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- EMDB-46448: CryoEM structure of PAR2 with endogenous tethered ligand. -

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Entry
Database: EMDB / ID: EMD-46448
TitleCryoEM structure of PAR2 with endogenous tethered ligand.
Map data
Sample
  • Complex: Complex of PAR2 with G proteins
    • Protein or peptide: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
    • Protein or peptide: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
    • Protein or peptide: scFv16
    • Protein or peptide: Proteinase-activated receptor 2
    • Protein or peptide: Guanine nucleotide-binding protein G(q) subunit alpha, Guanine nucleotide-binding protein G(s) subunit alpha isoforms short chimera,Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
KeywordsPAR2 / endogenous ligand / IMMUNE SYSTEM
Function / homology
Function and homology information


positive regulation of neutrophil mediated killing of gram-negative bacterium / positive regulation of renin secretion into blood stream / positive regulation of eosinophil degranulation / mature conventional dendritic cell differentiation / leukocyte proliferation / positive regulation of glomerular filtration / regulation of chemokine (C-X-C motif) ligand 2 production / negative regulation of toll-like receptor 3 signaling pathway / positive regulation of toll-like receptor 3 signaling pathway / thrombin-activated receptor activity ...positive regulation of neutrophil mediated killing of gram-negative bacterium / positive regulation of renin secretion into blood stream / positive regulation of eosinophil degranulation / mature conventional dendritic cell differentiation / leukocyte proliferation / positive regulation of glomerular filtration / regulation of chemokine (C-X-C motif) ligand 2 production / negative regulation of toll-like receptor 3 signaling pathway / positive regulation of toll-like receptor 3 signaling pathway / thrombin-activated receptor activity / positive regulation of toll-like receptor 2 signaling pathway / positive regulation of actin filament depolymerization / cell-cell junction maintenance / Fatty Acids bound to GPR40 (FFAR1) regulate insulin secretion / Acetylcholine regulates insulin secretion / phospholipase C-activating G protein-coupled glutamate receptor signaling pathway / positive regulation of toll-like receptor 4 signaling pathway / positive regulation of pseudopodium assembly / phospholipase C-activating serotonin receptor signaling pathway / T cell activation involved in immune response / PLC beta mediated events / positive regulation of cytokine production involved in immune response / regulation of platelet activation / entrainment of circadian clock / positive regulation of phagocytosis, engulfment / negative regulation of chemokine production / neutrophil activation / positive regulation of leukocyte chemotaxis / positive regulation of chemotaxis / establishment of endothelial barrier / positive regulation of positive chemotaxis / regulation of JNK cascade / regulation of canonical NF-kappaB signal transduction / regulation of canonical Wnt signaling pathway / negative regulation of JNK cascade / leukocyte migration / glutamate receptor signaling pathway / regulation of blood coagulation / positive regulation of Rho protein signal transduction / pseudopodium / positive regulation of interleukin-10 production / phototransduction, visible light / positive regulation of GTPase activity / PKA activation in glucagon signalling / developmental growth / G-protein alpha-subunit binding / hair follicle placode formation / photoreceptor outer segment / negative regulation of tumor necrosis factor-mediated signaling pathway / neuropeptide signaling pathway / D1 dopamine receptor binding / intracellular transport / postsynaptic cytosol / vascular endothelial cell response to laminar fluid shear stress / renal water homeostasis / activation of adenylate cyclase activity / Hedgehog 'off' state / positive regulation of chemokine production / positive regulation of superoxide anion generation / adenylate cyclase-activating adrenergic receptor signaling pathway / enzyme regulator activity / response to prostaglandin E / cellular response to glucagon stimulus / regulation of insulin secretion / GTPase activator activity / Peptide ligand-binding receptors / adenylate cyclase activator activity / Turbulent (oscillatory, disturbed) flow shear stress activates signaling by PIEZO1 and integrins in endothelial cells / positive regulation of interleukin-1 beta production / trans-Golgi network membrane / positive regulation of interleukin-8 production / positive regulation of JNK cascade / negative regulation of inflammatory response to antigenic stimulus / negative regulation of insulin secretion / G protein-coupled receptor binding / G protein-coupled receptor activity / bone development / positive regulation of interleukin-6 production / platelet aggregation / G-protein beta/gamma-subunit complex binding / vasodilation / cognition / positive regulation of type II interferon production / Olfactory Signaling Pathway / Activation of the phototransduction cascade / adenylate cyclase-activating G protein-coupled receptor signaling pathway / 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 / Prostacyclin signalling through prostacyclin receptor / G beta:gamma signalling through CDC42 / Glucagon signaling in metabolic regulation / G beta:gamma signalling through BTK / blood coagulation / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / ADP signalling through P2Y purinoceptor 12
Similarity search - Function
Protease-activated receptor 2 / Protease-activated receptor / G-protein alpha subunit, group Q / 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 ...Protease-activated receptor 2 / Protease-activated receptor / G-protein alpha subunit, group Q / 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-coupled receptor, rhodopsin-like / GPCR, rhodopsin-like, 7TM / G-protein coupled receptors family 1 profile. / 7 transmembrane receptor (rhodopsin family) / 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
Guanine nucleotide-binding protein G(q) subunit alpha / Proteinase-activated receptor 2 / 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) / unidentified (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsLyu Z / Lyu X / McGrath AP / Kang Y
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: Nat Commun / Year: 2025
Title: Structural basis for the activation of proteinase-activated receptors PAR1 and PAR2.
Authors: Zongyang Lyu / Xiaoxuan Lyu / Andrey G Malyutin / Guliang Xia / Daniel Carney / Vinicius M Alves / Matthew Falk / Nidhi Arora / Hua Zou / Aaron P McGrath / Yanyong Kang /
Abstract: Members of the proteinase-activated receptor (PAR) subfamily of G protein-coupled receptors (GPCRs) play critical roles in processes like hemostasis, thrombosis, development, wound healing, ...Members of the proteinase-activated receptor (PAR) subfamily of G protein-coupled receptors (GPCRs) play critical roles in processes like hemostasis, thrombosis, development, wound healing, inflammation, and cancer progression. Comprising PAR1-PAR4, these receptors are specifically activated by protease cleavage at their extracellular amino terminus, revealing a 'tethered ligand' that self-activates the receptor. This triggers complex intracellular signaling via G proteins and beta-arrestins, linking external protease signals to cellular functions. To date, direct structural visualization of these ligand-receptor complexes has been limited. Here, we present structural snapshots of activated PAR1 and PAR2 bound to their endogenous tethered ligands, revealing a shallow and constricted orthosteric binding pocket. Comparisons with antagonist-bound structures show minimal conformational changes in the TM6 helix and larger movements of TM7 upon activation. These findings reveal a common activation mechanism for PAR1 and PAR2, highlighting critical residues involved in ligand recognition. Additionally, the structure of PAR2 bound to a pathway selective antagonist, GB88, demonstrates how potent orthosteric engagement can be achieved by a small molecule mimicking the endogenous tethered ligand's interactions.
History
DepositionAug 6, 2024-
Header (metadata) releaseMay 7, 2025-
Map releaseMay 7, 2025-
UpdateMay 28, 2025-
Current statusMay 28, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_46448.png

Downloads & links

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Map

FileDownload / File: emd_46448.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.92 Å/pix.
x 240 pix.
= 220.8 Å		0.92 Å/pix.
x 240 pix.
= 220.8 Å		0.92 Å/pix.
x 240 pix.
= 220.8 Å

Surface

Projections

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.92 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.1
Minimum - Maximum-0.0016830396 - 1.9832155
Average (Standard dev.)0.0018773864 (±0.02974142)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 220.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_46448_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

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Histogram (log scale)

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Half map: #1

Fileemd_46448_half_map_2.map
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Sample components

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Entire : Complex of PAR2 with G proteins

EntireName: Complex of PAR2 with G proteins
Components
  • Complex: Complex of PAR2 with G proteins
    • Protein or peptide: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
    • Protein or peptide: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
    • Protein or peptide: scFv16
    • Protein or peptide: Proteinase-activated receptor 2
    • Protein or peptide: Guanine nucleotide-binding protein G(q) subunit alpha, Guanine nucleotide-binding protein G(s) subunit alpha isoforms short chimera,Guanine nucleotide-binding protein G(s) subunit alpha isoforms short

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Supramolecule #1: Complex of PAR2 with G proteins

SupramoleculeName: Complex of PAR2 with G proteins / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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

MacromoleculeName: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 37.285734 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: SELDQLRQEA EQLKNQIRDA RKACADATLS QITNNIDPVG RIQMRTRRTL RGHLAKIYAM HWGTDSRLLV SASQDGKLII WDSYTTNKV HAIPLRSSWV MTCAYAPSGN YVACGGLDNI CSIYNLKTRE GNVRVSRELA GHTGYLSCCR FLDDNQIVTS S GDTTCALW ...String:
SELDQLRQEA EQLKNQIRDA RKACADATLS QITNNIDPVG RIQMRTRRTL RGHLAKIYAM HWGTDSRLLV SASQDGKLII WDSYTTNKV HAIPLRSSWV MTCAYAPSGN YVACGGLDNI CSIYNLKTRE GNVRVSRELA GHTGYLSCCR FLDDNQIVTS S GDTTCALW DIETGQQTTT FTGHTGDVMS LSLAPDTRLF VSGACDASAK LWDVREGMCR QTFTGHESDI NAICFFPNGN AF ATGSDDA TCRLFDLRAD QELMTYSHDN IICGITSVSF SKSGRLLLAG YDDFNCNVWD ALKADRAGVL AGHDNRVSCL GVT DDGMAV ATGSWDSFLK IWN

UniProtKB: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1

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Macromolecule #2: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2

MacromoleculeName: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 6.504446 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString:
NTASIAQARK LVEQLKMEAN IDRIKVSKAA ADLMAYCEAH AKEDPLLTPV PASENPFRE

UniProtKB: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2

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Macromolecule #3: scFv16

MacromoleculeName: scFv16 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: unidentified (others)
Molecular weightTheoretical: 26.679721 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: DVQLVESGGG LVQPGGSRKL SCSASGFAFS SFGMHWVRQA PEKGLEWVAY ISSGSGTIYY ADTVKGRFTI SRDDPKNTLF LQMTSLRSE DTAMYYCVRS IYYYGSSPFD FWGQGTTLTV SSGGGGSGGG GSGGGGSDIV MTQATSSVPV TPGESVSISC R SSKSLLHS ...String:
DVQLVESGGG LVQPGGSRKL SCSASGFAFS SFGMHWVRQA PEKGLEWVAY ISSGSGTIYY ADTVKGRFTI SRDDPKNTLF LQMTSLRSE DTAMYYCVRS IYYYGSSPFD FWGQGTTLTV SSGGGGSGGG GSGGGGSDIV MTQATSSVPV TPGESVSISC R SSKSLLHS NGNTYLYWFL QRPGQSPQLL IYRMSNLASG VPDRFSGSGS GTAFTLTISR LEAEDVGVYY CMQHLEYPLT FG AGTKLEL KAAA

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Macromolecule #4: Proteinase-activated receptor 2

MacromoleculeName: Proteinase-activated receptor 2 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 44.173203 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MRSPSAAWLL GAAILLAASL SCSGTIQGTN RSSKGRSLIG KVDGTSHVTG KGVTVETVFS VDEFSASVLT GKLTTVFLPI VYTIVFVVG LPSNGMALWV FLFRTKKKHP AVIYMANLAL ADLLSVIWFP LKIAYHIHGN NWIYGEALCN VLIGFFYGNM Y CSILFMTC ...String:
MRSPSAAWLL GAAILLAASL SCSGTIQGTN RSSKGRSLIG KVDGTSHVTG KGVTVETVFS VDEFSASVLT GKLTTVFLPI VYTIVFVVG LPSNGMALWV FLFRTKKKHP AVIYMANLAL ADLLSVIWFP LKIAYHIHGN NWIYGEALCN VLIGFFYGNM Y CSILFMTC LSVQRYWVIV NPMGHSRKKA NIAIGISLAI WLLILLVTIP LYVVKQTIFI PALNITTCHD VLPEQLLVGD MF NYFLSLA IGVFLFPAFL TASAYVLMIR MLRSSAMDEN SEKKRKRAIK LIVTVLAMYL ICFTPSNLLL VVHYFLIKSQ GQS HVYALY IVALCLSTLN SCIDPFVYYF VSHDFRDHAK NALLCRSVRT VKQMQVSLTS KKHSRKSSSY SSSSTTVKTS Y

UniProtKB: Proteinase-activated receptor 2

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Macromolecule #5: Guanine nucleotide-binding protein G(q) subunit alpha, Guanine nu...

MacromoleculeName: Guanine nucleotide-binding protein G(q) subunit alpha, Guanine nucleotide-binding protein G(s) subunit alpha isoforms short chimera,Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 41.972578 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MGCTLSAEDK AAVERSKMID RNLREDGEKA RRELKLLLLG TGESGKSTFI KQMRIIHGSG YSDEDKRGFT KLVYQNIFTA MQAMIRAMD TLKIPYKYEH NKAHAQLVRE VDVEKVSAFE NPYVDAIKSL WNDPGIQECY DRRREYQLSD STKYYLNDLD R VADPAYLP ...String:
MGCTLSAEDK AAVERSKMID RNLREDGEKA RRELKLLLLG TGESGKSTFI KQMRIIHGSG YSDEDKRGFT KLVYQNIFTA MQAMIRAMD TLKIPYKYEH NKAHAQLVRE VDVEKVSAFE NPYVDAIKSL WNDPGIQECY DRRREYQLSD STKYYLNDLD R VADPAYLP TQQDVLRVRV PTTGIIEYPF DLQKVNFHMF DVGGQRSERR KWIQCFNDVT AIIFVVDSSD YNRLQEALND FK SIWNNRW LRTISVILFL NKQDLLAEKV LAGKSKIEDY FPEFARYTTP EDATPEPGED PRVTRAKYFI RKEFVDISTA SGD GRHICY PHFTCAVDTE NARRIFNDCK DIILQMNLRE YNLV

UniProtKB: Guanine nucleotide-binding protein G(q) subunit alpha, Guanine nucleotide-binding protein G(s) subunit alpha isoforms short

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7
GridModel: Quantifoil / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Support film - Film thickness: 50 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec.
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS GLACIOS
Image recordingFilm or detector model: TFS FALCON 4i (4k x 4k) / Average electron dose: 45.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 404635
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
FSC plot (resolution estimation)

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