PACAP is a 38-amino acid C-terminally amidated polypeptide (PACAP38) that was discovered as a hypothalamic neuropeptide to potentially induce cAMP levels in anterior pituitary cells. The N-terminal 27 residues of PACAP38, highly conserved in almost all vertebrate species and responsible for the physiological activity of the peptide, undergo internal cleavage-amidation to generate the PACAP27 fragment. Because of the 68% sequence identity between PACAP27 and vasoactive intestinal polypeptide (VIP), PACAP is identified as a member of the glucagon/gastric inhibitory polypeptide (GIP)/secretin/VIP family—a hormone family consisting of evolutionarily related peptides that regulate the activity of class B G-protein coupled receptor (GPCR) family, also known as secretin receptor family.
The receptors that recognize PACAP are characterized into three distinct subtypes based on their relative affinities to PACAP and VIP: the pituitary adenylate cyclase-activating polypeptide type I receptor (PAC1R) with two orders of magnitude higher affinity to PACAP than to VIP; the vasoactive intestinal polypeptide receptor 1 (VPACR1) and receptor 2 (VPACR2) with similar PACAP/VIP affinities.
PACAP and its receptors are broadly expressed in the central nervous system (CNS) and in most peripheral organs, and have been found to exert a variety of functions including control of neurotransmitter release, vasodilation, bronchodilation, activation of intestinal motility, neuroprotection, immune modulation, and stimulation of cell proliferation and/or differentiation.
As the major sensory and vasodilator neuropeptides, PACAP38 and VIP are involved in parasympathetic communication with the cranial vasculature. Abnormal activation and sensitization of the central trigeminovascular pain pathway mediate migraine and the release of these peptides. Intravenous infusion of PACAP38 but not VIP induces delayed migraine-like headaches, indicating that PAC1R is playing a major role in migraine and suggesting PACAP38-PAC1R as a potential therapeutic target for migraine treatment.
Maxadilan, another natural PAC1R agonist, is a 61-amino acid polypeptide isolated from the salivary gland of the blood-feeding sand fly Lutzomia lingipalpis. Maxadilan is an immunomodulator and has been shown to facilitate the transmission and establishment of leishmaniasis. Despite the low sequence homology between maxadilan and PACAP38, they both potently activate PAC1R. The structural basis of receptor ligand recognition and activation mechanism remains unknown.
Recent development in cryo-electron microscopy (cryo-EM) has enabled the determination of full-length class B GPCR structures in complex with their peptide ligand and G protein complex. However, no structural information is available for PAC1R. Here we report the cryo-EM structures of G s-protein coupled PAC1R in complex with PACAP38 and maxadilan, respectively, and uncover the underlying mechanism of convergent activity from distinct ligands on a class B GPCR. Structure-guided mutagenesis elucidates the key residues responsible for ligand binding and receptor activation. These results further strengthen our mechanistic understanding of PAC1R regulation and will benefit future rational design of therapeutic molecules for migraine.
For cryo-EM structure determination purpose, we modified the human PAC1R with a short C-terminal truncation (439–468), seven mutations (T163L, T167A, T169L and T170L on TM1; T276A, T278L and C280F on TM4), replacement of the native signal peptide by that of haemagglutinin (HA), and addition of affinity tags (an N-terminal FLAG tag and a C-terminal 10× His tag) (Supplementary information, Fig. S1). These modifications did not alter receptor ligand binding and pharmacological properties (Supplementary information, Fig. S2). We generated by site-directed mutagenesis the dominant-negative Gα s construct, including mutations that reduce nucleotide affinity (S54N and G226A) and improve the dominant-negative effect (E268A, N271K, K274D, R280K, T284D, and I285T) to improve the complex stability.
Human PAC1R, Gα s, Gβ 1, and Gγ 2 were co-expressed in High Five insect cells using baculovirus transfection to form the GPCR complex. Agonist peptide, PACAP38 or maxadilan, and Nanobody-35 (Nb35) were added during purification to enable a stable complex formation. The complex was solubilized in lauryl maltose neopentyl glycol (LMNG) and cholesteryl hemisuccinate, and then purified by nickel affinity and size exclusion chromatography to yield a monodisperse complex that contained all the components (Supplementary information, Fig. S2).
Single-particle cryo-EM analysis of the complexes in vitreous ice yielded a final map at a resolution of 3.5 Å for PACAP38-PAC1R-G s (reconstructed from 82,970 particles) and that of 3.6 Å for maxadilan-PAC1R-G s (reconstructed from 58,451 particles) (Fig. 1; Supplementary information, Figs. S3, 4, Table S1). The density for the seven transmembrane helices (TMs), the ligands, and G protein complex are unambiguously determined based on the well-traced α-helices and aromatic side chains. The extracellular domain (ECD) is incomplete due to flexibility, but we were able to utilize the previously solved crystal structure to place it according to the partial density (Supplementary information, Fig. S5).
a, b Cryo-EM density map (left), the structure model (middle) and local resolution distribution map (right) of PAC1R-Gs in complex with PACAP38 (a) and maxadilan (b). The structures of PAC1R (green in PACAP38-PAC1R-G s structure, orange in maxadilan-PAC1R-G s structure), PACAP38 (pink), maxadilan (purple), Gα (red), Gβ (blue), Gγ (yellow) and Nb35 (gray) are represented in cartoon.
PAC1R-G s complexes, with PACAP38 or maxadilan bound at the extracellular side and G s protein complex bound at the intracellular side of PAC1R, are in an active state (Fig. 1) with conformation reminiscent of the other class B GPCR-G s complex structures. The overall reported resolution is 3.5 Å and 3.6 Å for the PACAP38-PAC1R-G s and the maxadilan-PAC1R-G s complexes, respectively (Fig. 1a, b). Although both structures represent G protein-bound active state of PAC1R, the ligands PACAP38 and maxadilan, with their dramatically different sequences and stereo structures, adopted disparate modes of binding to the ECD and the orthosteric site. To accommodate differences in ligand size, the TMs and extracellular loops (ECLs) that consist of the orthosteric site shift slightly with a root-mean-square deviation (r.m.s.d.) of 1.11 Å in the 7-TM region between the two structures.
The PAC1R construct used in the cryo-EM study retains ligand binding and pharmacological properties comparable to the wild-type (WT) receptor (Supplementary information, Fig. S2). The peptide forms an α-helix with its N-terminus inserted into the orthosteric site. Structure of the N-terminal 27 residues, conserved and responsible for receptor activation, are unambiguously resolved (Fig. 2a), while the C-terminal 11 residues are not visible in the map probably due to flexibility and omitted from the final reported structure.
a Slice view of the PACAP38-PAC1R-G s structure and a schematic helical sequence for PACAP38 with unresolved residues in red. b Close view of the C-terminal part of PACAP38 showing detailed interactions. c Close views of the N-terminal part of PACAP38 from two angles with 180° rotation. The effective residues in mutagenesis assay are presented by spheres. d The PAC1R-PACAP38 interaction diagrams. PACAP38 (H1-Y13) is shown as sticks. Residues are represented as spheres and colored by interaction type. Interactions between the residues and the ligand atoms are drawn as dashed lines, colored by interaction type. The solvent accessible surface of an interacting residue and of an atom are represented by haloes around the residue and the atom. The diameter of the circle is proportional to the solvent accessible surface.
Outside the orthosteric site, PACAP38 mainly forms hydrophobic interactions
