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- PDB-8cml: Cryo-EM structure of complement C5 in complex with nanobodies UNb... -

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Basic information

Entry
Database: PDB / ID: 8cml
TitleCryo-EM structure of complement C5 in complex with nanobodies UNbC5-1 and UNbC5-2
Components
  • Complement C5 alpha chain
  • Complement C5 beta chain
  • Nanobody UNbC5-1
  • Nanobody UNbC5-2
KeywordsIMMUNE SYSTEM / nanobody / inhibitor / complement / C5 / inhibition / convertase / classical pathway / nanobody-antigen complex / alternative pathway
Function / homology
Function and homology information


Terminal pathway of complement / membrane attack complex / Activation of C3 and C5 / negative regulation of macrophage chemotaxis / complement activation, alternative pathway / chemokine activity / endopeptidase inhibitor activity / positive regulation of vascular endothelial growth factor production / positive regulation of chemokine production / Peptide ligand-binding receptors ...Terminal pathway of complement / membrane attack complex / Activation of C3 and C5 / negative regulation of macrophage chemotaxis / complement activation, alternative pathway / chemokine activity / endopeptidase inhibitor activity / positive regulation of vascular endothelial growth factor production / positive regulation of chemokine production / Peptide ligand-binding receptors / complement activation, classical pathway / Regulation of Complement cascade / chemotaxis / G alpha (i) signalling events / killing of cells of another organism / cell surface receptor signaling pathway / inflammatory response / G protein-coupled receptor signaling pathway / signaling receptor binding / extracellular space / extracellular exosome / extracellular region
Similarity search - Function
: / Complement component 5, CUB domain / Complement C3/4/5, macroglobulin domain MG1 / Macroglobulin domain MG1 / Anaphylatoxin, complement system domain / Anaphylatoxin domain signature. / Anaphylatoxin/fibulin / Anaphylatoxin, complement system / Anaphylotoxin-like domain / Anaphylatoxin domain profile. ...: / Complement component 5, CUB domain / Complement C3/4/5, macroglobulin domain MG1 / Macroglobulin domain MG1 / Anaphylatoxin, complement system domain / Anaphylatoxin domain signature. / Anaphylatoxin/fibulin / Anaphylatoxin, complement system / Anaphylotoxin-like domain / Anaphylatoxin domain profile. / Anaphylatoxin homologous domain / Netrin C-terminal Domain / Netrin module, non-TIMP type / UNC-6/NTR/C345C module / Alpha-macroglobulin, receptor-binding / Alpha-macroglobulin, receptor-binding domain superfamily / Macroglobulin domain MG4 / Macroglobulin domain MG3 / A-macroglobulin receptor binding domain / Macroglobulin domain MG4 / Macroglobulin domain MG3 / A-macroglobulin receptor / Netrin domain / NTR domain profile. / 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 / Terpenoid cyclases/protein prenyltransferase alpha-alpha toroid / Immunoglobulin-like fold
Similarity search - Domain/homology
Biological speciesLama glama (llama)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsDe la O Becerra, K.I. / Gros, P.
Funding support Netherlands, European Union, Mexico, 4items
OrganizationGrant numberCountry
Netherlands Organisation for Scientific Research (NWO)NWA.ID.17.036 Netherlands
European Research Council (ERC)101001937European Union
European Research Council (ERC)787241European Union
Consejo Nacional de Ciencia y Tecnologia (CONACYT)604718 Mexico
CitationJournal: J Biol Chem / Year: 2023
Title: Inhibition of cleavage of human complement component C5 and the R885H C5 variant by two distinct high affinity anti-C5 nanobodies.
Authors: Eva M Struijf / Karla I De la O Becerra / Maartje Ruyken / Carla J C de Haas / Fleur van Oosterom / Danique Y Siere / Joanne E van Keulen / Dani A C Heesterbeek / Edward Dolk / Raimond ...Authors: Eva M Struijf / Karla I De la O Becerra / Maartje Ruyken / Carla J C de Haas / Fleur van Oosterom / Danique Y Siere / Joanne E van Keulen / Dani A C Heesterbeek / Edward Dolk / Raimond Heukers / Bart W Bardoel / Piet Gros / Suzan H M Rooijakkers /
Abstract: The human complement system plays a crucial role in immune defense. However, its erroneous activation contributes to many serious inflammatory diseases. Since most unwanted complement effector ...The human complement system plays a crucial role in immune defense. However, its erroneous activation contributes to many serious inflammatory diseases. Since most unwanted complement effector functions result from C5 cleavage into C5a and C5b, development of C5 inhibitors, such as clinically approved monoclonal antibody eculizumab, are of great interest. Here, we developed and characterized two anti-C5 nanobodies, UNbC5-1 and UNbC5-2. Using surface plasmon resonance, we determined a binding affinity of 119.9 pM for UNbC5-1 and 7.7 pM for UNbC5-2. Competition experiments determined that the two nanobodies recognize distinct epitopes on C5. Both nanobodies efficiently interfered with C5 cleavage in a human serum environment, as they prevented red blood cell lysis via membrane attack complexes (C5b-9) and the formation of chemoattractant C5a. The cryo-EM structure of UNbC5-1 and UNbC5-2 in complex with C5 (3.6 Å resolution) revealed that the binding interfaces of UNbC5-1 and UNbC5-2 overlap with known complement inhibitors eculizumab and RaCI3, respectively. UNbC5-1 binds to the MG7 domain of C5, facilitated by a hydrophobic core and polar interactions, and UNbC5-2 interacts with the C5d domain mostly by salt bridges and hydrogen bonds. Interestingly, UNbC5-1 potently binds and inhibits C5 R885H, a genetic variant of C5 that is not recognized by eculizumab. Altogether, we identified and characterized two different, high affinity nanobodies against human C5. Both nanobodies could serve as diagnostic and/or research tools to detect C5 or inhibit C5 cleavage. Furthermore, the residues targeted by UNbC5-1 hold important information for therapeutic inhibition of different polymorphic variants of C5.
History
DepositionFeb 20, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 27, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Nanobody UNbC5-2
E: Complement C5 beta chain
B: Complement C5 alpha chain
C: Nanobody UNbC5-1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)219,2745
Polymers218,8504
Non-polymers4241
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: surface plasmon resonance
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Antibody Nanobody UNbC5-2


Mass: 16095.776 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Plasmid: pPQ81 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21 DE3 Rosetta 2
#2: Protein Complement C5 beta chain


Mass: 73361.453 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P01031
#3: Protein Complement C5 alpha chain


Mass: 112635.008 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P01031
#4: Antibody Nanobody UNbC5-1


Mass: 16757.295 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Plasmid: pPQ81 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21 DE3 Rosetta 2
#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
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

ComponentName: Complement C5 bound to nanobodies UNbC5-1 and UNbC5-2 / Type: COMPLEX / Entity ID: #1, #4 / Source: NATURAL
Molecular weightValue: 0.218 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.4 / Details: 1x PBS
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMphosphateNaPO41
2145 mMsodium chlorideNaCl1
SpecimenConc.: 0.26 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Purified protein were mixed before vitrification in
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277.15 K / Details: Blot 4 seconds at blot force 1

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 2600 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 55 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 3810
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 40 / Used frames/image: 1-40

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
EM software
IDNameVersionCategory
1cryoSPARCv3.2/3particle selection
2EPUimage acquisition
4cryoSPARCv3.2/3CTF correction
7UCSF ChimeraX1.2.5model fitting
9cryoSPARCv3.2/3initial Euler assignment
10cryoSPARCv3.2/3final Euler assignment
11cryoSPARCv3.2/3classification
12cryoSPARCv3.2/33D reconstruction
13PHENIX1.20.1model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 193009 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model building

3D fitting-ID: 1

IDPDB-IDPdb chain-IDAccession codeChain-IDChain residue rangeDetailsInitial refinement model-IDPdb chain residue rangeSource nameType
13CU7

3cu7

B3CU7B20-1676C5 complement120-1676PDBexperimental model
2A2-136UNbC5-2AlphaFoldin silico model
3C2-144UNbC5-1AlphaFoldin silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.01113883
ELECTRON MICROSCOPYf_angle_d1.02418827
ELECTRON MICROSCOPYf_dihedral_angle_d5.851879
ELECTRON MICROSCOPYf_chiral_restr0.0452133
ELECTRON MICROSCOPYf_plane_restr0.0052405

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