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- PDB-7la4: Integrin AlphaIIbBeta3-PT25-2 Complex -

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Basic information

Entry
Database: PDB / ID: 7la4
TitleIntegrin AlphaIIbBeta3-PT25-2 Complex
Components
  • Integrin alpha-IIb
  • Integrin beta-3
  • PT25-2 heavy chain
  • PT25-2 light chain
KeywordsCELL ADHESION / PT25-2
Function / homology
Function and homology information


tube development / regulation of serotonin uptake / positive regulation of adenylate cyclase-inhibiting opioid receptor signaling pathway / alpha9-beta1 integrin-ADAM8 complex / regulation of trophoblast cell migration / regulation of postsynaptic neurotransmitter receptor diffusion trapping / alphav-beta3 integrin-vitronectin complex / regulation of extracellular matrix organization / platelet alpha granule membrane / positive regulation of glomerular mesangial cell proliferation ...tube development / regulation of serotonin uptake / positive regulation of adenylate cyclase-inhibiting opioid receptor signaling pathway / alpha9-beta1 integrin-ADAM8 complex / regulation of trophoblast cell migration / regulation of postsynaptic neurotransmitter receptor diffusion trapping / alphav-beta3 integrin-vitronectin complex / regulation of extracellular matrix organization / platelet alpha granule membrane / positive regulation of glomerular mesangial cell proliferation / integrin alphav-beta3 complex / negative regulation of lipoprotein metabolic process / alphav-beta3 integrin-PKCalpha complex / maintenance of postsynaptic specialization structure / fibrinogen binding / alphav-beta3 integrin-HMGB1 complex / blood coagulation, fibrin clot formation / negative regulation of lipid transport / vascular endothelial growth factor receptor 2 binding / glycinergic synapse / negative regulation of low-density lipoprotein receptor activity / regulation of release of sequestered calcium ion into cytosol / angiogenesis involved in wound healing / Elastic fibre formation / mesodermal cell differentiation / cell-substrate junction assembly / alphav-beta3 integrin-IGF-1-IGF1R complex / platelet-derived growth factor receptor binding / filopodium membrane / extracellular matrix binding / positive regulation of fibroblast migration / regulation of postsynaptic neurotransmitter receptor internalization / positive regulation of vascular endothelial growth factor receptor signaling pathway / apolipoprotein A-I-mediated signaling pathway / regulation of bone resorption / apoptotic cell clearance / wound healing, spreading of epidermal cells / positive regulation of cell adhesion mediated by integrin / heterotypic cell-cell adhesion / integrin complex / Molecules associated with elastic fibres / positive regulation of leukocyte migration / positive regulation of cell-matrix adhesion / cellular response to insulin-like growth factor stimulus / smooth muscle cell migration / microvillus membrane / cell adhesion mediated by integrin / negative chemotaxis / Syndecan interactions / p130Cas linkage to MAPK signaling for integrins / activation of protein kinase activity / cellular response to platelet-derived growth factor stimulus / protein disulfide isomerase activity / cell-substrate adhesion / positive regulation of smooth muscle cell migration / positive regulation of osteoblast proliferation / TGF-beta receptor signaling activates SMADs / PECAM1 interactions / lamellipodium membrane / GRB2:SOS provides linkage to MAPK signaling for Integrins / negative regulation of macrophage derived foam cell differentiation / negative regulation of lipid storage / platelet-derived growth factor receptor signaling pathway / fibronectin binding / ECM proteoglycans / positive regulation of T cell migration / positive regulation of bone resorption / Integrin cell surface interactions / coreceptor activity / negative regulation of endothelial cell apoptotic process / positive regulation of substrate adhesion-dependent cell spreading / cell adhesion molecule binding / positive regulation of endothelial cell proliferation / embryo implantation / positive regulation of endothelial cell migration / Integrin signaling / substrate adhesion-dependent cell spreading / cell-matrix adhesion / response to activity / Signal transduction by L1 / integrin-mediated signaling pathway / regulation of actin cytoskeleton organization / protein kinase C binding / positive regulation of smooth muscle cell proliferation / Signaling by high-kinase activity BRAF mutants / wound healing / RUNX1 regulates genes involved in megakaryocyte differentiation and platelet function / MAP2K and MAPK activation / platelet activation / ruffle membrane / platelet aggregation / VEGFA-VEGFR2 Pathway / cell-cell adhesion / cellular response to mechanical stimulus / positive regulation of angiogenesis / Signaling by RAF1 mutants / Signaling by moderate kinase activity BRAF mutants / Paradoxical activation of RAF signaling by kinase inactive BRAF / Signaling downstream of RAS mutants / regulation of protein localization
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 cytoplasmic domain / Integrin_b_cyt / : / Integrin alpha Ig-like domain 3 / Integrin beta tail domain / Integrin beta subunit, tail / Integrin beta tail domain superfamily ...Integrin beta, epidermal growth factor-like domain 1 / Integrin beta epidermal growth factor like domain 1 / Integrin beta subunit, cytoplasmic domain / Integrin beta cytoplasmic domain / Integrin_b_cyt / : / Integrin alpha Ig-like domain 3 / Integrin beta tail domain / Integrin beta subunit, tail / Integrin beta tail domain superfamily / Integrin_B_tail / Integrin beta subunit, VWA domain / Integrin beta subunit / Integrin beta N-terminal / Integrin beta chain VWA domain / Integrin plexin domain / Integrins beta chain cysteine-rich domain signature. / Integrin beta subunits (N-terminal portion of extracellular region) / Integrin alpha cytoplasmic region / EGF-like domain, extracellular / EGF-like domain / Integrin alpha-2 / Integrin alpha Ig-like domain 1 / : / Integrin alpha Ig-like domain 2 / Integrin alpha chain / Integrin alpha beta-propellor / Integrin alpha chain, C-terminal cytoplasmic region, conserved site / Integrins alpha chain signature. / 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 / von Willebrand factor A-like domain superfamily / EGF-like domain signature 2. / EGF-like domain signature 1.
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
AuthorsBush, M.W. / Walz, T. / Coller, B. / Filizola, M. / Spasic, A. / Nesic, D. / Li, J.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)2R01HL019278-43 United States
CitationJournal: Blood Adv / Year: 2021
Title: Electron microscopy shows that binding of monoclonal antibody PT25-2 primes integrin αIIbβ3 for ligand binding.
Authors: Dragana Nešić / Martin Bush / Aleksandar Spasic / Jihong Li / Tetsuji Kamata / Makoto Handa / Marta Filizola / Thomas Walz / Barry S Coller /
Abstract: The murine monoclonal antibody (mAb) PT25-2 induces αIIbβ3 to bind ligand and initiate platelet aggregation. The underlying mechanism is unclear, because previous mutagenesis studies suggested that ...The murine monoclonal antibody (mAb) PT25-2 induces αIIbβ3 to bind ligand and initiate platelet aggregation. The underlying mechanism is unclear, because previous mutagenesis studies suggested that PT25-2 binds to the αIIb β propeller, a site distant from the Arg-Gly-Asp-binding pocket. To elucidate the mechanism, we studied the αIIbβ3-PT25-2 Fab complex by negative-stain and cryo-electron microscopy (EM). We found that PT25-2 binding results in αIIbβ3 partially exposing multiple ligand-induced binding site epitopes and adopting extended conformations without swing-out of the β3 hybrid domain. The cryo-EM structure showed PT25-2 binding to the αIIb residues identified by mutagenesis but also to 2 additional regions. Overlay of the cryo-EM structure with the bent αIIbβ3 crystal structure showed that binding of PT25-2 creates clashes with the αIIb calf-1/calf-2 domains, suggesting that PT25-2 selectively binds to partially or fully extended receptor conformations and prevents a return to its bent conformation. Kinetic studies of the binding of PT25-2 compared with mAbs 10E5 and 7E3 support this hypothesis. We conclude that PT25-2 induces αIIbβ3 ligand binding by binding to extended conformations and by preventing the interactions between the αIIb and β3 leg domains and subsequently the βI and β3 leg domains required for the bent-closed conformation.
History
DepositionJan 5, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 12, 2022Provider: repository / Type: Initial release
Revision 1.1Jul 27, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

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Assembly

Deposited unit
H: PT25-2 heavy chain
L: PT25-2 light chain
A: Integrin alpha-IIb
B: Integrin beta-3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)249,59115
Polymers247,3454
Non-polymers2,24611
Water724
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, 2 molecules AB

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


Mass: 113477.523 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ITGA2B, GP2B, ITGAB / Production host: Homo sapiens (human) / References: UniProt: P08514
#4: Protein Integrin beta-3 / Platelet membrane glycoprotein IIIa / GPIIIa


Mass: 87150.773 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ITGB3, GP3A / Production host: Homo sapiens (human) / References: UniProt: P05106

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Antibody , 2 types, 2 molecules HL

#1: Antibody PT25-2 heavy chain


Mass: 23079.824 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)
#2: Antibody PT25-2 light chain


Mass: 23636.932 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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Sugars , 4 types, 4 molecules

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


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}}LINUCSPDB-CARE
#6: Polysaccharide alpha-D-mannopyranose-(1-3)-alpha-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1- ...alpha-D-mannopyranose-(1-3)-alpha-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 748.682 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-3DManpa1-4DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/2,4,3/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1a_1-5]/1-1-2-2/a4-b1_b4-c1_c3-d1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][a-D-Manp]{[(3+1)][a-D-Manp]{}}}}}LINUCSPDB-CARE
#7: Polysaccharide alpha-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta- ...alpha-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
DManpa1-4DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/2,3,2/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1a_1-5]/1-1-2/a4-b1_b4-c1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][a-D-Manp]{}}}}LINUCSPDB-CARE
#9: 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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Non-polymers , 3 types, 11 molecules

#8: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Ca
#10: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#11: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Formula: H2O

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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
1Human aIIBb3 integrin headpiece with PT25-2 antibody fragment boundCELL#1-#40MULTIPLE SOURCES
2PT25-2 heavy and light chainsCELL#1-#21RECOMBINANT
3aIIBb3 integrin headpieceCELL#3-#41RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Mus musculus (house mouse)10090
23Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Mus musculus (house mouse)10090
23Homo sapiens (human)9606
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / 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
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 80 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 166124 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0058456
ELECTRON MICROSCOPYf_angle_d0.7111488
ELECTRON MICROSCOPYf_dihedral_angle_d13.9731219
ELECTRON MICROSCOPYf_chiral_restr0.0491293
ELECTRON MICROSCOPYf_plane_restr0.0041485

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