), is crucial for activation of the hypothalamic-pituitary-adrenal axis in response to stress, and has been a target of intense drug design for the treatment of anxiety, depression, and related disorders. As a class B GPCR, CRFR1 contains an N-terminal extracellular domain (ECD) that provides the primary ligand binding determinants. Here we present three crystal structures of the human CRFR1 ECD, one in a ligand-free form and two in distinct CRF-bound states. The CRFR1 ECD adopts the α-β-βα fold observed for other class B GPCR ECDs, but the N-terminal α-helix is significantly shorter and does not contact CRF. CRF adopts a continuous α-helix that docks in a hydrophobic surface of the ECD that is distinct from the peptide-binding site of other class B GPCRs, thereby providing a basis for the specificity of ligand recognition between CRFR1 and other class B GPCRs. The binding of CRF is accompanied by clamp-like conformational changes of two loops of the receptor that anchor the CRF C terminus, including the C-terminal amide group. These structural studies provide a molecular framework for understanding peptide binding and specificity by the CRF receptors as well as a template for designing potent and selective CRFR1 antagonists for therapeutic applications. The bimolecular interaction between corticotropin-releasing factor (CRF), a neuropeptide, and its type 1 receptor (CRFR1), a class B G-protein-coupled receptor (GPCR), is crucial for activation of the hypothalamic-pituitary-adrenal axis in response to stress, and has been a target of intense drug design for the treatment of anxiety, depression, and related disorders. As a class B GPCR, CRFR1 contains an N-terminal extracellular domain (ECD) that provides the primary ligand binding determinants. Here we present three crystal structures of the human CRFR1 ECD, one in a ligand-free form and two in distinct CRF-bound states. The CRFR1 ECD adopts the α-β-βα fold observed for other class B GPCR ECDs, but the N-terminal α-helix is significantly shorter and does not contact CRF. CRF adopts a continuous α-helix that docks in a hydrophobic surface of the ECD that is distinct from the peptide-binding site of other class B GPCRs, thereby providing a basis for the specificity of ligand recognition between CRFR1 and other class B GPCRs. The binding of CRF is accompanied by clamp-like conformational changes of two loops of the receptor that anchor the CRF C terminus, including the C-terminal amide group. These structural studies provide a molecular framework for understanding peptide binding and specificity by the CRF receptors as well as a template for designing potent and selective CRFR1 antagonists for therapeutic applications. Corticotropin-releasing factor (CRF) 3The abbreviations used are: CRF, corticotropin releasing factor; CRFR, CRF receptor; Ucn, urocortin; PTH, parathyroid hormone; PTH1R, PTH receptor type 1; GIP, glucose-dependent insulinotropic peptide; GIPR, GIP receptor; GLP1R, glucagon-like peptide 1 receptor; GPCR, G-protein-coupled receptor; PDB, Protein Data Bank; MBP, maltose-binding protein; ECD, extracellular domain; MOPS, 4-morpholinepropanesulfonic acid; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; SCR, short consensus repeat; MR, molecular replacement; PEG, polyethylene glycol; TLS, translation, libration, screw rotation. is a 41-amino acid, C-terminally amidated neuropeptide originally isolated from sheep hypothalami based on its ability to stimulate secretion of adrenocorticotropin from pituitary cells (1Vale W. Spiess J. Rivier C. Rivier J. Science. 1981; 213: 1394-1397Crossref PubMed Scopus (4080) Google Scholar). Several other CRF-related peptides have since been identified, including the urocortins (Ucn) I, II, an