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- PDB-8enu: Structure of the C3bB proconvertase in complex with lufaxin -

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Basic information

Entry
Database: PDB / ID: 8enu
TitleStructure of the C3bB proconvertase in complex with lufaxin
Components
  • Complement C3 beta chain
  • Complement C3b alpha' chain
  • Complement factor B
  • 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 / positive regulation of activation of membrane attack complex / complement binding / 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 / positive regulation of activation of membrane attack complex / complement binding / vertebrate eye-specific patterning / positive regulation of apoptotic cell clearance / complement-mediated synapse pruning / Alternative complement activation / Activation of C3 and C5 / positive regulation of phagocytosis, engulfment / positive regulation of lipid storage / positive regulation of G protein-coupled receptor signaling pathway / complement receptor mediated signaling pathway / positive regulation of type IIa hypersensitivity / complement-dependent cytotoxicity / positive regulation of D-glucose transmembrane transport / complement activation / complement activation, alternative pathway / endopeptidase inhibitor activity / neuron remodeling / amyloid-beta clearance / B cell activation / positive regulation of vascular endothelial growth factor production / complement activation, classical pathway / Purinergic signaling in leishmaniasis infection / Peptide ligand-binding receptors / Regulation of Complement cascade / fatty acid metabolic process / Post-translational protein phosphorylation / response to bacterium / positive regulation of receptor-mediated endocytosis / Regulation of Insulin-like Growth Factor (IGF) transport and uptake by Insulin-like Growth Factor Binding Proteins (IGFBPs) / Immunoregulatory interactions between a Lymphoid and a non-Lymphoid cell / positive regulation of angiogenesis / azurophil granule lumen / positive regulation of protein phosphorylation / toxin activity / G alpha (i) signalling events / secretory granule lumen / blood microparticle / immune response / G protein-coupled receptor signaling pathway / inflammatory response / receptor ligand activity / endoplasmic reticulum lumen / signaling receptor binding / serine-type endopeptidase activity / 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 / Netrin domain / NTR domain profile. / Alpha-macroglobulin, receptor-binding / Alpha-macroglobulin, receptor-binding domain superfamily / Macroglobulin domain MG3 / : / A-macroglobulin receptor binding domain / Macroglobulin domain MG3 / A-macroglobulin receptor / Tissue inhibitor of metalloproteinases-like, OB-fold / Alpha-2-macroglobulin / Macroglobulin domain / 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) / 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 / Glycosyltransferase / Alpha/alpha barrel / von Willebrand factor (vWF) type A domain / VWFA domain profile. / von Willebrand factor, type A / von Willebrand factor A-like domain superfamily / 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 / Immunoglobulin-like fold / Peptidase S1, PA clan / Immunoglobulins / Beta Barrel / Immunoglobulin-like / Sandwich / Mainly Beta / Mainly Alpha
Similarity search - Domain/homology
Complement factor B / Complement C3 / Lufaxin
Similarity search - Component
Biological speciesLutzomyia longipalpis (insect)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.22 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
DepositionSep 30, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 9, 2023Provider: repository / Type: Initial release
Revision 1.1Feb 21, 2024Group: Data collection / Database references
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / 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
Revision 1.2Oct 23, 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

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Assembly

Deposited unit
G: Complement C3 beta chain
H: Complement C3b alpha' chain
D: Complement factor B
A: Lufaxin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)296,14811
Polymers293,5044
Non-polymers2,6447
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 , 4 types, 4 molecules GHDA

#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: 104074.148 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: 32525.715 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

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Sugars , 3 types, 7 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.: 2
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
#6: Polysaccharide beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-6)-2-acetamido-2-deoxy-beta- ...beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-6)-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
DManpb1-4DGlcpNAcb1-6DGlcpNAcb1-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/a6-b1_b4-c1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(6+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{}}}LINUCSPDB-CARE
#7: 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.: 4
Source method: isolated from a genetically manipulated source
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

ComponentName: The C3 proconvertase from the alternative pathway of complement in complex with lufaxin, a complement inhibitor
Type: COMPLEX / Entity ID: #1-#4 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.3023 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 mesh size: 300 divisions/in. / 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 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
IDNameCategory
1Gautomatchparticle selection
13FREALIGN3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.22 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 80727 / Symmetry type: POINT

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