Crystal structure of the PAC1R extracellular domain unifies a consensus fold for hormone recognition by class B G-protein coupled receptors

Abstract

Pituitary adenylate cyclase activating polypeptide (PACAP) is a member of the PACAP/glucagon family of peptide hormones, which controls many physiological functions in the immune, nervous, endocrine, and muscular systems. It activates adenylate cyclase by binding to its receptor, PAC1R, a member of class B G-protein coupled receptors (GPCR). Crystal structures of a number of Class B GPCR extracellular domains (ECD) bound to their respective peptide hormones have revealed a consensus mechanism of hormone binding. However, the mechanism of how PACAP binds to its receptor remains controversial as an NMR structure of the PAC1R ECD/PACAP complex reveals a different topology of the ECD and a distinct mode of ligand recognition. Here we report a 1.9 Å crystal structure of the PAC1R ECD, which adopts the same fold as commonly observed for other members of Class B GPCR. Binding studies and cell-based assays with alanine-scanned peptides and mutated receptor support a model that PAC1R uses the same conserved fold of Class B GPCR ECD for PACAP binding, thus unifying the consensus mechanism of hormone binding for this family of receptors.

Figure 1. Biophysical characterization of PAC1R.

(a) Non-reducing native PAGE of PAC1R. MBP-PAC1R gives a smear at the initial steps but attains a homogenous conformation after refolding. DsbC, which appears as an extra band after refolding, was removed by Ni Column-2 purification. (b) Competitive Alphascreen with unlabelled PACAP as competitor for binding of 40 nM biotin-PACAP (6–38)-NH2 and 40 nM MBP-PAC1R (25–140)-His6 in the presence of 5 µg/ml beads. Dashed, dotted and solid curves represent un-labeled PACAP (8–38), PACAP (12–27) and PACAP (15–31) as competitors, respectively. (c) Binding affinities of different Ala scanning mutants of PACAP (15–31). K20A′ mutation completely abrogates PACAP binding while L27A′ and V19A′ also seem to play important roles in the binding. PACAP residues 18, 24, 25 are Ala and 28 is Gly and, therefore, were not mutated to another residue.

Figure 2. Structure of PAC1R.

(a)(i) The ribbon diagram of the PAC1R ECD. Strands β1 and β2 make an anti-parallel β-sheet and strands β3, β4 and β5 make another anti-parallel β-sheet. (b) The stereo view of the electron density around the conserved residues that form the core of the PAC1R structure. The density is contoured at the 2σ level. (c) Stereo view of the electron density for the C77–C113 disulphide bond and the residues around it contoured at 1 σ. The residues are as labelled. The electron density obtained for the β3–β4 loop is clean allowing even the side chains to be discerned clearly.

Figure 3. Superimposition of the backbone of the ECD of class B members.

(a) Superimposition of the backbone of the ECD of class B members GIP1R (black, PDB code:2QKH), GLP1R (red, PDB code:3C5T), PAC1R (green, PDB code:3N94) and VIP2R (blue, PDB code:2X57). The disulphide bridges C34–C63, C54–C97 and C77–C113 are labelled. (b) superimposition of conserved residues. The color scheme is as in panel A. (c) Ligand orientation in class B GPCRs. The ligands incretin of GIP1R, glucagon of GLP1R and PTH of PTH1R are shown in black, orangeand yellow, respectively. For simplicity the surface diagram of PAC1R ECD alone is shown in white with the trace of PAC1R in green. PACAP from the NMR structure is shown in magenta. While all the class B ligands follow the same binding site and orientation, PACAP of the NMR structure is shown to bind PAC1R at a different location. Furthermore, its polarity does not follow that of the other ligands.

Figure 4. Superimposition of PAC1R/VIP2R and PAC1R X-ray and NMR structures.

(a) the C-terminal portion of PAC1R (green) and VIP2R (blue) depicts the expected similarity in (i) the backbone and (ii) the position of the conserved residues. (b)(i) Superimposition of the backbone of the X-ray (green) and NMR (magenta) structures of the PAC1R ECD. The two molecules were aligned using Pymol and laterally separated (ii) the close up of selected residues in the two structures. Note the unexpected dissimilarity in the position of the conserved residues and near the C77–C118 disulphide linkage, which would warrant its disruption to allow such massive displacement in solution. Furthermore, the region around Pro78 is very different between the X-ray and NMR structures. However, this region of the X-ray PAC1R structure and the VIP2R structure superimposes well and shows the expected conformational similarity.

Figure 5. PAC1R and PACAP interaction model.

(a) (i) The surface charge distribution of PAC1R is depicted with red and blue potentials, ranging from −10 KeT to +10 KeT. The potentials were calculated using APBS . PACAP is shown in cyan. Our docking result correlates well with other published class B GPCR ECD:ligand complex structures. (ii) The polarity of the N-terminal α-helix of PAC1R and the PACAP is in the same direction. Following other structures, PACAP residues 1–8 are not expected to make any contact with the ECD of PAC1R and hence not included in our docking study. (b) A close-up view of the interaction. The residues that are likely to make important contacts are shown as sticks and are labeled. K20′ and E104 form a salt bridge with a distance of 2.6 Å. (c) (i) Alphascreen of mutations in PAC1R affecting the binding to PACAP. All the mutations were made on the outer surface of the receptor so that the structural core of PAC1R is unaffected. The mutants and the wildtype receptor ECDs were assayed for interaction with biotin-PACAP(6–38) at increasing equimolar concentration to enable the reading of the assay to reach saturation. (ii) The location of the mutated residues on the surface of PAC1R ECD. ECD is coloured in green while the side chains of the mutated residues are shown as blue sticks.

Figure 6. Stimulation of cAMP signaling by PACAP38-MBP.

AD293 cells transiently transfected with PAC1R were stimulated with PACAP38-MBP for 4 hrs at 37°C after which the cells were lysed and assayed for cAMP content. All data points are average of duplicate samples and normalized to the stimulation level of wild-type receptor and peptide. (a) 0.03 nM PACAP38-MBP mutants were used to stimulate AD293 cells transiently expressing full length PAC1R-wt. (b) 0.03 nM PACAP38-MBP wt was used to stimulate cells transiently expressing PAC1R mutants.