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- PDB-8gw8: the human PTH1 receptor bound to an intracellular biased agonist -

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Basic information

Entry
Database: PDB / ID: 8gw8
Titlethe human PTH1 receptor bound to an intracellular biased agonist
Components
  • (Guanine nucleotide-binding protein ...) x 2
  • Isoform Gnas-2 of Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
  • Nanobody-35Single-domain antibody
  • Parathyroid hormone/parathyroid hormone-related peptide receptor
KeywordsSIGNALING PROTEIN / G protein-coupled receptor / membrane protein
Function / homology
Function and homology information


parathyroid hormone receptor 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 ...parathyroid hormone receptor 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 / Ca2+ pathway / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / G protein-coupled peptide receptor activity / G alpha (z) signalling events / Vasopressin regulates renal water homeostasis via Aquaporins / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / Adrenaline,noradrenaline inhibits insulin secretion / ADP signalling through P2Y purinoceptor 12 / G alpha (q) signalling events / G alpha (i) signalling events / Class B/2 (Secretin family receptors) / Thrombin signalling through proteinase activated receptors (PARs) / 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 / Glucagon-type ligand receptors / alkylglycerophosphoethanolamine phosphodiesterase activity / Adrenaline,noradrenaline inhibits insulin secretion / G alpha (12/13) signalling events / G beta:gamma signalling through BTK / ADP signalling through P2Y purinoceptor 12 / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / Thrombin signalling through proteinase activated receptors (PARs) / Ca2+ pathway / osteoblast development / G alpha (z) signalling events / Extra-nuclear estrogen signaling / G alpha (s) signalling events / G alpha (q) signalling events / positive regulation of inositol phosphate biosynthetic process / photoreceptor outer segment membrane / G alpha (i) signalling events / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / spectrin binding / Vasopressin regulates renal water homeostasis via Aquaporins / bone mineralization / peptide hormone binding / PKA activation in glucagon signalling / G protein-coupled receptor signaling pathway, coupled to cyclic nucleotide second messenger / hair follicle placode formation / photoreceptor outer segment / developmental growth / intracellular transport / D1 dopamine receptor binding / positive regulation of cAMP-mediated signaling / chondrocyte differentiation / Hedgehog 'off' state / adenylate cyclase-activating adrenergic receptor signaling pathway / cell maturation / bone resorption / cardiac muscle cell apoptotic process / activation of adenylate cyclase activity / adenylate cyclase activator activity / photoreceptor inner segment / trans-Golgi network membrane / skeletal system development / G-protein beta/gamma-subunit complex binding / bone development / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / Prostacyclin signalling through prostacyclin receptor / Glucagon signaling in metabolic regulation / adenylate cyclase-activating G protein-coupled receptor signaling pathway / cognition / platelet aggregation / positive regulation of GTPase activity / Glucagon-type ligand receptors / Vasopressin regulates renal water homeostasis via Aquaporins / G alpha (z) signalling events / cellular response to catecholamine stimulus / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / intracellular calcium ion homeostasis / ADORA2B mediated anti-inflammatory cytokines production / sensory perception of taste / adenylate cyclase-activating dopamine receptor signaling pathway / cellular response to prostaglandin E stimulus / GPER1 signaling / G-protein beta-subunit binding / heterotrimeric G-protein complex / : / sensory perception of smell / signaling receptor complex adaptor activity / retina development in camera-type eye / GTPase binding
Similarity search - Function
GPCR, family 2, parathyroid hormone receptor / 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 ...GPCR, family 2, parathyroid hormone receptor / 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 / 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 / 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. / WD domain, G-beta repeat / 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
PCO-371 / 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 / Parathyroid hormone/parathyroid hormone-related peptide receptor
Similarity search - Component
Biological speciesHomo sapiens (human)
Rattus norvegicus (Norway rat)
Bos taurus (cattle)
unidentified (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsKobayashi, K. / Kusakizako, T. / Okamoto, H.H. / Nureki, O.
Funding support Japan, 1items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS) Japan
CitationJournal: Nature / Year: 2023
Title: Class B1 GPCR activation by an intracellular agonist.
Authors: Kazuhiro Kobayashi / Kouki Kawakami / Tsukasa Kusakizako / Atsuhiro Tomita / Michihiro Nishimura / Kazuhiro Sawada / Hiroyuki H Okamoto / Suzune Hiratsuka / Gaku Nakamura / Riku Kuwabara / ...Authors: Kazuhiro Kobayashi / Kouki Kawakami / Tsukasa Kusakizako / Atsuhiro Tomita / Michihiro Nishimura / Kazuhiro Sawada / Hiroyuki H Okamoto / Suzune Hiratsuka / Gaku Nakamura / Riku Kuwabara / Hiroshi Noda / Hiroyasu Muramatsu / Masaru Shimizu / Tomohiko Taguchi / Asuka Inoue / Takeshi Murata / Osamu Nureki /
Abstract: G protein-coupled receptors (GPCRs) generally accommodate specific ligands in the orthosteric-binding pockets. Ligand binding triggers a receptor allosteric conformational change that leads to the ...G protein-coupled receptors (GPCRs) generally accommodate specific ligands in the orthosteric-binding pockets. Ligand binding triggers a receptor allosteric conformational change that leads to the activation of intracellular transducers, G proteins and β-arrestins. Because these signals often induce adverse effects, the selective activation mechanism for each transducer must be elucidated. Thus, many orthosteric-biased agonists have been developed, and intracellular-biased agonists have recently attracted broad interest. These agonists bind within the receptor intracellular cavity and preferentially tune the specific signalling pathway over other signalling pathways, without allosteric rearrangement of the receptor from the extracellular side. However, only antagonist-bound structures are currently available, and there is no evidence to support that biased agonist binding occurs within the intracellular cavity. This limits the comprehension of intracellular-biased agonism and potential drug development. Here we report the cryogenic electron microscopy structure of a complex of G and the human parathyroid hormone type 1 receptor (PTH1R) bound to a PTH1R agonist, PCO371. PCO371 binds within an intracellular pocket of PTH1R and directly interacts with G. The PCO371-binding mode rearranges the intracellular region towards the active conformation without extracellularly induced allosteric signal propagation. PCO371 stabilizes the significantly outward-bent conformation of transmembrane helix 6, which facilitates binding to G proteins rather than β-arrestins. Furthermore, PCO371 binds within the highly conserved intracellular pocket, activating 7 out of the 15 class B1 GPCRs. Our study identifies a new and conserved intracellular agonist-binding pocket and provides evidence of a biased signalling mechanism that targets the receptor-transducer interface.
History
DepositionSep 16, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jun 14, 2023Provider: repository / Type: Initial release
Revision 1.1Jun 21, 2023Group: Database references / Category: citation / citation_author / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Jul 12, 2023Group: Database references / Refinement description / Category: citation / citation_author / refine
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID / _refine.ls_d_res_high / _refine.ls_d_res_low
Revision 1.3Sep 27, 2023Group: Data collection / Derived calculations / Structure summary
Category: chem_comp / chem_comp_atom ...chem_comp / chem_comp_atom / chem_comp_bond / entity / pdbx_entity_nonpoly
Item: _chem_comp.name / _chem_comp.pdbx_synonyms ..._chem_comp.name / _chem_comp.pdbx_synonyms / _entity.pdbx_description / _pdbx_entity_nonpoly.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Isoform Gnas-2 of 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: Nanobody-35
R: Parathyroid hormone/parathyroid hormone-related peptide receptor
hetero molecules


Theoretical massNumber of molelcules
Total (without water)157,1516
Polymers156,5155
Non-polymers6361
Water0
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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Protein , 2 types, 2 molecules AR

#1: Protein Isoform Gnas-2 of Guanine nucleotide-binding protein G(s) subunit alpha isoforms short / Adenylate cyclase-stimulating G alpha protein


Mass: 42073.574 Da / Num. of mol.: 1 / Mutation: G49D,E50N,A249D,S252D,I362A,V365I
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNAS, GNAS1, GSP / Production host: Escherichia coli (E. coli) / References: UniProt: P63092
#5: Protein Parathyroid hormone/parathyroid hormone-related peptide receptor / PTH/PTHrP type I receptor / PTH/PTHr receptor / Parathyroid hormone 1 receptor / PTH1 receptor


Mass: 54264.359 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PTH1R, PTHR, PTHR1 / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: Q03431

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Guanine nucleotide-binding protein ... , 2 types, 2 molecules BG

#2: 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 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P54311
#3: 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 (cattle) / Gene: GNG2 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P63212

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

#4: Antibody Nanobody-35 / Single-domain antibody


Mass: 13885.439 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) unidentified (others) / Production host: Escherichia coli (E. coli)
#6: Chemical ChemComp-KHF / PCO-371 / 1-[3,5-dimethyl-4-[2-[[4-oxidanylidene-2-[4-(trifluoromethyloxy)phenyl]-1,3,8-triazaspiro[4.5]dec-1-en-8-yl]sulfonyl]ethyl]phenyl]-5,5-dimethyl-imidazolidine-2,4-dione


Mass: 635.654 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C29H32F3N5O6S

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Details

Has ligand of interestN

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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
1the human PTH1 receptor bound to an intracellular biased agonistCOMPLEX#1-#50RECOMBINANT
2Guanine nucleotide-binding protein G(s) subunit alpha isoforms shortCOMPLEX#11RECOMBINANT
3Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1COMPLEX#21RECOMBINANT
4Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2COMPLEX#31RECOMBINANT
5nanobody Nb35COMPLEX#41RECOMBINANT
6Parathyroid hormone/parathyroid hormone-related peptide receptorCOMPLEX#51RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Rattus norvegicus (Norway rat)10116
34Bos taurus (cattle)9913
45unidentified (others)32644
56Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli (E. coli)562
23Spodoptera frugiperda (fall armyworm)7108
34Spodoptera frugiperda (fall armyworm)7108
46Homo sapiens (human)9606
55Escherichia coli (E. coli)562
Buffer solutionpH: 9
SpecimenConc.: 7 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: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1500 nm / Nominal defocus min: 600 nm
Image recordingElectron dose: 54.578 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19_4092: / Classification: refinement
EM software
IDNameVersionCategory
1RELION3.1particle selection
4CTFFIND4.1CTF correction
10RELION3.1initial Euler assignment
11RELION3.1final Euler assignment
12RELION3.1classification
13RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 109422 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
RefinementResolution: 2.9→2.9 Å / Cor.coef. Fo:Fc: 0.896 / SU B: 7.491 / SU ML: 0.144 / ESU R: 0.208
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.33401 --
obs0.33401 165424 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 92.918 Å2
Refinement stepCycle: 1 / Total: 7716
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0027873
ELECTRON MICROSCOPYf_angle_d0.45810674
ELECTRON MICROSCOPYf_dihedral_angle_d4.6691102
ELECTRON MICROSCOPYf_chiral_restr0.0391199
ELECTRON MICROSCOPYf_plane_restr0.0041361
LS refinement shellResolution: 2.6→2.668 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork0.955 12199 -
obs--100 %

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