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- EMDB-16730: Cryo-EM structure of complement C5 in complex with nanobodies UNb... -

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Basic information

Entry
Database: EMDB / ID: EMD-16730
TitleCryo-EM structure of complement C5 in complex with nanobodies UNbC5-1 and UNbC5-2
Map datamap
Sample
  • Complex: Complement C5 bound to nanobodies UNbC5-1 and UNbC5-2
    • Protein or peptide: Nanobody UNbC5-2
    • Protein or peptide: Nanobody UNbC5-1
  • Protein or peptide: Complement C5 beta chain
  • Protein or peptide: Complement C5 alpha chain
Keywordsnanobody / inhibitor / complement / C5 / inhibition / convertase / classical pathway / nanobody-antigen complex / alternative pathway / IMMUNE SYSTEM
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 / complement activation, classical pathway / positive regulation of chemokine production ...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 / complement activation, classical pathway / positive regulation of chemokine production / Peptide ligand-binding receptors / 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 speciesHomo sapiens (human) / Lama glama (llama)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsDe la O Becerra KI / Gros P
Funding support Netherlands, European Union, Mexico, 4 items
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, 2023-
Header (metadata) releaseSep 27, 2023-
Map releaseSep 27, 2023-
UpdateSep 27, 2023-
Current statusSep 27, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16730.png

Downloads & links

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Map

FileDownload / File: emd_16730.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationmap
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
1.04 Å/pix.
x 320 pix.
= 332.8 Å		1.04 Å/pix.
x 320 pix.
= 332.8 Å		1.04 Å/pix.
x 320 pix.
= 332.8 Å

Surface

Projections

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.04 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.186
Minimum - Maximum-1.3636078 - 2.0704834
Average (Standard dev.)0.00017733297 (±0.031740237)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 332.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_16730_msk_1.map
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Histogram

Histogram (log scale)

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Half map: half map b

Fileemd_16730_half_map_1.map
Annotationhalf map b
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: half map a

Fileemd_16730_half_map_2.map
Annotationhalf map a
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Sample components

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Entire : Complement C5 bound to nanobodies UNbC5-1 and UNbC5-2

EntireName: Complement C5 bound to nanobodies UNbC5-1 and UNbC5-2
Components
  • Complex: Complement C5 bound to nanobodies UNbC5-1 and UNbC5-2
    • Protein or peptide: Nanobody UNbC5-2
    • Protein or peptide: Nanobody UNbC5-1
  • Protein or peptide: Complement C5 beta chain
  • Protein or peptide: Complement C5 alpha chain

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Supramolecule #1: Complement C5 bound to nanobodies UNbC5-1 and UNbC5-2

SupramoleculeName: Complement C5 bound to nanobodies UNbC5-1 and UNbC5-2 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1, #4
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 218 KDa

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Macromolecule #1: Nanobody UNbC5-2

MacromoleculeName: Nanobody UNbC5-2 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Lama glama (llama)
Molecular weightTheoretical: 16.095776 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MEVQLVESGG GLVQPGGSLR LSCAASGRTF STNTMGWFRQ APGQEREFVA LISGNGRILD YSDSAKGRFT ISRDNAKNTV YLQMNSLKP EDTGVYFCAA EFRGRTLASY WGQGTQVTVS SAAASGSLEQ KLISEEDLNG AAHHHHHHGA A

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Macromolecule #2: Complement C5 beta chain

MacromoleculeName: Complement C5 beta chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 73.361453 KDa
SequenceString: QEQTYVISAP KIFRVGASEN IVIQVYGYTE AFDATISIKS YPDKKFSYSS GHVHLSSENK FQNSAILTIQ PKQLPGGQNP VSYVYLEVV SKHFSKSKRM PITYDNGFLF IHTDKPVYTP DQSVKVRVYS LNDDLKPAKR ETVLTFIDPE GSEVDMVEEI D HIGIISFP ...String:
QEQTYVISAP KIFRVGASEN IVIQVYGYTE AFDATISIKS YPDKKFSYSS GHVHLSSENK FQNSAILTIQ PKQLPGGQNP VSYVYLEVV SKHFSKSKRM PITYDNGFLF IHTDKPVYTP DQSVKVRVYS LNDDLKPAKR ETVLTFIDPE GSEVDMVEEI D HIGIISFP DFKIPSNPRY GMWTIKAKYK EDFSTTGTAY FEVKEYVLPH FSVSIEPEYN FIGYKNFKNF EITIKARYFY NK VVTEADV YITFGIREDL KDDQKEMMQT AMQNTMLING IAQVTFDSET AVKELSYYSL EDLNNKYLYI AVTVIESTGG FSE EAEIPG IKYVLSPYKL NLVATPLFLK PGIPYPIKVQ VKDSLDQLVG GVPVTLNAQT IDVNQETSDL DPSKSVTRVD DGVA SFVLN LPSGVTVLEF NVKTDAPDLP EENQAREGYR AIAYSSLSQS YLYIDWTDNH KALLVGEHLN IIVTPKSPYI DKITH YNYL ILSKGKIIHF GTREKFSDAS YQSINIPVTQ NMVPSSRLLV YYIVTGEQTA ELVSDSVWLN IEEKCGNQLQ VHLSPD ADA YSPGQTVSLN MATGMDSWVA LAAVDSAVYG VQRGAKKPLE RVFQFLEKSD LGCGAGGGLN NANVFHLAGL TFLTNAN AD DSQENDEPCK EIL

UniProtKB: Complement C5

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Macromolecule #3: Complement C5 alpha chain

MacromoleculeName: Complement C5 alpha chain / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 112.635008 KDa
SequenceString: TLQKKIEEIA AKYKHSVVKK CCYDGACVNN DETCEQRAAR ISLGPRCIKA FTECCVVASQ LRANISHKDM QLGRLHMKTL LPVSKPEIR SYFPESWLWE VHLVPRRKQL QFALPDSLTT WEIQGVGISN TGICVADTVK AKVFKDVFLE MNIPYSVVRG E QIQLKGTV ...String:
TLQKKIEEIA AKYKHSVVKK CCYDGACVNN DETCEQRAAR ISLGPRCIKA FTECCVVASQ LRANISHKDM QLGRLHMKTL LPVSKPEIR SYFPESWLWE VHLVPRRKQL QFALPDSLTT WEIQGVGISN TGICVADTVK AKVFKDVFLE MNIPYSVVRG E QIQLKGTV YNYRTSGMQF CVKMSAVEGI CTSESPVIDH QGTKSSKCVR QKVEGSSSHL VTFTVLPLEI GLHNINFSLE TW FGKEILV KTLRVVPEGV KRESYSGVTL DPRGIYGTIS RRKEFPYRIP LDLVPKTEIK RILSVKGLLV GEILSAVLSQ EGI NILTHL PKGSAEAELM SVVPVFYVFH YLETGNHWNI FHSDPLIEKQ KLKKKLKEGM LSIMSYRNAD YSYSVWKGGS ASTW LTAFA LRVLGQVNKY VEQNQNSICN SLLWLVENYQ LDNGSFKENS QYQPIKLQGT LPVEARENSL YLTAFTVIGI RKAFD ICPL VKIDTALIKA DNFLLENTLP AQSTFTLAIS AYALSLGDKT HPQFRSIVSA LKREALVKGN PPIYRFWKDN LQHKDS SVP NTGTARMVET TAYALLTSLN LKDINYVNPV IKWLSEEQRY GGGFYSTQDT INAIEGLTEY SLLVKQLRLS MDIDVSY KH KGALHNYKMT DKNFLGRPVE VLLNDDLIVS TGFGSGLATV HVTTVVHKTS TSEEVCSFYL KIDTQDIEAS HYRGYGNS D YKRIVACASY KPSREESSSG SSHAVMDISL PTGISANEED LKALVEGVDQ LFTDYQIKDG HVILQLNSIP SSDFLCVRF RIFELFEVGF LSPATFTVYE YHRPDKQCTM FYSTSNIKIQ KVCEGAACKC VEADCGQMQE ELDLTISAET RKQTACKPEI AYAYKVSIT SITVENVFVK YKATLLDIYK TGEAVAEKDS EITFIKKVTC TNAELVKGRQ YLIMGKEALQ IKYNFSFRYI Y PLDSLTWI EYWPRDTTCS SCQAFLANLD EFAEDIFLNG C

UniProtKB: Complement C5

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Macromolecule #4: Nanobody UNbC5-1

MacromoleculeName: Nanobody UNbC5-1 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Lama glama (llama)
Molecular weightTheoretical: 16.757295 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MEVQLVESGG GLVQAGGSLR LSCAASGFTF DDYAIGWFRQ APGKEREGVS CISTSDGSTY YADSVKGRFT ISSDNAKNTV YLQMNSLKP EDTAVYYCAA DPYLPIRGRG IESTDFGSWG QGTQVTVSSA AASGSLEQKL ISEEDLNGAA HHHHHHGAA

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.26 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
10.0 mMNaPO4phosphate
145.0 mMNaClSodium chloridesodium chloride

Details: 1x PBS
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV / Details: Blot 4 seconds at blot force 1.
DetailsPurified protein were mixed before vitrification in

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.6 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 130000
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 1-40 / Number grids imaged: 2 / Number real images: 3810 / Average electron dose: 55.0 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

3cu7


Details: First C5 crystal structure
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v3.2/3) / Details: performed in cryoSPARC
Final 3D classificationSoftware - Name: cryoSPARC (ver. v3.2/3)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v3.2/3) / Details: performed in cryoSPARC
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v3.2/3) / Number images used: 193009
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChainDetails

3cu7

chain_id: B, residue_range: 20-1676, source_name: PDB, initial_model_type: experimental modelC5 complement
chain_id: A, residue_range: 2-136, source_name: AlphaFold, initial_model_type: in silico modelUNbC5-2
chain_id: C, residue_range: 2-144, source_name: AlphaFold, initial_model_type: in silico modelUNbC5-1
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-8cml:
Cryo-EM structure of complement C5 in complex with nanobodies UNbC5-1 and UNbC5-2

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