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- PDB-8g94: Structure of CD69-bound S1PR1 coupled to heterotrimeric Gi -

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Entry
Database: PDB / ID: 8g94
TitleStructure of CD69-bound S1PR1 coupled to heterotrimeric Gi
Components
  • (Guanine nucleotide-binding protein ...) x 3
  • Early activation antigen CD69
  • Sphingosine 1-phosphate receptor 1
  • scFv16
KeywordsGuanine nucleotide-binding protein / CD69 / S1PR1 / Sphingosine-1-phosphate / lymphocyte egress / GPCR
Function / homology
Function and homology information


negative regulation of T cell migration / cardiac muscle tissue growth involved in heart morphogenesis / blood vessel maturation / sphingolipid binding / sphingosine-1-phosphate receptor activity / Lysosphingolipid and LPA receptors / negative regulation of T-helper 17 cell lineage commitment / endothelial cell differentiation / heart trabecula morphogenesis / regulation of bone mineralization ...negative regulation of T cell migration / cardiac muscle tissue growth involved in heart morphogenesis / blood vessel maturation / sphingolipid binding / sphingosine-1-phosphate receptor activity / Lysosphingolipid and LPA receptors / negative regulation of T-helper 17 cell lineage commitment / endothelial cell differentiation / heart trabecula morphogenesis / regulation of bone mineralization / sphingosine-1-phosphate receptor signaling pathway / leukocyte chemotaxis / regulation of bone resorption / positive regulation of positive chemotaxis / negative regulation of stress fiber assembly / lamellipodium assembly / regulation of metabolic process / transmission of nerve impulse / molecular sequestering activity / adenylate cyclase inhibitor activity / regulation of cell adhesion / positive regulation of protein localization to cell cortex / T cell migration / Adenylate cyclase inhibitory pathway / D2 dopamine receptor binding / response to prostaglandin E / adenylate cyclase regulator activity / G protein-coupled serotonin receptor binding / adenylate cyclase-inhibiting serotonin receptor signaling pathway / positive regulation of smooth muscle cell proliferation / cellular response to forskolin / regulation of mitotic spindle organization / Regulation of insulin secretion / positive regulation of cholesterol biosynthetic process / negative regulation of insulin secretion / G protein-coupled receptor activity / G protein-coupled receptor binding / response to peptide hormone / adenylate cyclase-inhibiting G protein-coupled receptor signaling pathway / brain development / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / centriolar satellite / G-protein beta/gamma-subunit complex binding / Olfactory Signaling Pathway / Activation of the phototransduction cascade / cellular response to xenobiotic stimulus / G beta:gamma signalling through PLC beta / Presynaptic function of Kainate receptors / Thromboxane signalling through TP receptor / G protein-coupled acetylcholine receptor signaling pathway / neuron differentiation / 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 / chemotaxis / G beta:gamma signalling through BTK / 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 / GDP binding / G alpha (z) signalling events / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / ADP signalling through P2Y purinoceptor 1 / cellular response to catecholamine stimulus / ADORA2B mediated anti-inflammatory cytokines production / G beta:gamma signalling through PI3Kgamma / transmembrane signaling receptor activity / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / adenylate cyclase-activating dopamine receptor signaling pathway / cell migration / 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 / sensory perception of taste / extracellular vesicle / presynapse / signaling receptor complex adaptor activity / Thrombin signalling through proteinase activated receptors (PARs) / retina development in camera-type eye / G protein activity / GTPase binding / carbohydrate binding / Ca2+ pathway / actin cytoskeleton organization / midbody / fibroblast proliferation / High laminar flow shear stress activates signaling by PIEZO1 and PECAM1:CDH5:KDR in endothelial cells / cell cortex / angiogenesis
Similarity search - Function
: / EDG-1 sphingosine 1-phosphate receptor / Sphingosine 1-phosphate receptor / Natural killer cell receptor-like, C-type lectin-like domain / Lectin C-type domain / C-type lectin domain profile. / C-type lectin-like / C-type lectin (CTL) or carbohydrate-recognition domain (CRD) / C-type lectin-like/link domain superfamily / Rhopdopsin 7-helix transmembrane proteins ...: / EDG-1 sphingosine 1-phosphate receptor / Sphingosine 1-phosphate receptor / Natural killer cell receptor-like, C-type lectin-like domain / Lectin C-type domain / C-type lectin domain profile. / C-type lectin-like / C-type lectin (CTL) or carbohydrate-recognition domain (CRD) / C-type lectin-like/link domain superfamily / Rhopdopsin 7-helix transmembrane proteins / Rhodopsin 7-helix transmembrane proteins / C-type lectin fold / G-protein alpha subunit, group I / YVTN repeat-like/Quinoprotein amine dehydrogenase / Serpentine type 7TM GPCR chemoreceptor Srsx / 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 coupled receptors family 1 signature. / 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 / Immunoglobulins / Up-down Bundle / Immunoglobulin-like / Sandwich / P-loop containing nucleoside triphosphate hydrolase / Mainly Beta / Mainly Alpha
Similarity search - Domain/homology
Sphingosine 1-phosphate receptor 1 / 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(i) subunit alpha-1 / Early activation antigen CD69
Similarity search - Component
Biological speciesHomo sapiens (human)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.15 Å
AuthorsChen, H. / Li, X.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM135343 United States
Welch FoundationI-1957 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)P01 HL160487-01 United States
CitationJournal: Elife / Year: 2023
Title: Transmembrane protein CD69 acts as an S1PR1 agonist.
Authors: Hongwen Chen / Yu Qin / Marissa Chou / Jason G Cyster / Xiaochun Li /
Abstract: The activation of Sphingosine-1-phosphate receptor 1 (S1PR1) by S1P promotes lymphocyte egress from lymphoid organs, a process critical for immune surveillance and T cell effector activity. Multiple ...The activation of Sphingosine-1-phosphate receptor 1 (S1PR1) by S1P promotes lymphocyte egress from lymphoid organs, a process critical for immune surveillance and T cell effector activity. Multiple drugs that inhibit S1PR1 function are in use clinically for the treatment of autoimmune diseases. Cluster of Differentiation 69 (CD69) is an endogenous negative regulator of lymphocyte egress that interacts with S1PR1 in cis to facilitate internalization and degradation of the receptor. The mechanism by which CD69 causes S1PR1 internalization has been unclear. Moreover, although there are numerous class A GPCR structures determined with different small molecule agonists bound, it remains unknown whether a transmembrane protein per se can act as a class A GPCR agonist. Here, we present the cryo-EM structure of CD69-bound S1PR1 coupled to the heterotrimeric G complex. The transmembrane helix (TM) of one protomer of CD69 homodimer contacts the S1PR1-TM4. This interaction allosterically induces the movement of S1PR1-TMs 5-6, directly activating the receptor to engage the heterotrimeric G. Mutations in key residues at the interface affect the interactions between CD69 and S1PR1, as well as reduce the receptor internalization. Thus, our structural findings along with functional analyses demonstrate that CD69 acts in cis as a protein agonist of S1PR1, thereby promoting G-dependent S1PR1 internalization, loss of S1P gradient sensing, and inhibition of lymphocyte egress.
History
DepositionFeb 21, 2023Deposition site: RCSB / Processing site: RCSB
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Revision 1.1Apr 26, 2023Group: Database references / Category: citation
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Sphingosine 1-phosphate receptor 1
B: Guanine nucleotide-binding protein G(i) subunit alpha-1
C: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
D: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
E: scFv16
F: Early activation antigen CD69
G: Early activation antigen CD69


Theoretical massNumber of molelcules
Total (without water)201,7297
Polymers201,7297
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 2 types, 3 molecules AFG

#1: Protein Sphingosine 1-phosphate receptor 1 / S1P receptor 1 / S1P1 / Endothelial differentiation G-protein coupled receptor 1 / Sphingosine 1- ...S1P receptor 1 / S1P1 / Endothelial differentiation G-protein coupled receptor 1 / Sphingosine 1-phosphate receptor Edg-1 / S1P receptor Edg-1


Mass: 40083.781 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: S1PR1, CHEDG1, EDG1 / Cell line (production host): HEK293S GnTI / Production host: Homo sapiens (human) / References: UniProt: P21453
#6: Protein Early activation antigen CD69 / Activation inducer molecule / AIM / BL-AC/P26 / C-type lectin domain family 2 member C / EA1 / ...Activation inducer molecule / AIM / BL-AC/P26 / C-type lectin domain family 2 member C / EA1 / Early T-cell activation antigen p60 / GP32/28 / Leukocyte surface antigen Leu-23 / MLR-3


Mass: 23912.793 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CD69, CLEC2C / Production host: Homo sapiens (human) / References: UniProt: Q07108

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

#2: Protein Guanine nucleotide-binding protein G(i) subunit alpha-1 / Adenylate cyclase-inhibiting G alpha protein


Mass: 40445.059 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNAI1 / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P63096
#3: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Transducin beta chain 1


Mass: 37728.152 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNB1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P62873
#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: Spodoptera frugiperda (fall armyworm) / References: UniProt: P59768

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Antibody , 1 types, 1 molecules E

#5: Antibody scFv16


Mass: 27784.896 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Spodoptera frugiperda (fall armyworm)

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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: Complex of CD69-bound S1PR1 coupled to heterotrimeric Gi
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.2 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293
Buffer solutionpH: 7.5
SpecimenConc.: 10 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 293 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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.16_3549: / Classification: refinement
EM software
IDNameVersionCategory
7UCSF Chimera1.15model fitting
13PHENIX1.16model refinement
CTF correctionType: NONE
3D reconstructionResolution: 3.15 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 293516 / Symmetry type: POINT

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