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- PDB-9axl: Structure of the semi-extended AlphaIIbBeta3 in complex with R21D... -

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Basic information

Entry
Database: PDB / ID: 9axl
TitleStructure of the semi-extended AlphaIIbBeta3 in complex with R21D10 Fab
Components
  • Integrin alpha-IIb
  • Integrin beta-3
  • R21D10 Fab heavy chain
  • R21D10 Fab light chain
KeywordsBLOOD CLOTTING/IMMUNE SYSTEM / monoclonal antibody / integrin receptor / allosteric inhibitor / platelets / BLOOD CLOTTING / BLOOD CLOTTING-IMMUNE SYSTEM complex
Function / homology
Function and homology information


regulation of serotonin uptake / positive regulation of adenylate cyclase-inhibiting opioid receptor signaling pathway / tube development / alpha9-beta1 integrin-ADAM8 complex / response to platelet-derived growth factor / integrin alphaIIb-beta3 complex / regulation of postsynaptic neurotransmitter receptor diffusion trapping / maintenance of postsynaptic specialization structure / alphav-beta3 integrin-vitronectin complex / platelet alpha granule membrane ...regulation of serotonin uptake / positive regulation of adenylate cyclase-inhibiting opioid receptor signaling pathway / tube development / alpha9-beta1 integrin-ADAM8 complex / response to platelet-derived growth factor / integrin alphaIIb-beta3 complex / regulation of postsynaptic neurotransmitter receptor diffusion trapping / maintenance of postsynaptic specialization structure / alphav-beta3 integrin-vitronectin complex / platelet alpha granule membrane / positive regulation of glomerular mesangial cell proliferation / integrin alphav-beta3 complex / negative regulation of lipoprotein metabolic process / smooth muscle cell migration / alphav-beta3 integrin-PKCalpha complex / fibrinogen binding / positive regulation of leukocyte migration / alphav-beta3 integrin-HMGB1 complex / negative regulation of lipid transport / vascular endothelial growth factor receptor 2 binding / angiogenesis involved in wound healing / positive regulation of vascular endothelial growth factor signaling pathway / regulation of release of sequestered calcium ion into cytosol / Elastic fibre formation / mesodermal cell differentiation / positive regulation of bone resorption / alphav-beta3 integrin-IGF-1-IGF1R complex / platelet-derived growth factor receptor binding / cell-cell adhesion mediated by integrin / filopodium membrane / extracellular matrix binding / glycinergic synapse / positive regulation of fibroblast migration / positive regulation of cell adhesion mediated by integrin / positive regulation of vascular endothelial growth factor receptor signaling pathway / apolipoprotein A-I-mediated signaling pathway / regulation of bone resorption / negative regulation of low-density lipoprotein particle clearance / apoptotic cell clearance / wound healing, spreading of epidermal cells / positive regulation of smooth muscle cell migration / integrin complex / Molecules associated with elastic fibres / heterotypic cell-cell adhesion / cell adhesion mediated by integrin / negative chemotaxis / positive regulation of osteoblast proliferation / Mechanical load activates signaling by PIEZO1 and integrins in osteocytes / cellular response to insulin-like growth factor stimulus / Syndecan interactions / p130Cas linkage to MAPK signaling for integrins / regulation of postsynaptic neurotransmitter receptor internalization / cell-substrate adhesion / protein disulfide isomerase activity / microvillus membrane / PECAM1 interactions / GRB2:SOS provides linkage to MAPK signaling for Integrins / negative regulation of endothelial cell apoptotic process / TGF-beta receptor signaling activates SMADs / lamellipodium membrane / fibronectin binding / negative regulation of macrophage derived foam cell differentiation / negative regulation of lipid storage / Integrin cell surface interactions / ECM proteoglycans / substrate adhesion-dependent cell spreading / positive regulation of T cell migration / cell-matrix adhesion / embryo implantation / coreceptor activity / Integrin signaling / positive regulation of endothelial cell proliferation / positive regulation of smooth muscle cell proliferation / positive regulation of endothelial cell migration / cell adhesion molecule binding / integrin-mediated signaling pathway / Turbulent (oscillatory, disturbed) flow shear stress activates signaling by PIEZO1 and integrins in endothelial cells / response to activity / protein kinase C binding / wound healing / Signal transduction by L1 / cellular response to xenobiotic stimulus / regulation of actin cytoskeleton organization / cell-cell adhesion / cellular response to mechanical stimulus / platelet activation / Signaling by high-kinase activity BRAF mutants / MAP2K and MAPK activation / RUNX1 regulates genes involved in megakaryocyte differentiation and platelet function / platelet aggregation / VEGFA-VEGFR2 Pathway / integrin binding / ruffle membrane / blood coagulation / positive regulation of angiogenesis / Signaling by RAF1 mutants / cell-cell junction / Signaling by moderate kinase activity BRAF mutants / Paradoxical activation of RAF signaling by kinase inactive BRAF / Signaling downstream of RAS mutants
Similarity search - Function
Integrin beta, epidermal growth factor-like domain 1 / Integrin beta epidermal growth factor like domain 1 / Integrin beta subunit, cytoplasmic domain / : / Integrin beta tail domain / Integrin beta cytoplasmic domain / Integrin alpha Ig-like domain 3 / Integrin_b_cyt / : / Integrin EGF domain ...Integrin beta, epidermal growth factor-like domain 1 / Integrin beta epidermal growth factor like domain 1 / Integrin beta subunit, cytoplasmic domain / : / Integrin beta tail domain / Integrin beta cytoplasmic domain / Integrin alpha Ig-like domain 3 / Integrin_b_cyt / : / Integrin EGF domain / EGF-like domain, extracellular / EGF-like domain / Integrin beta subunit, tail / Integrin beta tail domain superfamily / Integrin_B_tail / Integrin alpha cytoplasmic region / : / Integrins beta chain EGF (I-EGF) domain profile. / Integrin beta subunit, VWA domain / Integrin beta subunit / Integrin beta N-terminal / Integrin beta chain VWA domain / Integrin plexin domain / Integrins beta chain EGF (I-EGF) domain signature. / Integrin beta subunits (N-terminal portion of extracellular region) / Integrin alpha-2 / Integrin alpha Ig-like domain 1 / Integrin alpha chain, C-terminal cytoplasmic region, conserved site / Integrins alpha chain signature. / Integrin alpha chain / Integrin alpha beta-propellor / : / Integrin alpha Ig-like domain 2 / FG-GAP repeat profile. / Integrin alpha (beta-propellor repeats). / FG-GAP repeat / FG-GAP repeat / Integrin domain superfamily / Integrin alpha, N-terminal / PSI domain / domain found in Plexins, Semaphorins and Integrins / EGF-like domain signature 1. / von Willebrand factor A-like domain superfamily / EGF-like domain signature 2.
Similarity search - Domain/homology
Integrin beta-3 / Integrin alpha-IIb
Similarity search - Component
Biological speciesMus musculus (house mouse)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsWang, J.L. / Walz, T. / Coller, B. / Wang, L. / Li, J.H.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)5R01HL019278-48 United States
CitationJournal: Blood Adv / Year: 2024
Title: An αIIbβ3 monoclonal antibody traps a semiextended conformation and allosterically inhibits large ligand binding.
Authors: Lu Wang / Jialing Wang / Jihong Li / Thomas Walz / Barry S Coller /
Abstract: Monoclonal antibodies (mAbs) have provided valuable information regarding the structure and function of platelet αIIbβ3. Protein disulfide isomerase (PDI) has been implicated in αIIbβ3 activation ...Monoclonal antibodies (mAbs) have provided valuable information regarding the structure and function of platelet αIIbβ3. Protein disulfide isomerase (PDI) has been implicated in αIIbβ3 activation and binds to thrombin-activated αIIbβ3. Using human platelets as the immunogen, we identified a new mAb (R21D10) that inhibits the binding of PDI to platelets activated with thrombin receptor-activating peptide (T6). R21D10 also partially inhibited T6-induced fibrinogen and PAC-1 binding to platelets, as well as T6- and adenosine 5'-diphosphate-induced platelet aggregation. Mutual competition experiments showed that R21D10 does not inhibit the binding of mAbs 10E5 (anti-αIIb cap domain) or 7E3 (anti-β3 β-I domain), and immunoblot studies indicated that R21D10 binds to β3. The dissociation of αIIbβ3 by EDTA had a minimal effect on R21D10 binding. Cryogenic electron microscopy of the αIIbβ3-R21D10 Fab complex revealed that R21D10 binds to the β3 integrin-epidermal growth factor 1 (I-EGF1) domain and traps an intermediate conformation of αIIbβ3 with semiextended leg domains. The binding of R21D10 produces a major structural change in the β3 I-EGF2 domain associated with a new interaction between the β3 I-EGF2 and αIIb thigh domains, which may prevent the swing-out motion of the β3 hybrid domain required for high-affinity ligand binding and protect αIIbβ3 from EDTA-induced dissociation. R21D10 partially reversed the ligand binding priming effect of eptifibatide, suggesting that it could convert the swung-out conformation into a semiextended conformation. We concluded that R21D10 inhibits ligand binding to αIIbβ3 via a unique allosteric mechanism, which may or may not be related to its inhibition of PDI binding.
History
DepositionMar 6, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 13, 2024Provider: repository / Type: Initial release
Revision 1.1Nov 27, 2024Group: Data collection / Category: em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Integrin alpha-IIb
B: Integrin beta-3
H: R21D10 Fab heavy chain
L: R21D10 Fab light chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)248,77711
Polymers248,5124
Non-polymers2657
Water1267
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 2 types, 2 molecules AB

#1: Protein Integrin alpha-IIb / GPalpha IIb / GPIIb / Platelet membrane glycoprotein IIb


Mass: 113477.523 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P08514
#2: Protein Integrin beta-3 / Platelet membrane glycoprotein IIIa / GPIIIa


Mass: 87150.773 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P05106

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Antibody , 2 types, 2 molecules HL

#3: Antibody R21D10 Fab heavy chain


Mass: 24539.334 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Mus musculus (house mouse)
#4: Antibody R21D10 Fab light chain


Mass: 23344.779 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Mus musculus (house mouse)

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Non-polymers , 3 types, 14 molecules

#5: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#7: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 7 / Source method: isolated from a natural source / Formula: H2O

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Details

Compound detailsMurine monoclonal antibody R21D10 stabilizes a semi-extended conformation of the platelet ...Murine monoclonal antibody R21D10 stabilizes a semi-extended conformation of the platelet AlphaIIbBeta3 integrin receptor and serves as an allosteric inhibitor of ligand binding and platelet aggregation. It also is the first mAb reported to inhibit the binding of protein disulfide isomerase.
Has ligand of interestY
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: The semi-extended AlphaIIbBeta3 in complex with R21D10 Fab
Type: COMPLEX / Entity ID: #1-#4 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.25 MDa / Experimental value: YES
Buffer solutionpH: 7.4
SpecimenConc.: 0.05 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GRAPHENE OXIDE / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK I / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 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 magnification: 81000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1500 nm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 54 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARCv4.4.0particle selection
4cryoSPARCv4.4.0CTF correction
7UCSF Chimeraversion 1.16model fitting
9cryoSPARCv4.4.0initial Euler assignment
10cryoSPARCv4.4.0final Euler assignment
11cryoSPARCv4.4.0classification
12cryoSPARCv4.4.03D reconstruction
13PHENIXversion 1.20.1-4487model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1332000
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 596272 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model building

3D fitting-ID: 1 / Source name: PDB / Type: experimental model

IDPDB-IDPdb chain-IDAccession codeChain-IDInitial refinement model-ID
13FSC

3fsc

A3FSCA1
23FSC

3fsc

B3FSCB1
37LA4

7la4

H7LA4H2
47LA4

7la4

L7LA4L2

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