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- PDB-8eok: Structure of the C3bB proconvertase in complex with lufaxin and f... -

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Entry
Database: PDB / ID: 8eok
TitleStructure of the C3bB proconvertase in complex with lufaxin and factor Xa
Components
  • Activated factor Xa heavy chain
  • Complement C3 beta chain
  • Complement C3b alpha' chain
  • Complement factor B
  • Factor X light chain
  • Lufaxin
KeywordsIMMUNE SYSTEM / Complement / Alternative pathway / inhibitor / sand fly
Function / homology
Function and homology information


alternative-complement-pathway C3/C5 convertase / C5L2 anaphylatoxin chemotactic receptor binding / oviduct epithelium development / regulation of triglyceride biosynthetic process / complement binding / positive regulation of activation of membrane attack complex / vertebrate eye-specific patterning / positive regulation of apoptotic cell clearance / complement-mediated synapse pruning / Alternative complement activation ...alternative-complement-pathway C3/C5 convertase / C5L2 anaphylatoxin chemotactic receptor binding / oviduct epithelium development / regulation of triglyceride biosynthetic process / complement binding / positive regulation of activation of membrane attack complex / vertebrate eye-specific patterning / positive regulation of apoptotic cell clearance / complement-mediated synapse pruning / Alternative complement activation / coagulation factor Xa / Activation of C3 and C5 / positive regulation of phagocytosis, engulfment / positive regulation of lipid storage / positive regulation of G protein-coupled receptor signaling pathway / positive regulation of type IIa hypersensitivity / complement receptor mediated signaling pathway / Defective factor IX causes thrombophilia / Defective cofactor function of FVIIIa variant / Defective F9 variant does not activate FX / complement-dependent cytotoxicity / positive regulation of D-glucose transmembrane transport / complement activation / complement activation, alternative pathway / Extrinsic Pathway of Fibrin Clot Formation / endopeptidase inhibitor activity / neuron remodeling / amyloid-beta clearance / B cell activation / positive regulation of vascular endothelial growth factor production / complement activation, classical pathway / positive regulation of TOR signaling / Transport of gamma-carboxylated protein precursors from the endoplasmic reticulum to the Golgi apparatus / Gamma-carboxylation of protein precursors / Common Pathway of Fibrin Clot Formation / Removal of aminoterminal propeptides from gamma-carboxylated proteins / Purinergic signaling in leishmaniasis infection / Intrinsic Pathway of Fibrin Clot Formation / Peptide ligand-binding receptors / Regulation of Complement cascade / Post-translational protein phosphorylation / response to bacterium / fatty acid metabolic process / phospholipid binding / positive regulation of receptor-mediated endocytosis / Golgi lumen / Regulation of Insulin-like Growth Factor (IGF) transport and uptake by Insulin-like Growth Factor Binding Proteins (IGFBPs) / positive regulation of protein phosphorylation / positive regulation of angiogenesis / Immunoregulatory interactions between a Lymphoid and a non-Lymphoid cell / blood coagulation / azurophil granule lumen / toxin activity / secretory granule lumen / G alpha (i) signalling events / blood microparticle / immune response / positive regulation of cell migration / G protein-coupled receptor signaling pathway / receptor ligand activity / endoplasmic reticulum lumen / inflammatory response / signaling receptor binding / external side of plasma membrane / serine-type endopeptidase activity / calcium ion binding / Neutrophil degranulation / cell surface / signal transduction / protein-containing complex / proteolysis / extracellular space / extracellular exosome / extracellular region / plasma membrane
Similarity search - Function
Complement factor B / Complement B/C2 / OB fold (Dihydrolipoamide Acetyltransferase, E2P) - #120 / Complement C3-like, NTR domain / : / : / Complement component 3, CUB domain, second segment / Complement component 3, CUB domain, first segment / Alpha-2-macroglobulin, conserved site / Alpha-2-macroglobulin family thiolester region signature. ...Complement factor B / Complement B/C2 / OB fold (Dihydrolipoamide Acetyltransferase, E2P) - #120 / Complement C3-like, NTR domain / : / : / Complement component 3, CUB domain, second segment / Complement component 3, CUB domain, first segment / Alpha-2-macroglobulin, conserved site / Alpha-2-macroglobulin family thiolester region signature. / Complement C3/4/5, macroglobulin domain MG1 / Macroglobulin domain MG1 / Anaphylatoxin, complement system domain / : / Anaphylatoxin domain signature. / Alpha-macro-globulin thiol-ester bond-forming region / Anaphylatoxin, complement system / Anaphylatoxin/fibulin / Anaphylotoxin-like domain / Anaphylatoxin domain profile. / Anaphylatoxin homologous domain / Netrin C-terminal Domain / Netrin module, non-TIMP type / UNC-6/NTR/C345C module / Macroglobulin domain MG4 / Macroglobulin domain MG4 / Glycosyltransferase - #20 / Alpha-macroglobulin, receptor-binding / Alpha-macroglobulin, receptor-binding domain superfamily / Macroglobulin domain MG3 / : / A-macroglobulin receptor binding domain / Macroglobulin domain MG3 / A-macroglobulin receptor / Netrin domain / NTR domain profile. / Alpha-2-macroglobulin / Macroglobulin domain / Tissue inhibitor of metalloproteinases-like, OB-fold / Alpha-2-macroglobulin, bait region domain / Alpha-macroglobulin-like, TED domain / Alpha-2-macroglobulin family / MG2 domain / A-macroglobulin TED domain / Alpha-2-macroglobulin bait region domain / Alpha-2-Macroglobulin / Alpha-2-macroglobulin family / Sushi repeat (SCR repeat) / Domain abundant in complement control proteins; SUSHI repeat; short complement-like repeat (SCR) / Peptidase S1A, coagulation factor VII/IX/X/C/Z / : / Coagulation factor-like, Gla domain superfamily / von Willebrand factor type A domain / Sushi/SCR/CCP domain / Sushi/CCP/SCR domain profile. / Sushi/SCR/CCP superfamily / Terpenoid cyclases/protein prenyltransferase alpha-alpha toroid / Coagulation Factor Xa inhibitory site / Glycosyltransferase / EGF-like domain / Alpha/alpha barrel / EGF-type aspartate/asparagine hydroxylation site / EGF-like calcium-binding, conserved site / Calcium-binding EGF-like domain signature. / Aspartic acid and asparagine hydroxylation site. / VWFA domain profile. / von Willebrand factor (vWF) type A domain / EGF-like calcium-binding domain / Calcium-binding EGF-like domain / Vitamin K-dependent carboxylation/gamma-carboxyglutamic (GLA) domain / Gamma-carboxyglutamic acid-rich (GLA) domain / Gamma-carboxyglutamic acid-rich (GLA) domain superfamily / Vitamin K-dependent carboxylation domain. / Gla domain profile. / Domain containing Gla (gamma-carboxyglutamate) residues. / von Willebrand factor, type A / Epidermal growth factor-like domain. / EGF-like domain profile. / von Willebrand factor A-like domain superfamily / EGF-like domain signature 1. / EGF-like domain signature 2. / EGF-like domain / OB fold (Dihydrolipoamide Acetyltransferase, E2P) / Serine proteases, trypsin family, histidine active site / Serine proteases, trypsin family, serine active site / Serine proteases, trypsin family, histidine active site. / Peptidase S1A, chymotrypsin family / Serine proteases, trypsin family, serine active site. / Serine proteases, trypsin domain profile. / Trypsin-like serine protease / Serine proteases, trypsin domain / Trypsin / Trypsin-like serine proteases / Thrombin, subunit H / Immunoglobulin-like fold / Peptidase S1, PA clan, chymotrypsin-like fold / Immunoglobulins / Peptidase S1, PA clan / Beta Barrel / Immunoglobulin-like
Similarity search - Domain/homology
Coagulation factor X / Complement factor B / Complement C3 / Lufaxin
Similarity search - Component
Biological speciesLutzomyia longipalpis (insect)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.53 Å
AuthorsAndersen, J.F. / Lei, H.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID) United States
CitationJournal: Blood / Year: 2023
Title: A bispecific inhibitor of complement and coagulation blocks activation in complementopathy models via a novel mechanism.
Authors: John F Andersen / Haotian Lei / Ethan C Strayer / Tapan Kanai / Van Pham / Xiang-Zuo Pan / Patricia Hessab Alvarenga / Gloria F Gerber / Oluwatoyin A Asojo / Ivo M B Francischetti / Robert A ...Authors: John F Andersen / Haotian Lei / Ethan C Strayer / Tapan Kanai / Van Pham / Xiang-Zuo Pan / Patricia Hessab Alvarenga / Gloria F Gerber / Oluwatoyin A Asojo / Ivo M B Francischetti / Robert A Brodsky / Jesus G Valenzuela / José M C Ribeiro /
Abstract: Inhibitors of complement and coagulation are present in the saliva of a variety of blood-feeding arthropods that transmit parasitic and viral pathogens. Here, we describe the structure and mechanism ...Inhibitors of complement and coagulation are present in the saliva of a variety of blood-feeding arthropods that transmit parasitic and viral pathogens. Here, we describe the structure and mechanism of action of the sand fly salivary protein lufaxin, which inhibits the formation of the central alternative C3 convertase (C3bBb) and inhibits coagulation factor Xa (fXa). Surface plasmon resonance experiments show that lufaxin stabilizes the binding of serine protease factor B (FB) to C3b but does not detectably bind either C3b or FB alone. The crystal structure of the inhibitor reveals a novel all β-sheet fold containing 2 domains. A structure of the lufaxin-C3bB complex obtained via cryo-electron microscopy (EM) shows that lufaxin binds via its N-terminal domain at an interface containing elements of both C3b and FB. By occupying this spot, the inhibitor locks FB into a closed conformation in which proteolytic activation of FB by FD cannot occur. C3bB-bound lufaxin binds fXa at a separate site in its C-terminal domain. In the cryo-EM structure of a C3bB-lufaxin-fXa complex, the inhibitor binds to both targets simultaneously, and lufaxin inhibits fXa through substrate-like binding of a C-terminal peptide at the active site as well as other interactions in this region. Lufaxin inhibits complement activation in ex vivo models of atypical hemolytic uremic syndrome (aHUS) and paroxysmal nocturnal hemoglobinuria (PNH) as well as thrombin generation in plasma, providing a rationale for the development of a bispecific inhibitor to treat complement-related diseases in which thrombosis is a prominent manifestation.
History
DepositionOct 3, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 30, 2023Provider: repository / Type: Initial release
Revision 1.1Oct 30, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
G: Complement C3 beta chain
H: Complement C3b alpha' chain
D: Complement factor B
A: Lufaxin
L: Factor X light chain
C: Activated factor Xa heavy chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)339,14610
Polymers338,0936
Non-polymers1,0534
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 , 6 types, 6 molecules GHDALC

#1: Protein Complement C3 beta chain


Mass: 71393.320 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P01024
#2: Protein Complement C3b alpha' chain


Mass: 104073.164 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P01024
#3: Protein Complement factor B / C3/C5 convertase / Glycine-rich beta glycoprotein / GBG / PBF2 / Properdin factor B


Mass: 85510.617 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human)
References: UniProt: P00751, alternative-complement-pathway C3/C5 convertase
#4: Protein Lufaxin / 32.4 kDa salivary protein / Lutzomyia longipalpis FXa inhibitor


Mass: 33354.586 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lutzomyia longipalpis (insect) / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: Q5WPU8
#5: Protein Factor X light chain


Mass: 15210.793 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P00742
#6: Protein Activated factor Xa heavy chain


Mass: 28550.596 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P00742

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Sugars , 2 types, 3 molecules

#7: 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-ROHGlycam 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
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-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.: 2 / 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 , 1 types, 1 molecules

#8: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg

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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

ComponentName: The C3 proconvertase from the alternative pathway of complement in complex with lufaxin, a complement/coagulation inhibitor and coagulation factor Xa
Type: COMPLEX / Entity ID: #1-#3, #5-#6 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.3483 MDa / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK 293 / Plasmid: VR2001
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMHEPESC8H18N2O4S1
2150 mMSodium ChlorideNaCl1
35 mMMagnesium ChlorideMgCl21
SpecimenConc.: 1.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: The sample was monodisperse
Specimen supportGrid material: GOLD / Grid type: C-flat-1.2/1.3
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 45000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 300 nm
Image recordingElectron dose: 58.31 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1Gautomatchparticle selection
4RELION4.0.0CTF correction
10RELION4.0.0initial Euler assignment
11RELION4.0.0final Euler assignment
13RELION4.0.03D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 713201
3D reconstructionResolution: 3.53 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 248281 / Symmetry type: POINT

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