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

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-R2beta 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.

Fig. 1.

3D NMR structure of ECD1–CRF-R2β in complex with astressin. (A) Superposition of 20 energy-minimized conformers representing the 3D NMR structure of free ECD1–CRF-R2β (the backbone C^α atoms of residues 57–83 and 99–120 were superimposed). (B) Superposition of 20 energy-minimized conformers of ECD1–CRF-R2β in complex with astressin (the backbone C^α atoms of residues 57–120 of ECD1 and 30–41 of astressin were superimposed). The backbone of residues 44–122 of ECD1 is shown in magenta, and the backbone of residues Leu-27–Ile-41 of astressin is colored in green. In A and B the disulfide bonds are shown in yellow. (C) Ribbon diagram of the lowest energy conformer representing the 3D NMR structure of the ECD–CRF-R2β–astressin complex. The β-sheets are shown in cyan, and the side chains of the core residues Trp-71 and Trp-109 along with the disulfide bonds are shown in yellow. The salt bridge Arg-101 (in blue)–Asp-65 (in red) is shown as dashed spine in orange. The backbone of astressin from Leu-27–Ile-41 is shown in green. (D) Side view of the ribbon diagram shown in C. MOLMOL was used to generate the figures (49).

Fig. 2.

Structural and dynamical characterization of the ECD–CRF-R2β–astressin complex. (A) Superposition of the 20 conformers of free ECD1–CRF-R2β depicted as a cyan sausage with the lowest energy conformer of the complex ECD1–CRF-R2β–astressin shown as a magenta ribbon. The disulfide bonds are shown in yellow. (B) [¹⁵N,¹H]-TROSY spectra of the free (in red contours) and complex (in black contours) ECD1–CRF-R2β. Residues enclosed in rectangular boxes are from loop 2 (residues 84–98). The peaks from free ECD1–CRF-R2β are missing for most of the residues in loop 2 because of slow dynamics. (C) Surface representation of ECD1–CRF-R2β showing the residues in pink, whose chemical shift perturbations are >0.5 ppm upon complex formation with astressin (see also SI Fig. 4C). Astressin is shown in green along with the side chains interacting with the ECD1. (D) Amino acid sequence differences of mouse CRF-R1 and CRF-R2 are mapped in purple onto the ECD1 surface of the complex structure. The residues are labeled with single letter code for CRF-R1, followed by CRF-R2. Astressin is shown as a green–yellow ribbon. Highlighted in yellow are the sequence differences of CRF ligands (see also SI Fig. 6).

Fig. 3.

Molecular anatomy of the residues in the interaction site between astressin and ECD1–CRF-R2β. Shown are front (A) and back (B) stereo views of the side chain interactions between the ECD1 and astressin. The backbones of astressin and ECD1 are shown as green and magenta ribbons, respectively. Hydrophobic side chains are shown in yellow, hydrogen bonds are shown in cyan, and the salt bridges Arg-35–Glu-86 and Lys-36–Glu-39 are shown in gray. All of the residues in the interaction surface are marked for clarity. “X” refers to norleucine residue (Nle).