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- PDB-9p94: Cryo-EM structure of the PAC1sR-PACAP27-Gs complex -

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Entry
Database: PDB / ID: 9p94
TitleCryo-EM structure of the PAC1sR-PACAP27-Gs complex
Components
  • (Guanine nucleotide-binding protein ...) x 3
  • Isoform S of Pituitary adenylate cyclase-activating polypeptide type I receptor
  • Nanobody35
  • Pituitary adenylate cyclase-activating polypeptide
KeywordsMEMBRANE PROTEIN / drug discovery / G protein coupled receptor / signalling
Function / homology
Function and homology information


negative regulation of response to reactive oxygen species / pituitary adenylate cyclase activating polypeptide activity / development of primary female sexual characteristics / type 1 vasoactive intestinal polypeptide receptor binding / type 2 vasoactive intestinal polypeptide receptor binding / pituitary adenylate cyclase-activating polypeptide receptor activity / vasoactive intestinal polypeptide receptor activity / positive regulation of growth hormone secretion / positive regulation of chemokine (C-C motif) ligand 5 production / NGF-independant TRKA activation ...negative regulation of response to reactive oxygen species / pituitary adenylate cyclase activating polypeptide activity / development of primary female sexual characteristics / type 1 vasoactive intestinal polypeptide receptor binding / type 2 vasoactive intestinal polypeptide receptor binding / pituitary adenylate cyclase-activating polypeptide receptor activity / vasoactive intestinal polypeptide receptor activity / positive regulation of growth hormone secretion / positive regulation of chemokine (C-C motif) ligand 5 production / NGF-independant TRKA activation / neuropeptide hormone activity / regulation of G protein-coupled receptor signaling pathway / positive regulation of small GTPase mediated signal transduction / G protein-coupled peptide receptor activity / neuropeptide binding / insulin secretion / peptide hormone receptor binding / positive regulation of inositol phosphate biosynthetic process / negative regulation of cell cycle / positive regulation of cAMP/PKA signal transduction / positive regulation of protein kinase activity / peptide hormone binding / positive regulation of calcium ion transport into cytosol / adenylate cyclase binding / cAMP/PKA signal transduction / PKA activation in glucagon signalling / developmental growth / hair follicle placode formation / positive regulation of GTPase activity / bicellular tight junction / D1 dopamine receptor binding / neuropeptide signaling pathway / intracellular transport / vascular endothelial cell response to laminar fluid shear stress / multicellular organismal response to stress / renal water homeostasis / activation of adenylate cyclase activity / Hedgehog 'off' state / adenylate cyclase-activating adrenergic receptor signaling pathway / regulation of insulin secretion / cellular response to glucagon stimulus / adenylate cyclase activator activity / trans-Golgi network membrane / negative regulation of inflammatory response to antigenic stimulus / female pregnancy / bone development / caveola / small GTPase binding / platelet aggregation / cognition / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / G-protein beta/gamma-subunit complex binding / Olfactory Signaling Pathway / Activation of the phototransduction cascade / adenylate cyclase-activating G protein-coupled receptor signaling pathway / neuron projection development / G beta:gamma signalling through PLC beta / Presynaptic function of Kainate receptors / Thromboxane signalling through TP receptor / G protein-coupled acetylcholine receptor signaling pathway / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / G-protein activation / G beta:gamma signalling through CDC42 / Prostacyclin signalling through prostacyclin receptor / Glucagon signaling in metabolic regulation / G beta:gamma signalling through BTK / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / ADP signalling through P2Y purinoceptor 12 / photoreceptor disc membrane / Sensory perception of sweet, bitter, and umami (glutamate) taste / Glucagon-type ligand receptors / Adrenaline,noradrenaline inhibits insulin secretion / Vasopressin regulates renal water homeostasis via Aquaporins / sensory perception of smell / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / G alpha (z) signalling events / ADP signalling through P2Y purinoceptor 1 / cellular response to catecholamine stimulus / ADORA2B mediated anti-inflammatory cytokines production / G beta:gamma signalling through PI3Kgamma / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / adenylate cyclase-activating dopamine receptor signaling pathway / response to estradiol / cell-cell signaling / GPER1 signaling / Inactivation, recovery and regulation of the phototransduction cascade / G-protein beta-subunit binding / cellular response to prostaglandin E stimulus / heterotrimeric G-protein complex / signaling receptor activity / G alpha (12/13) signalling events / extracellular vesicle / sensory perception of taste / regulation of protein localization / positive regulation of cold-induced thermogenesis / Thrombin signalling through proteinase activated receptors (PARs) / signaling receptor complex adaptor activity / positive regulation of cytosolic calcium ion concentration / retina development in camera-type eye
Similarity search - Function
GPCR, family 2, pituitary adenylate cyclase activating polypeptide type 1 receptor / : / Glucagon/GIP/secretin/VIP / Peptide hormone / Glucagon / GIP / secretin / VIP family signature. / Glucagon like hormones / G-protein coupled receptors family 2 signature 1. / : / GPCR, family 2, extracellular hormone receptor domain / G-protein coupled receptors family 2 profile 1. ...GPCR, family 2, pituitary adenylate cyclase activating polypeptide type 1 receptor / : / Glucagon/GIP/secretin/VIP / Peptide hormone / Glucagon / GIP / secretin / VIP family signature. / Glucagon like hormones / G-protein coupled receptors family 2 signature 1. / : / GPCR, family 2, extracellular hormone receptor domain / G-protein coupled receptors family 2 profile 1. / Domain present in hormone receptors / Hormone receptor domain / GPCR family 2, extracellular hormone receptor domain superfamily / G-protein coupled receptors family 2 signature 2. / GPCR, family 2, secretin-like, conserved site / GPCR, family 2, secretin-like / 7 transmembrane receptor (Secretin family) / GPCR, family 2-like / G-protein coupled receptors family 2 profile 2. / G-protein alpha subunit, group S / Guanine nucleotide binding protein (G-protein), alpha subunit / G protein alpha subunit, helical insertion / G-protein alpha subunit / G-alpha domain profile. / G protein alpha subunit / G-protein, gamma subunit / G-protein gamma subunit domain profile. / G-protein gamma-like domain / G-protein gamma-like domain superfamily / GGL domain / G protein gamma subunit-like motifs / GGL domain / G protein beta WD-40 repeat protein / Guanine nucleotide-binding protein, beta subunit / G-protein, beta subunit / G-protein beta WD-40 repeat / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Pituitary adenylate cyclase-activating polypeptide / Pituitary adenylate cyclase-activating polypeptide type I receptor / Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 / Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
Similarity search - Component
Biological speciesHomo sapiens (human)
Lama glama (llama)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsPiper, S.J. / Sexton, P. / Wootten, D.
Funding support Australia, 6items
OrganizationGrant numberCountry
National Health and Medical Research Council (NHMRC, Australia)1155302 Australia
National Health and Medical Research Council (NHMRC, Australia)1150083 Australia
National Health and Medical Research Council (NHMRC, Australia)1154434 Australia
National Health and Medical Research Council (NHMRC, Australia)2025694 Australia
National Health and Medical Research Council (NHMRC, Australia)2026300 Australia
Australian Research Council (ARC)230102776 Australia
CitationJournal: Proc Natl Acad Sci U S A / Year: 2025
Title: Structural basis of modified ligand selectivity from N-terminal PAC1R alternative splicing.
Authors: Jessica J Lu / Giuseppe Deganutti / Miaomiao Li / Laura J Humphrys / Yandi Li / Theodore J Nettleton / Hariprasad Venugopal / Villy Julita / George Christopoulos / Christopher A Reynolds / ...Authors: Jessica J Lu / Giuseppe Deganutti / Miaomiao Li / Laura J Humphrys / Yandi Li / Theodore J Nettleton / Hariprasad Venugopal / Villy Julita / George Christopoulos / Christopher A Reynolds / Patrick M Sexton / Denise Wootten / Peishen Zhao / Sarah J Piper /
Abstract: The pituitary adenylate cyclase-activating polypeptide (PACAP) 1 receptor (PAC1R) is a class B1 G protein-coupled receptor activated by the endogenous peptide agonists PACAP and vasoactive intestinal ...The pituitary adenylate cyclase-activating polypeptide (PACAP) 1 receptor (PAC1R) is a class B1 G protein-coupled receptor activated by the endogenous peptide agonists PACAP and vasoactive intestinal peptide (VIP). Alternate splicing within the receptor extracellular domain (ECD) generates the PAC1R short variant (PAC1sR) that has selectively enhanced VIP function compared to the full-length, PAC1R null variant (PAC1nR). However, to date, a comprehensive pharmacological assessment of the downstream signaling outcomes of PAC1sR activation compared to PAC1nR has not been performed, and little information is available to mechanistically understand how ECD splicing may alter ligand engagement. Here, we demonstrated that VIP, but not PACAP, has globally enhanced activity across a broad range of functional endpoints at PAC1sR compared to PAC1nR. Cryo-EM structures of VIP-bound, stimulatory G protein (G)-coupled PAC1sR and PAC1nR, supported by molecular dynamics (MD) simulations, demonstrate transient engagement of the null loop in PAC1nR, which is absent in PAC1sR, with residues in extracellular loop 2 (ECL2) and the N-terminal helix of the ECD. These interactions result in differential engagement of VIP with these domains and the top of TM2/ECL1 with PAC1sR and PAC1nR. Moreover, MD simulations predicted differential interactions of the G protein with the two PAC1R variants when bound by VIP that correlate with a greater allosteric influence of the G protein on VIP affinity at the PAC1sR, relative to PAC1nR. Our study provides insights into the structural basis and functional consequences of PAC1R ECD splicing, increasing understanding of PAC1R ligand selectivity and signaling.
History
DepositionJun 24, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 19, 2025Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
B: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
G: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
N: Nanobody35
P: Pituitary adenylate cyclase-activating polypeptide
R: Isoform S of Pituitary adenylate cyclase-activating polypeptide type I receptor
A: Guanine nucleotide-binding protein G(s) subunit alpha isoforms short


Theoretical massNumber of molelcules
Total (without water)163,5466
Polymers163,5466
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Guanine nucleotide-binding protein ... , 3 types, 3 molecules BGA

#1: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Transducin beta chain 1


Mass: 38534.062 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: N-terminal HIS tag / Source: (gene. exp.) Homo sapiens (human) / Gene: GNB1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P62873
#2: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 / G gamma-I


Mass: 7861.143 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNG2 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P59768
#6: Protein Guanine nucleotide-binding protein G(s) subunit alpha isoforms short / Adenylate cyclase-stimulating G alpha protein


Mass: 45699.434 Da / Num. of mol.: 1
Mutation: S54N G226A E268A N271K K274D R280K T284D I285T A366S
Source method: isolated from a genetically manipulated source
Details: Molecule contains the following mutations as dominant-negative Gs mutations: S54N G226A E268A N271K K274D R280K T284D I285T A366S Reference: doi: 10.1021/acsptsci.8b00017
Source: (gene. exp.) Homo sapiens (human) / Gene: GNAS, GNAS1, GSP / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P63092

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Antibody / Protein/peptide / Protein , 3 types, 3 molecules NPR

#3: Antibody Nanobody35


Mass: 15140.742 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: C-terminal HIS tag / Source: (gene. exp.) Lama glama (llama) / Production host: Escherichia coli (E. coli)
#4: Protein/peptide Pituitary adenylate cyclase-activating polypeptide / PACAP


Mass: 3154.642 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human) / References: UniProt: P18509
#5: Protein Isoform S of Pituitary adenylate cyclase-activating polypeptide type I receptor / PAC1 receptor / PAC1R / PACAP type I receptor / PACAP-R-1 / PACAP-R1


Mass: 53155.906 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: N-terminal FLAG tag, C-terminal HIS tag / Source: (gene. exp.) Homo sapiens (human) / Gene: ADCYAP1R1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P41586

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Details

Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1Gs coupled Isoform S of Pituitary adenylate cyclase-activating polypeptide type I receptor complex with PACAP27 peptideCOMPLEXall0MULTIPLE SOURCES
2Guanine nucleotide-binding protein G(s) subunit alpha isoforms short, Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1, Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2, Isoform S of Pituitary adenylate cyclase-activating polypeptide type I receptorCOMPLEX#1-#2, #5-#61RECOMBINANT
3Nanobody 35COMPLEX#31RECOMBINANT
4PACAP27 peptideCOMPLEX#41RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Lama glama (llama)9844
34Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Spodoptera frugiperda (fall armyworm)7108
23Escherichia coli (E. coli)562
34synthetic construct (others)32630
Buffer solutionpH: 7.4
SpecimenConc.: 4.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1300 nm / Nominal defocus min: 700 nm
Image recordingElectron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

EM software
IDNameCategory
1crYOLOparticle selection
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 583096 / Symmetry type: POINT
RefinementHighest resolution: 3 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)

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