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- PDB-8vti: Latrophilin-3 (ADGRL3) HormR and GAIN domains in the context of t... -

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Basic information

Entry
Database: PDB / ID: 8vti
TitleLatrophilin-3 (ADGRL3) HormR and GAIN domains in the context of the holoreceptor
Components
  • (Isoform 4 of Adhesion G protein-coupled receptor ...) x 2
  • sAB Heavy Chain
  • sAB Light Chain
KeywordsSIGNALING PROTEIN / adhesion GPCR / GAIN domain / cell-cell adhesion / synapse
Function / homology
Function and homology information


cell-cell adhesion via plasma-membrane adhesion molecules / synapse assembly / neuron migration / G protein-coupled receptor activity / adenylate cyclase-activating G protein-coupled receptor signaling pathway / cell-cell junction / carbohydrate binding / cell surface receptor signaling pathway / G protein-coupled receptor signaling pathway / axon ...cell-cell adhesion via plasma-membrane adhesion molecules / synapse assembly / neuron migration / G protein-coupled receptor activity / adenylate cyclase-activating G protein-coupled receptor signaling pathway / cell-cell junction / carbohydrate binding / cell surface receptor signaling pathway / G protein-coupled receptor signaling pathway / axon / calcium ion binding / glutamatergic synapse / membrane / plasma membrane
Similarity search - Function
GPCR, family 2, latrophilin, C-terminal / GPCR, family 2, latrophilin / Latrophilin Cytoplasmic C-terminal region / D-galactoside/L-rhamnose binding SUEL lectin domain superfamily / D-galactoside/L-rhamnose binding SUEL lectin domain / D-galactoside/L-rhamnose binding SUEL lectin domain / SUEL-type lectin domain profile. / GAIN domain, N-terminal / GPCR-Autoproteolysis INducing (GAIN) domain / Olfactomedin-like domain ...GPCR, family 2, latrophilin, C-terminal / GPCR, family 2, latrophilin / Latrophilin Cytoplasmic C-terminal region / D-galactoside/L-rhamnose binding SUEL lectin domain superfamily / D-galactoside/L-rhamnose binding SUEL lectin domain / D-galactoside/L-rhamnose binding SUEL lectin domain / SUEL-type lectin domain profile. / GAIN domain, N-terminal / GPCR-Autoproteolysis INducing (GAIN) domain / Olfactomedin-like domain / Olfactomedin-like domain / Olfactomedin-like domain profile. / Olfactomedin-like domains / GAIN domain superfamily / GPCR proteolysis site, GPS, motif / GPS motif / GAIN-B domain profile. / G-protein-coupled receptor proteolytic site domain / 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.
Similarity search - Domain/homology
Adhesion G protein-coupled receptor L3
Similarity search - Component
Biological speciesHomo sapiens (human)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsKordon, S.P. / Bandekar, S.J. / Arac, D.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35 GM148412 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM134035-01 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)F32 GM142266 United States
CitationJournal: Nat Commun / Year: 2024
Title: Conformational coupling between extracellular and transmembrane domains modulates holo-adhesion GPCR function.
Authors: Szymon P Kordon / Kristina Cechova / Sumit J Bandekar / Katherine Leon / Przemysław Dutka / Gracie Siffer / Anthony A Kossiakoff / Reza Vafabakhsh / Demet Araç /
Abstract: Adhesion G Protein-Coupled Receptors (aGPCRs) are key cell-adhesion molecules involved in numerous physiological functions. aGPCRs have large multi-domain extracellular regions (ECRs) containing a ...Adhesion G Protein-Coupled Receptors (aGPCRs) are key cell-adhesion molecules involved in numerous physiological functions. aGPCRs have large multi-domain extracellular regions (ECRs) containing a conserved GAIN domain that precedes their seven-pass transmembrane domain (7TM). Ligand binding and mechanical force applied on the ECR regulate receptor function. However, how the ECR communicates with the 7TM remains elusive, because the relative orientation and dynamics of the ECR and 7TM within a holoreceptor is unclear. Here, we describe the cryo-EM reconstruction of an aGPCR, Latrophilin3/ADGRL3, and reveal that the GAIN domain adopts a parallel orientation to the transmembrane region and has constrained movement. Single-molecule FRET experiments unveil three slow-exchanging FRET states of the ECR relative to the transmembrane region within the holoreceptor. GAIN-targeted antibodies, and cancer-associated mutations at the GAIN-7TM interface, alter FRET states, cryo-EM conformations, and receptor signaling. Altogether, this data demonstrates conformational and functional coupling between the ECR and 7TM, suggesting an ECR-mediated mechanism for aGPCR activation.
History
DepositionJan 26, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 11, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Isoform 4 of Adhesion G protein-coupled receptor L3
B: Isoform 4 of Adhesion G protein-coupled receptor L3
C: sAB Light Chain
D: sAB Heavy Chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)126,8406
Polymers126,0324
Non-polymers8082
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable, gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Isoform 4 of Adhesion G protein-coupled receptor ... , 2 types, 2 molecules AB

#1: Protein Isoform 4 of Adhesion G protein-coupled receptor L3 / Calcium-independent alpha-latrotoxin receptor 3 / CIRL-3 / Latrophilin-3 / Lectomedin-3


Mass: 41641.762 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ADGRL3, KIAA0768, LEC3, LPHN3 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q9HAR2
#2: Protein Isoform 4 of Adhesion G protein-coupled receptor L3 / Calcium-independent alpha-latrotoxin receptor 3 / CIRL-3 / Latrophilin-3 / Lectomedin-3


Mass: 35839.250 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ADGRL3, KIAA0768, LEC3, LPHN3 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q9HAR2

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Antibody , 2 types, 2 molecules CD

#3: Antibody sAB Light Chain


Mass: 23329.863 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)
#4: Antibody sAB Heavy Chain


Mass: 25221.016 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)

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Sugars , 2 types, 2 molecules

#5: Polysaccharide beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta- ...beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 586.542 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/2,3,2/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5]/1-1-2/a4-b1_b4-c1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{}}}}LINUCSPDB-CARE
#6: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0

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Details

Has ligand of interestN
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
1Latrophilin3/ADGRL3 HormR+GAIN/7TM holoreceptor in complex with synthetic antibody fragmentCOMPLEX#1-#40RECOMBINANT
2Latrophilin3/ADGRL3COMPLEX#1-#21RECOMBINANT
3Synthetic antibody fragmentCOMPLEX#3-#41RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.126 MDaNO
210.076 MDaNO
310.050 MDaNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Homo sapiens (human)9606
32Homo sapiens (human)9606
43synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Spodoptera frugiperda (fall armyworm)7108
32Spodoptera frugiperda (fall armyworm)7108
43Escherichia coli (E. coli)562
Buffer solutionpH: 7.5
Buffer component
IDConc.NameBuffer-ID
130 mMHEPES1
2150 mMNaCl1
30.00075 %LMNG1
40.00025 %GDN1
50.0001 %CHS1
SpecimenConc.: 2.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Monodisperse protein sample in detergent buffer
Specimen supportGrid material: GOLD / 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: 283.15 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 / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 1.1 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategory
4cryoSPARC4.4CTF correction
10cryoSPARC4.4initial Euler assignment
11cryoSPARC4.4final Euler assignment
12cryoSPARC4.4classification
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2621532
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 96958 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0124787
ELECTRON MICROSCOPYf_angle_d1.4286506
ELECTRON MICROSCOPYf_dihedral_angle_d15.2431737
ELECTRON MICROSCOPYf_chiral_restr0.244753
ELECTRON MICROSCOPYf_plane_restr0.012826

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