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- PDB-6m1i: CryoEM structure of human PAC1 receptor in complex with PACAP38 -

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Basic information

Entry
Database: PDB / ID: 6m1i
TitleCryoEM structure of human PAC1 receptor in complex with PACAP38
Components
  • (Guanine nucleotide-binding protein ...) x 3
  • (Pituitary adenylate cyclase-activating ...) x 2
  • Nanobody 35Single-domain antibody
KeywordsPROTEIN BINDING / GPCR
Function / homology
Function and homology information


negative regulation of response to reactive oxygen species / development of primary female sexual characteristics / pituitary adenylate cyclase activating polypeptide activity / vasoactive intestinal polypeptide receptor activity / positive regulation of chemokine (C-C motif) ligand 5 production / positive regulation of growth hormone secretion / NGF-independant TRKA activation / neuropeptide hormone activity / regulation of G protein-coupled receptor signaling pathway / neuropeptide binding ...negative regulation of response to reactive oxygen species / development of primary female sexual characteristics / pituitary adenylate cyclase activating polypeptide activity / vasoactive intestinal polypeptide receptor activity / positive regulation of chemokine (C-C motif) ligand 5 production / positive regulation of growth hormone secretion / NGF-independant TRKA activation / neuropeptide hormone activity / regulation of G protein-coupled receptor signaling pathway / neuropeptide binding / G protein-coupled peptide receptor activity / positive regulation of small GTPase mediated signal transduction / insulin secretion / positive regulation of inositol phosphate biosynthetic process / peptide hormone receptor binding / positive regulation of calcium ion transport into cytosol / PKA activation in glucagon signalling / peptide hormone binding / cAMP-mediated signaling / hair follicle placode formation / adenylate cyclase binding / intracellular transport / negative regulation of cell cycle / D1 dopamine receptor binding / developmental growth / bicellular tight junction / neuropeptide signaling pathway / positive regulation of protein kinase activity / multicellular organismal response to stress / Hedgehog 'off' state / positive regulation of cAMP-mediated signaling / adenylate cyclase-activating adrenergic receptor signaling pathway / activation of adenylate cyclase activity / adenylate cyclase activator activity / trans-Golgi network membrane / female pregnancy / caveola / G-protein beta/gamma-subunit complex binding / Olfactory Signaling Pathway / Activation of the phototransduction cascade / G beta:gamma signalling through PLC beta / Presynaptic function of Kainate receptors / Thromboxane signalling through TP receptor / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / bone development / G-protein activation / G protein-coupled acetylcholine receptor signaling pathway / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / Prostacyclin signalling through prostacyclin receptor / Glucagon signaling in metabolic regulation / G beta:gamma signalling through CDC42 / adenylate cyclase-activating G protein-coupled receptor signaling pathway / ADP signalling through P2Y purinoceptor 12 / G beta:gamma signalling through BTK / Sensory perception of sweet, bitter, and umami (glutamate) taste / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / photoreceptor disc membrane / Adrenaline,noradrenaline inhibits insulin secretion / platelet aggregation / small GTPase binding / Glucagon-type ligand receptors / cognition / Vasopressin regulates renal water homeostasis via Aquaporins / positive regulation of GTPase activity / G alpha (z) signalling events / cellular response to catecholamine stimulus / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / ADORA2B mediated anti-inflammatory cytokines production / adenylate cyclase-activating dopamine receptor signaling pathway / ADP signalling through P2Y purinoceptor 1 / G beta:gamma signalling through PI3Kgamma / cellular response to prostaglandin E stimulus / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / sensory perception of taste / GPER1 signaling / neuron projection development / G-protein beta-subunit binding / heterotrimeric G-protein complex / Inactivation, recovery and regulation of the phototransduction cascade / extracellular vesicle / regulation of protein localization / G alpha (12/13) signalling events / signaling receptor complex adaptor activity / sensory perception of smell / Thrombin signalling through proteinase activated receptors (PARs) / response to estradiol / retina development in camera-type eye / cell-cell signaling / signaling receptor activity / GTPase binding / Ca2+ pathway / phospholipase C-activating G protein-coupled receptor signaling pathway / positive regulation of cold-induced thermogenesis / positive regulation of cytosolic calcium ion concentration / G alpha (i) signalling events / fibroblast proliferation / perikaryon / G alpha (s) signalling events / G alpha (q) signalling events
Similarity search - Function
GPCR, family 2, pituitary adenylate cyclase activating polypeptide type 1 receptor / : / Transducin (heterotrimeric G protein), gamma chain / G Protein Gi Gamma 2 / Glucagon/GIP/secretin/VIP / Peptide hormone / Glucagon / GIP / secretin / VIP family signature. / Glucagon like hormones / G-protein coupled receptors family 2 signature 1. / Hormone receptor domain ...GPCR, family 2, pituitary adenylate cyclase activating polypeptide type 1 receptor / : / Transducin (heterotrimeric G protein), gamma chain / G Protein Gi Gamma 2 / Glucagon/GIP/secretin/VIP / Peptide hormone / Glucagon / GIP / secretin / VIP family signature. / Glucagon like hormones / G-protein coupled receptors family 2 signature 1. / Hormone receptor domain / GPCR, family 2, extracellular hormone receptor domain / G-protein coupled receptors family 2 profile 1. / Domain present in hormone receptors / 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 / YVTN repeat-like/Quinoprotein amine dehydrogenase / 7 Propeller / Methylamine Dehydrogenase; Chain H / G-alpha domain profile. / Guanine nucleotide binding protein (G-protein), alpha subunit / G protein alpha subunit, helical insertion / G-protein alpha subunit / G protein alpha subunit / G-protein, gamma subunit / G-protein gamma subunit domain profile. / GGL domain / G-protein gamma-like domain superfamily / G-protein gamma-like domain / GGL domain / G protein gamma subunit-like motifs / Guanine nucleotide-binding protein, beta subunit / G-protein, beta subunit / Few Secondary Structures / Irregular / G-protein beta WD-40 repeat / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / P-loop containing nucleoside triphosphate hydrolase / Mainly Beta
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: 3.5 Å
AuthorsSong, X. / Wang, J. / Zhang, D. / Wang, H.W. / Ma, Y.
CitationJournal: Cell Res / Year: 2020
Title: Cryo-EM structures of PAC1 receptor reveal ligand binding mechanism.
Authors: Jia Wang / Xianqiang Song / Dandan Zhang / Xiaoqing Chen / Xun Li / Yaping Sun / Cui Li / Yunpeng Song / Yao Ding / Ruobing Ren / Essa Hu Harrington / Liaoyuan A Hu / Wenge Zhong / Cen Xu / ...Authors: Jia Wang / Xianqiang Song / Dandan Zhang / Xiaoqing Chen / Xun Li / Yaping Sun / Cui Li / Yunpeng Song / Yao Ding / Ruobing Ren / Essa Hu Harrington / Liaoyuan A Hu / Wenge Zhong / Cen Xu / Xin Huang / Hong-Wei Wang / Yingli Ma /
Abstract: The pituitary adenylate cyclase-activating polypeptide type I receptor (PAC1R) belongs to the secretin receptor family and is widely distributed in the central neural system and peripheral organs. ...The pituitary adenylate cyclase-activating polypeptide type I receptor (PAC1R) belongs to the secretin receptor family and is widely distributed in the central neural system and peripheral organs. Abnormal activation of the receptor mediates trigeminovascular activation and sensitization, which is highly related to migraine, making PAC1R a potential therapeutic target. Elucidation of PAC1R activation mechanism would benefit discovery of therapeutic drugs for neuronal disorders. PAC1R activity is governed by pituitary adenylate cyclase-activating polypeptide (PACAP), known as a major vasodilator neuropeptide, and maxadilan, a native peptide from the sand fly, which is also capable of activating the receptor with similar potency. These peptide ligands have divergent sequences yet initiate convergent PAC1R activity. It is of interest to understand the mechanism of PAC1R ligand recognition and receptor activity regulation through structural biology. Here we report two near-atomic resolution cryo-EM structures of PAC1R activated by PACAP38 or maxadilan, providing structural insights into two distinct ligand binding modes. The structures illustrate flexibility of the extracellular domain (ECD) for ligands with distinct conformations, where ECD accommodates ligands in different orientations while extracellular loop 1 (ECL1) protrudes to further anchor the ligand bound in the orthosteric site. By structure-guided molecular modeling and mutagenesis, we tested residues in the ligand-binding pockets and identified clusters of residues that are critical for receptor activity. The structures reported here for the first time elucidate the mechanism of specificity and flexibility of ligand recognition and binding for PAC1R, and provide insights toward the design of therapeutic molecules targeting PAC1R.
History
DepositionFeb 26, 2020Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Mar 11, 2020Provider: repository / Type: Initial release
Revision 1.1May 27, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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Structure visualization

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Assembly

Deposited unit
A: Pituitary adenylate cyclase-activating polypeptide type I receptor
B: Pituitary adenylate cyclase-activating polypeptide
C: Nanobody 35
D: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
E: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
F: Guanine nucleotide-binding protein G(s) subunit alpha isoforms short


Theoretical massNumber of molelcules
Total (without water)157,5966
Polymers157,5966
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area15060 Å2
ΔGint-97 kcal/mol
Surface area52480 Å2

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Components

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Pituitary adenylate cyclase-activating ... , 2 types, 2 molecules AB

#1: Protein Pituitary adenylate cyclase-activating polypeptide type I receptor


Mass: 47315.629 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Insect BA phytoplasma (bacteria) / References: UniProt: P41586*PLUS
#2: Protein/peptide Pituitary adenylate cyclase-activating polypeptide / Pituitary adenylate cyclase-activating peptide / PACAP


Mass: 4547.336 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human) / References: UniProt: P18509

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

#4: 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: Insect BA phytoplasma (bacteria) / References: UniProt: P59768
#5: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Transducin beta chain 1


Mass: 37473.980 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNB1 / Production host: Insect BA phytoplasma (bacteria) / References: UniProt: P62873
#6: Protein Guanine nucleotide-binding protein G(s) subunit alpha isoforms short


Mass: 45683.434 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Insect BA phytoplasma (bacteria) / References: UniProt: P63092*PLUS

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Antibody , 1 types, 1 molecules C

#3: Antibody Nanobody 35 / Single-domain antibody


Mass: 14714.320 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Production host: Escherichia coli (E. coli)

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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

ComponentName: PACAP38-PAC1R complex / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.15 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Cs: 0 mm / C2 aperture diameter: 50 µm
Image recordingElectron dose: 50 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
Image scansWidth: 3838 / Height: 3710

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Processing

SoftwareName: PHENIX / Version: 1.14_3260: / Classification: refinement
EM software
IDNameVersionCategory
4CTFFIND4.1CTF correction
7UCSF Chimeramodel fitting
9PHENIXmodel refinement
13RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 82970 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingPDB-ID: 5B16

5b16

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