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- PDB-6lpb: Cryo-EM structure of the human PAC1 receptor coupled to an engine... -

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Basic information

Entry
Database: PDB / ID: 6lpb
TitleCryo-EM structure of the human PAC1 receptor coupled to an engineered heterotrimeric G protein
Components
  • (Guanine nucleotide-binding protein ...) x 3
  • (Pituitary adenylate cyclase-activating ...) x 2
  • nanobody Nb35
KeywordsSIGNALING PROTEIN/HORMONE / CLASS B GPCR / PACAP / PAC1R / SIGNALING PROTEIN-HORMONE COMPLEX
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 / 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 / development of primary female sexual characteristics / pituitary adenylate cyclase activating polypeptide activity / 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 / regulation of G protein-coupled receptor signaling pathway / neuropeptide hormone activity / G-protein activation / Activation of the phototransduction cascade / Glucagon-type ligand receptors / Thromboxane signalling through TP receptor / Sensory perception of sweet, bitter, and umami (glutamate) taste / G beta:gamma signalling through PI3Kgamma / G beta:gamma signalling through CDC42 / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / Ca2+ pathway / neuropeptide binding / G alpha (z) signalling events / High laminar flow shear stress activates signaling by PIEZO1 and PECAM1:CDH5:KDR in endothelial cells / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / Vasopressin regulates renal water homeostasis via Aquaporins / positive regulation of small GTPase mediated signal transduction / G protein-coupled peptide receptor activity / Adrenaline,noradrenaline inhibits insulin secretion / ADP signalling through P2Y purinoceptor 12 / G alpha (q) signalling events / G alpha (i) signalling events / insulin secretion / Activation of G protein gated Potassium channels / G-protein activation / G beta:gamma signalling through PI3Kgamma / Prostacyclin signalling through prostacyclin receptor / G beta:gamma signalling through PLC beta / ADP signalling through P2Y purinoceptor 1 / Thromboxane signalling through TP receptor / Presynaptic function of Kainate receptors / G beta:gamma signalling through CDC42 / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / Thrombin signalling through proteinase activated receptors (PARs) / G alpha (12/13) signalling events / Glucagon-type ligand receptors / G beta:gamma signalling through BTK / ADP signalling through P2Y purinoceptor 12 / Adrenaline,noradrenaline inhibits insulin secretion / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / Ca2+ pathway / Thrombin signalling through proteinase activated receptors (PARs) / G alpha (z) signalling events / Extra-nuclear estrogen signaling / G alpha (s) signalling events / photoreceptor outer segment membrane / G alpha (q) signalling events / peptide hormone receptor binding / positive regulation of inositol phosphate biosynthetic process / G alpha (i) signalling events / spectrin binding / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / High laminar flow shear stress activates signaling by PIEZO1 and PECAM1:CDH5:KDR in endothelial cells / Vasopressin regulates renal water homeostasis via Aquaporins / positive regulation of calcium ion transport into cytosol / alkylglycerophosphoethanolamine phosphodiesterase activity / peptide hormone binding / positive regulation of cAMP/PKA signal transduction / cAMP/PKA signal transduction / negative regulation of cell cycle / adenylate cyclase binding / PKA activation in glucagon signalling / hair follicle placode formation / photoreceptor outer segment / positive regulation of protein kinase activity / bicellular tight junction / developmental growth / neuropeptide signaling pathway / D1 dopamine receptor binding / intracellular transport / multicellular organismal response to stress / vascular endothelial cell response to laminar fluid shear stress / renal water homeostasis / Hedgehog 'off' state / adenylate cyclase-activating adrenergic receptor signaling pathway / activation of adenylate cyclase activity / photoreceptor inner segment / cardiac muscle cell apoptotic process / positive regulation of GTPase activity / regulation of insulin secretion / cellular response to glucagon stimulus / adenylate cyclase activator activity / trans-Golgi network membrane / female pregnancy / negative regulation of inflammatory response to antigenic stimulus / bone development / caveola / small GTPase binding
Similarity search - Function
GPCR, family 2, pituitary adenylate cyclase activating polypeptide type 1 receptor / : / GPCR, family 2, extracellular hormone receptor domain / Hormone receptor fold / 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 ...GPCR, family 2, pituitary adenylate cyclase activating polypeptide type 1 receptor / : / GPCR, family 2, extracellular hormone receptor domain / Hormone receptor fold / 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. / : / 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. / Rhopdopsin 7-helix transmembrane proteins / Rhodopsin 7-helix transmembrane proteins / G-protein alpha subunit, group S / YVTN repeat-like/Quinoprotein amine dehydrogenase / 7 Propeller / Methylamine Dehydrogenase; Chain H / 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 / Few Secondary Structures / Irregular / 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 / 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. / P-loop containing nucleotide triphosphate hydrolases / WD domain, G-beta repeat / 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 / Up-down Bundle / Rossmann fold / P-loop containing nucleoside triphosphate hydrolase / 3-Layer(aba) Sandwich / Mainly Beta / Mainly Alpha / Alpha 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(T) subunit beta-1 / Guanine nucleotide-binding protein G(s) subunit alpha isoforms short / Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
Similarity search - Component
Biological speciesHomo sapiens (human)
Rattus norvegicus (Norway rat)
unidentified (others)
Bos taurus (domestic cattle)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsKobayashi, K. / Shihoya, W. / Nishizawa, T. / Nureki, O.
CitationJournal: Nat Struct Mol Biol / Year: 2020
Title: Cryo-EM structure of the human PAC1 receptor coupled to an engineered heterotrimeric G protein.
Authors: Kazuhiro Kobayashi / Wataru Shihoya / Tomohiro Nishizawa / Francois Marie Ngako Kadji / Junken Aoki / Asuka Inoue / Osamu Nureki /
Abstract: Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic neuropeptide hormone. The PACAP receptor PAC1R, which belongs to the class B G-protein-coupled receptors (GPCRs), is a drug ...Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic neuropeptide hormone. The PACAP receptor PAC1R, which belongs to the class B G-protein-coupled receptors (GPCRs), is a drug target for mental disorders and dry eye syndrome. Here, we present a cryo-EM structure of human PAC1R bound to PACAP and an engineered G heterotrimer. The structure revealed that transmembrane helix TM1 plays an essential role in PACAP recognition. The extracellular domain (ECD) of PAC1R tilts by ~40° compared with that of the glucagon-like peptide-1 receptor (GLP-1R) and thus does not cover the peptide ligand. A functional analysis demonstrated that the PAC1R ECD functions as an affinity trap and is not required for receptor activation, whereas the GLP-1R ECD plays an indispensable role in receptor activation, illuminating the functional diversity of the ECDs in class B GPCRs. Our structural information will facilitate the design and improvement of better PAC1R agonists for clinical applications.
History
DepositionJan 9, 2020Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Mar 11, 2020Provider: repository / Type: Initial release
Revision 1.1Mar 25, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Oct 9, 2024Group: Data collection / Database references ...Data collection / Database references / Refinement description / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / em_admin / pdbx_entry_details / pdbx_initial_refinement_model / pdbx_modification_feature
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_admin.last_update

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Structure visualization

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Assembly

Deposited unit
P: Pituitary adenylate cyclase-activating polypeptide
R: Pituitary adenylate cyclase-activating polypeptide type I receptor
B: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
A: Guanine nucleotide-binding protein G(s) subunit alpha isoforms short,Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
N: nanobody Nb35
G: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2


Theoretical massNumber of molelcules
Total (without water)141,4956
Polymers141,4956
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
SymmetryPoint symmetry: (Schoenflies symbol: C2 (2 fold cyclic))
Noncrystallographic symmetry (NCS)NCS oper:
IDCodeMatrixVector
1given(1), (1), (1)
2generate(-1), (-1), (1)250.38, 250.38

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Components

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

#1: Protein/peptide Pituitary adenylate cyclase-activating polypeptide / PACAP


Mass: 4547.336 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human) / References: UniProt: P18509
#2: Protein Pituitary adenylate cyclase-activating polypeptide type I receptor / PACAP-R1


Mass: 46675.520 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ADCYAP1R1 / Production host: Homo sapiens (human) / References: UniProt: P41586

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

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


Mass: 38744.371 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Gnb1 / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P54311
#4: Protein Guanine nucleotide-binding protein G(s) subunit alpha isoforms short,Guanine nucleotide-binding protein G(s) subunit alpha isoforms short


Mass: 28964.734 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli) / References: UniProt: P63092*PLUS
#6: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 / G gamma-I


Mass: 7547.685 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bos taurus (domestic cattle) / Gene: GNG2 / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P63212

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Antibody , 1 types, 1 molecules N

#5: Antibody nanobody Nb35


Mass: 15015.728 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) unidentified (others) / Production host: Escherichia coli (E. coli)

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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

ComponentName: PAC1R-Gs trimer / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER/RHODIUM / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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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: OTHER
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 64 e/Å2 / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.14_3260: / Classification: refinement
EM softwareName: PHENIX / Version: 1.14 / Category: model refinement / Details: phenix.real_space_refine
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 132808 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER
Atomic model buildingPDB-ID: 5VAI

5vai


Accession code: 5VAI / Source name: PDB / Type: experimental model

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