Cryo-EM structure of an activated VIP1 receptor-G protein complex revealed by a NanoBiT tethering strategy

Abstract

Vasoactive intestinal polypeptide receptor (VIP1R) is a widely expressed class B G protein-coupled receptor and a drug target for the treatment of neuronal, metabolic, and inflammatory diseases. However, our understanding of its mechanism of action and the potential of drug discovery targeting this receptor is limited by the lack of structural information of VIP1R. Here we report a cryo-electron microscopy structure of human VIP1R bound to PACAP27 and Gs heterotrimer, whose complex assembly is stabilized by a NanoBiT tethering strategy. Comparison with other class B GPCR structures reveals that PACAP27 engages VIP1R with its N-terminus inserting into the ligand binding pocket at the transmembrane bundle of the receptor, which subsequently couples to the G protein in a receptor-specific manner. This structure has provided insights into the molecular basis of PACAP27 binding and VIP receptor activation. The methodology of the NanoBiT tethering may help to provide structural information of unstable complexes.

Fig. 1. The NanoBiT strategy for stabilization of PACAP27–VIP1R–Gs protein complex.

a Schematic diagram of the NanoBiT aided assembly of the VIP1R–Gs complex. PACAP27 is colored in orange, VIP1R in green, Gαs in yellow, Gβ in blue, Gγ in purple, LgBiT in light blue, and HiBiT in red. b Respective size-exclusion chromatography elution profiles of the VIP1R–Gs and VIP1R-LgBiT-Gs-HiBiT complexes. c Dynamic light scattering (DLS) size distribution histograms of VIP1R–Gs and VIP1R-LgBiT-Gs-HiBiT complexes. Values of radius, % intensity of monomer, and ratio of monomer/aggregation (M/A) are listed. d Representative negative staining images of the corresponding complexes. The scale bar is 200 nm. Source data are provided as a Source Data file.

Fig. 2. The overall cryo-EM structure of PACAP27–VIP1R–Gs complex.

a A cut-through view of the cryo-EM map of PACAP27–VIP1R–Gs complex with a disc-shaped micelle. b A cartoon representation of the PACAP27–VIP1R–Gs complex. c Extracellular view of the PACAP27–VIP1R–Gs complex structure. PACAP27 is colored in orange; VIP1R in green; Gαs Ras-like domain in yellow; Gβ subunit in blue; Gγ subunit in purple; Nb35 in gray; and lipid molecules in cyan.

Fig. 3. Comparison of the binding mode of PACAPs to VIP1R and PAC1R.

a Sequence alignment of the VIP1R peptide ligands VIP, PACAP27, and PACAP38. b The binding mode of PACAP27 to VIP1R, showing that PACAP27 adopts α-helical conformation and interacts with all TM helices of VIP1R except TM4. c The cross-section view of the PACAP27 binding pocket in the TM bundle of VIP1R. Structural comparisons of PACAP binding pockets in VIP1R and PAC1R. Residues interact with peptide amino acids H1 and D3 (d), S2 (e), G4, I5, and F6 (f), as well as amino acids from T7 to R14 (g, h) are shown as sticks. The hydrogen bonds between PACAP27 and residues of VIP1R are marked as black dotted lines, and the hydrogen bonds between PACAP38 and residues of VIP1R are shown as red dotted lines. PACAP27 is colored in orange, and VIP1R in green. PACAP38 is shown in cyan, and PAC1R (PDB code: 6P9Y) in light blue.

Fig. 4. Structure comparisons of active VIP1R with inactive GCGR.

a The structural alignment of activated VIP1R with inactive GCGR showing the outward bending of the intracellular portion of TM6 of activated VIP1R, which results in a kink at the PxxG motif in TM6 and a ~90° angle between two portions of TM6 of the activated receptor. The TM6 kink in the active VIP1R structure is indicated by a dotted black line. The residues in the conserved PxxG motif in TM6 are shown in stick representation. b Polar and hydrophobic interactions that stabilize the kink at TM6 of activated VIP1R. The polar contacts are marked as black dotted lines. The positions of conserved polar residue networks located within VIP1R (green) and inactive GCGR (PDB code:4L6R, colored in salmon): central polar network (c), HETY network (d), and TM2–6–7–helix 8 network (e). Side chains of the residues are shown in stick representation.

Fig. 5. The interactions between VIP1R and Gs heterotrimer.

a, b The binding interface between the cavity on the intracellular side of VIP1R TMD (green) and α5 helix of the Gαs Ras-like domain (yellow). c The interface between ICL2 of VIP1R (green) and α5 and αN of the Gαs Ras-like domain (yellow). d The interface between helix 8 of VIP1R (green) and Gβ subunit (blue). Residues in VIP1R–Gs interfaces are shown in stick representation.