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- PDB-8e3x: Cryo-EM structure of the PAC1R-PACAP27-Gs complex -

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Entry
Database: PDB / ID: 8e3x
TitleCryo-EM structure of the PAC1R-PACAP27-Gs complex
Components
  • (Guanine nucleotide-binding protein ...) x 3
  • (Pituitary adenylate cyclase-activating ...) x 2
  • Nanobody35
KeywordsMEMBRANE PROTEIN / drug discovery / G protein coupled receptor / signalling
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 / neuropeptide hormone activity / regulation of G protein-coupled receptor signaling pathway / neuropeptide binding / positive regulation of small GTPase mediated signal transduction / G protein-coupled peptide receptor activity / insulin secretion / positive regulation of inositol phosphate biosynthetic process / peptide hormone receptor binding / positive regulation of calcium ion transport into cytosol / negative regulation of cell cycle / positive regulation of cAMP/PKA signal transduction / peptide hormone binding / positive regulation of protein kinase activity / cAMP/PKA signal transduction / adenylate cyclase binding / PKA activation in glucagon signalling / developmental growth / hair follicle placode formation / bicellular tight junction / neuropeptide signaling pathway / D1 dopamine receptor binding / multicellular organismal response to stress / intracellular transport / vascular endothelial cell response to laminar fluid shear stress / renal water homeostasis / activation of adenylate cyclase activity / Hedgehog 'off' state / adenylate cyclase-activating adrenergic receptor signaling pathway / positive regulation of GTPase activity / cellular response to glucagon stimulus / regulation of insulin secretion / adenylate cyclase activator activity / trans-Golgi network membrane / negative regulation of inflammatory response to antigenic stimulus / female pregnancy / bone development / caveola / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / small GTPase binding / G-protein beta/gamma-subunit complex binding / platelet aggregation / Olfactory Signaling Pathway / Activation of the phototransduction cascade / cognition / G beta:gamma signalling through PLC beta / Presynaptic function of Kainate receptors / Thromboxane signalling through TP receptor / G protein-coupled acetylcholine receptor signaling pathway / G-protein activation / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / adenylate cyclase-activating G protein-coupled receptor signaling pathway / Prostacyclin signalling through prostacyclin receptor / G beta:gamma signalling through CDC42 / Glucagon signaling in metabolic regulation / G beta:gamma signalling through BTK / neuron projection development / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / ADP signalling through P2Y purinoceptor 12 / Sensory perception of sweet, bitter, and umami (glutamate) taste / photoreceptor disc membrane / Glucagon-type ligand receptors / Adrenaline,noradrenaline inhibits insulin secretion / Vasopressin regulates renal water homeostasis via Aquaporins / G alpha (z) signalling events / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / cellular response to catecholamine stimulus / ADP signalling through P2Y purinoceptor 1 / ADORA2B mediated anti-inflammatory cytokines production / G beta:gamma signalling through PI3Kgamma / sensory perception of smell / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / adenylate cyclase-activating dopamine receptor signaling pathway / GPER1 signaling / Inactivation, recovery and regulation of the phototransduction cascade / cellular response to prostaglandin E stimulus / G-protein beta-subunit binding / heterotrimeric G-protein complex / G alpha (12/13) signalling events / cell-cell signaling / sensory perception of taste / response to estradiol / extracellular vesicle / signaling receptor activity / signaling receptor complex adaptor activity / Thrombin signalling through proteinase activated receptors (PARs) / regulation of protein localization / positive regulation of cold-induced thermogenesis / retina development in camera-type eye / G protein activity
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. / 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 / 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 / Immunoglobulins / Up-down Bundle / Immunoglobulin-like / Sandwich / P-loop containing nucleoside triphosphate hydrolase / Mainly Beta / Mainly Alpha
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: 2.3 Å
AuthorsPiper, S.J. / Danev, R. / Sexton, P. / Wootten, D.
Funding support Australia, United Kingdom, Japan, 5items
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
Royal Society United Kingdom
Japan Science and Technology Japan
CitationJournal: Nat Commun / Year: 2022
Title: Understanding VPAC receptor family peptide binding and selectivity.
Authors: Sarah J Piper / Giuseppe Deganutti / Jessica Lu / Peishen Zhao / Yi-Lynn Liang / Yao Lu / Madeleine M Fletcher / Mohammed Akhter Hossain / Arthur Christopoulos / Christopher A Reynolds / ...Authors: Sarah J Piper / Giuseppe Deganutti / Jessica Lu / Peishen Zhao / Yi-Lynn Liang / Yao Lu / Madeleine M Fletcher / Mohammed Akhter Hossain / Arthur Christopoulos / Christopher A Reynolds / Radostin Danev / Patrick M Sexton / Denise Wootten /
Abstract: The vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) receptors are key regulators of neurological processes. Despite recent structural data, a ...The vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) receptors are key regulators of neurological processes. Despite recent structural data, a comprehensive understanding of peptide binding and selectivity among different subfamily receptors is lacking. Here, we determine structures of active, Gs-coupled, VIP-VPAC1R, PACAP27-VPAC1R, and PACAP27-PAC1R complexes. Cryo-EM structural analyses and molecular dynamics simulations (MDSs) reveal fewer stable interactions between VPAC1R and VIP than for PACAP27, more extensive dynamics of VIP interaction with extracellular loop 3, and receptor-dependent differences in interactions of conserved N-terminal peptide residues with the receptor core. MD of VIP modelled into PAC1R predicts more transient VIP-PAC1R interactions in the receptor core, compared to VIP-VPAC1R, which may underlie the selectivity of VIP for VPAC1R over PAC1R. Collectively, our work improves molecular understanding of peptide engagement with the PAC1R and VPAC1R that may benefit the development of novel selective agonists.
History
DepositionAug 17, 2022Deposition site: RCSB / Processing site: RCSB
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
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: Pituitary adenylate cyclase-activating polypeptide type I receptor


Theoretical massNumber of molelcules
Total (without water)165,8136
Polymers165,8136
Non-polymers00
Water19811
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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

#1: Protein Guanine nucleotide-binding protein G(s) subunit alpha isoforms short / Adenylate cyclase-stimulating G alpha protein


Mass: 45683.434 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNAS, GNAS1, GSP / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P63092
#2: 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
Source: (gene. exp.) Homo sapiens (human) / Gene: GNB1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P62873
#3: 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: Trichoplusia ni (cabbage looper) / References: UniProt: P59768

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

#5: 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
#6: Protein Pituitary adenylate cyclase-activating polypeptide type I receptor / PACAP-R1


Mass: 55439.207 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ADCYAP1R1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P41586

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Antibody / Non-polymers , 2 types, 12 molecules N

#4: Antibody Nanobody35


Mass: 15140.742 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)
#7: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 11 / Source method: isolated from a natural source / Formula: H2O

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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 Pituitary adenylate cyclase-activating polypeptide type I receptor complex with PACAP27 peptideCOMPLEX#1-#60MULTIPLE 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, Pituitary adenylate cyclase-activating polypeptide type I receptorCOMPLEX#1-#3, #61RECOMBINANT
3Nanobody35COMPLEX#41RECOMBINANT
4Pituitary adenylate cyclase activating polypeptide-27COMPLEX#51RECOMBINANT
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
12Trichoplusia ni (cabbage looper)7111
23Escherichia coli (E. coli)562
34synthetic construct (others)32630
Buffer solutionpH: 7.4
SpecimenConc.: 5.15 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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 FIELD / Nominal defocus max: 1400 nm / Nominal defocus min: 500 nm
Image recordingElectron dose: 52.4 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM softwareName: PHENIX / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 440740 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0029554
ELECTRON MICROSCOPYf_angle_d0.44412935
ELECTRON MICROSCOPYf_dihedral_angle_d14.6181280
ELECTRON MICROSCOPYf_chiral_restr0.0381417
ELECTRON MICROSCOPYf_plane_restr0.0031657

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