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- EMDB-23909: Structural basis for CSPG4 as a receptor for TcdB and a therapeut... -

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Basic information

Entry
Database: EMDB / ID: EMD-23909
TitleStructural basis for CSPG4 as a receptor for TcdB and a therapeutic target in Clostridioides difficile infection
Map dataStructural basis for CSPG4 as a receptor for TcdB and a therapeutic target in Clostridioides difficile infection
Sample
  • Complex: EM map of TcdB in complex with CSPG4
    • Complex: TcdB
      • Protein or peptide: Toxin B
    • Complex: CSPG4
      • Protein or peptide: Chondroitin sulfate proteoglycan 4
  • Ligand: ZINC ION
Function / homology
Function and homology information


Chondroitin sulfate biosynthesis / Defective CHST3 causes SEDCJD / Defective CHST14 causes EDS, musculocontractural type / Defective CHSY1 causes TPBS / Dermatan sulfate biosynthesis / Defective B3GALT6 causes EDSP2 and SEMDJL1 / CS/DS degradation / Defective B4GALT7 causes EDS, progeroid type / Defective B3GAT3 causes JDSSDHD / substrate-dependent cell migration ...Chondroitin sulfate biosynthesis / Defective CHST3 causes SEDCJD / Defective CHST14 causes EDS, musculocontractural type / Defective CHSY1 causes TPBS / Dermatan sulfate biosynthesis / Defective B3GALT6 causes EDSP2 and SEMDJL1 / CS/DS degradation / Defective B4GALT7 causes EDS, progeroid type / Defective B3GAT3 causes JDSSDHD / substrate-dependent cell migration / A tetrasaccharide linker sequence is required for GAG synthesis / glial cell migration / tissue remodeling / ruffle assembly / glucosyltransferase activity / host cell cytosol / Transferases; Glycosyltransferases; Hexosyltransferases / lamellipodium membrane / platelet-derived growth factor receptor signaling pathway / coreceptor activity / cysteine-type peptidase activity / ruffle / lysosomal lumen / host cell endosome membrane / Golgi lumen / positive regulation of peptidyl-tyrosine phosphorylation / toxin activity / collagen-containing extracellular matrix / angiogenesis / Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases / positive regulation of MAPK cascade / intracellular signal transduction / apical plasma membrane / focal adhesion / lipid binding / protein kinase binding / host cell plasma membrane / cell surface / proteolysis / extracellular exosome / extracellular region / nucleoplasm / membrane / metal ion binding / plasma membrane
Similarity search - Function
Cadherin-like / CSPG repeat / CSPG repeat profile. / Dermonecrotic/RTX toxin, membrane localization domain / TcdA/TcdB toxin, N-terminal helical domain / TcdB toxin N-terminal helical domain / Membrane Localization Domain / TcdA/TcdB toxin, catalytic glycosyltransferase domain / TcdA/TcdB catalytic glycosyltransferase domain / TcdA/TcdB toxin, pore forming domain ...Cadherin-like / CSPG repeat / CSPG repeat profile. / Dermonecrotic/RTX toxin, membrane localization domain / TcdA/TcdB toxin, N-terminal helical domain / TcdB toxin N-terminal helical domain / Membrane Localization Domain / TcdA/TcdB toxin, catalytic glycosyltransferase domain / TcdA/TcdB catalytic glycosyltransferase domain / TcdA/TcdB toxin, pore forming domain / TcdA/TcdB pore forming domain / Laminin G domain / CGT/MARTX, cysteine protease (CPD) domain / CGT/MARTX, cysteine protease (CPD) domain superfamily / Peptidase C80 family / CGT/MARTX cysteine protease (CPD) domain profile. / Laminin G domain profile. / Choline-binding repeat / Putative cell wall binding repeat / Laminin G domain / Laminin G domain / Cell wall/choline-binding repeat / Cell wall-binding repeat profile. / Nucleotide-diphospho-sugar transferases / Concanavalin A-like lectin/glucanase domain superfamily
Similarity search - Domain/homology
Toxin B / Chondroitin sulfate proteoglycan 4
Similarity search - Component
Biological speciesClostridioides difficile (bacteria) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.17 Å
AuthorsChen P / Jin R
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI) United States
CitationJournal: Nat Commun / Year: 2021
Title: Structural basis for CSPG4 as a receptor for TcdB and a therapeutic target in Clostridioides difficile infection.
Authors: Peng Chen / Ji Zeng / Zheng Liu / Hatim Thaker / Siyu Wang / Songhai Tian / Jie Zhang / Liang Tao / Craig B Gutierrez / Li Xing / Ralf Gerhard / Lan Huang / Min Dong / Rongsheng Jin /
Abstract: C. difficile is a major cause of antibiotic-associated gastrointestinal infections. Two C. difficile exotoxins (TcdA and TcdB) are major virulence factors associated with these infections, and ...C. difficile is a major cause of antibiotic-associated gastrointestinal infections. Two C. difficile exotoxins (TcdA and TcdB) are major virulence factors associated with these infections, and chondroitin sulfate proteoglycan 4 (CSPG4) is a potential receptor for TcdB, but its pathophysiological relevance and the molecular details that govern recognition remain unknown. Here, we determine the cryo-EM structure of a TcdB-CSPG4 complex, revealing a unique binding site spatially composed of multiple discontinuous regions across TcdB. Mutations that selectively disrupt CSPG4 binding reduce TcdB toxicity in mice, while CSPG4-knockout mice show reduced damage to colonic tissues during C. difficile infections. We further show that bezlotoxumab, the only FDA approved anti-TcdB antibody, blocks CSPG4 binding via an allosteric mechanism, but it displays low neutralizing potency on many TcdB variants from epidemic hypervirulent strains due to sequence variations in its epitopes. In contrast, a CSPG4-mimicking decoy neutralizes major TcdB variants, suggesting a strategy to develop broad-spectrum therapeutics against TcdB.
History
DepositionApr 27, 2021-
Header (metadata) releaseJun 9, 2021-
Map releaseJun 9, 2021-
UpdateJun 30, 2021-
Current statusJun 30, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 5
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 5
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7ml7
  • Surface level: 5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23909.png

Downloads & links

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Map

FileDownload / File: emd_23909.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationStructural basis for CSPG4 as a receptor for TcdB and a therapeutic target in Clostridioides difficile infection
Voxel sizeX=Y=Z: 1.245 Å
Density
Contour LevelBy AUTHOR: 5.0 / Movie #1: 5
Minimum - Maximum-12.735268 - 26.157877
Average (Standard dev.)1.0222875e-11 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions192192192
Spacing192192192
CellA=B=C: 239.04001 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.2451.2451.245
M x/y/z192192192
origin x/y/z0.0000.0000.000
length x/y/z239.040239.040239.040
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ192192192
MAP C/R/S321
start NC/NR/NS000
NC/NR/NS192192192
D min/max/mean-12.73526.1580.000

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Supplemental data

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Sample components

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Entire : EM map of TcdB in complex with CSPG4

EntireName: EM map of TcdB in complex with CSPG4
Components
  • Complex: EM map of TcdB in complex with CSPG4
    • Complex: TcdB
      • Protein or peptide: Toxin B
    • Complex: CSPG4
      • Protein or peptide: Chondroitin sulfate proteoglycan 4
  • Ligand: ZINC ION

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Supramolecule #1: EM map of TcdB in complex with CSPG4

SupramoleculeName: EM map of TcdB in complex with CSPG4 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2

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Supramolecule #2: TcdB

SupramoleculeName: TcdB / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Clostridioides difficile (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Supramolecule #3: CSPG4

SupramoleculeName: CSPG4 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Mammalian expression vector BsrGI-MCS-pcDNA3.1 (others)

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Macromolecule #1: Toxin B

MacromoleculeName: Toxin B / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
EC number: Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases
Source (natural)Organism: Clostridioides difficile (bacteria)
Molecular weightTheoretical: 228.752531 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MSAWSHPQFE KGGGSGGGSG GSAWSHPQFE KLEVLFQGPM SLVNRKQLEK MANVRFRTQE DEYVAILDAL EEYHNMSENT VVEKYLKLK DINSLTDIYI DTYKKSGRNK ALKKFKEYLV TEVLELKNNN LTPVEKNLHF VAIGGQINDT AINYINQWKD V NSDYNVNV ...String:
MSAWSHPQFE KGGGSGGGSG GSAWSHPQFE KLEVLFQGPM SLVNRKQLEK MANVRFRTQE DEYVAILDAL EEYHNMSENT VVEKYLKLK DINSLTDIYI DTYKKSGRNK ALKKFKEYLV TEVLELKNNN LTPVEKNLHF VAIGGQINDT AINYINQWKD V NSDYNVNV FYDSNAFLIN TLKKTVVESA INDTLESFRE NLNDPRFDYN KFFRKRMEII YDKQKNFINY YKAQREENPE LI IDDIVKT YLSNEYSKEI DELNTYIEES LNKITQNSGN DVRNFEEFKN GESFNLYEQE LVERWNLAAA SDILRISALK EIG GMYLNV NMLPGIQPDL FESIEKPSSV TVDFWEMTKL EAIMKYKEYI PEYTSEHFDM LDEEVQSSFE SVLASKSDKS EIFS SLGDM EASPLEVKIA FNSKGIINQG LISVKDSYCS NLIVKQIENR YKILNNSLNP AISEDNDFNT TTNTFIDSIM AEANA DNGR FMMELGKYLR VGFFPDVKTT INLSGPEAYA AAYQDLLMFK EGSMNIHLIE ADLRNFEISK TNISQSTEQE MASLWS FDD ARAKAQFEEY KRNYFEGSAG EDDNLDFSQN IVVDKEYLLE KISSLARSSE RGYIHYIVQL QGDKISYEAA CNLFAKT PY DSVLFQKNIE DSEIAYYYNP GDGEIQEIDK YKIPSIISDR PKIKLTFIGH GKDEFNTDIF AGFDVDSLST EIEAAIDL A KEDISPKSIE INLLGCNMFS YSINVEETYP GKLLLKVKDK ISELMPSISQ DSIIVSANQY EVRINSEGRR ELLDHSGEW INKEESIIKD ISSKEYISFN PKENKITVKS KNLPELSTLL QEIRNNSNSS DIELEEKVML TECEINVISN IDTQIVEERI EEAKNLTSD SINYIKDEFK LIESISDALC DLKQQNELED SHFISFEDIS ETDEGFSIRF INKETGESIF VETEKTIFSE Y ANHITEEI SKIKGTIFDT VNGKLVKKVN LDTTHEVNTL NAAFFIQSLI EYNSSKESLS NLSVAMKVQV YAQLFSTGLN TI TDAAKVV ELVSTALDET IDLLPTLSEG LPIIATIIDG VSLGAAIKEL SETSDPLLRQ EIEAKIGIMA VNLTTATTAI ITS SLGIAS GFSILLVPLA GISAGIPSLV NNELVLRDKA TKVVDYFKHV SLVETEGVFT LLDDKIMMPQ DDLVISEIDF NNNS IVLGK CEIWRMEGGS GHTVTDDIDH FFSAPSITYR EPHLSIYDVL EVQKEELDLS KDLMVLPNAP NRVFAWETGW TPGLR SLEN DGTKLLDRIR DNYEGEFYWR YFAFIADALI TTLKPRYEDT NIRINLDSNT RSFIVPIITT EYIREKLSYS FYGSGG TYA LSLSQYNMGI NIELSESDVW IIDVDNVVRD VTIESDKIKK GDLIEGILST LSIEENKIIL NSHEINFSGE VNGSNGF VS LTFSILEGIN AIIEVDLLSK SYKLLISGEL KILMLNSNHI QQKIDYIGFN SELQKNIPYS FVDSEGKENG FINGSTKE G LFVSELPDVV LISKVYMDDS KPSFGYYSNN LKDVKVITKD NVNILTGYYL KDDIKISLSL TLQDEKTIKL NSVHLDESG VAEILKFMNR KGNTNTSDSL MSFLESMNIK SIFVNFLQSN IKFILDANFI ISGTTSIGQF EFICDENDNI QPYFIKFNTL ETNYTLYVG NRQNMIVEPN YDLDDSGDIS STVINFSQKY LYGIDSCVNK VVISPNIYTD EINITPVYET NNTYPEVIVL D ANYINEKI NVNINDLSIR YVWSNDGNDF ILMSTSEENK VSQVKIRFVN VFKDKTLANK LSFNFSDKQD VPVSEIILSF TP SYYEDGL IGYDLGLVSL YNEKFYINNF GMMVSGLIYI NDSLYYFKPP VNNLITGFVT VGDDKYYFNP INGGAASIGE TII DDKNYY FNQSGVLQTG VFSTEDGFKY FAPANTLDEN LEGEAIDFTG KLIIDENIYY FDDNYRGAVE WKELDGEMHY FSPE TGKAF KLEHHHHHH

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Macromolecule #2: Chondroitin sulfate proteoglycan 4

MacromoleculeName: Chondroitin sulfate proteoglycan 4 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 82.298305 KDa
Recombinant expressionOrganism: Mammalian expression vector BsrGI-MCS-pcDNA3.1 (others)
SequenceString: HHHHHHHHHS GGGSGGGIEG RPSGSASFFG ENHLEVPVAT ALTDIDLQLQ FSTSQPEALL LLAAGPADHL LLQLYSGRLQ VRLVLGQEE LRLQTPAETL LSDSIPHTVV LTVVEGWATL SVDGFLNASS AVPGAPLEVP YGLFVGGTGT LGLPYLRGTS R PLRGCLHA ...String:
HHHHHHHHHS GGGSGGGIEG RPSGSASFFG ENHLEVPVAT ALTDIDLQLQ FSTSQPEALL LLAAGPADHL LLQLYSGRLQ VRLVLGQEE LRLQTPAETL LSDSIPHTVV LTVVEGWATL SVDGFLNASS AVPGAPLEVP YGLFVGGTGT LGLPYLRGTS R PLRGCLHA ATLNGRSLLR PLTPDVHEGC AEEFSASDDV ALGFSGPHSL AAFPAWGTQD EGTLEFTLTT QSRQAPLAFQ AG GRRGDFI YVDIFEGHLR AVVEKGQGTV LLHNSVPVAD GQPHEVSVHI NAHRLEISVD QYPTHTSNRG VLSYLEPRGS LLL GGLDAE ASRHLQEHRL GLTPEATNAS LLGCMEDLSV NGQRRGLREA LLTRNMAAGC RLEEEEYEDD AYGHYEAFST LAPE AWPAM ELPEPCVPEP GLPPVFANFT QLLTISPLVV AEGGTAWLEW RHVQPTLDLM EAELRKSQVL FSVTRGARHG ELELD IPGA QARKMFTLLD VVNRKARFIH DGSEDTSDQL VLEVSVTARV PMPSCLRRGQ TYLLPIQVNP VNDPPHIIFP HGSLMV ILE HTQKPLGPEV FQAYDPDSAC EGLTFQVLGT SSGLPVERRD QPGEPATEFS CRELEAGSLV YVHRGGPAQD LTFRVSD GL QASPPATLKV VAIRPAIQIH RSTGLRLAQG SAMPILPANL SVETNAVGQD VSVLFRVTGA LQFGELQKQG AGGVEGAE W WATQAFHQRD VEQGRVRYLS TDPQHHAYDT VENLALEVQ

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Macromolecule #3: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 3 / Number of copies: 1 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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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

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 46.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

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

6oq5

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.17 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 177995
FSC plot (resolution estimation)

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