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- PDB-7ml7: Structural basis for CSPG4 as a receptor for TcdB and a therapeut... -

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Basic information

Entry
Database: PDB / ID: 7ml7
TitleStructural basis for CSPG4 as a receptor for TcdB and a therapeutic target in Clostridioides difficile infection
Components
  • Chondroitin sulfate proteoglycan 4
  • Toxin B
KeywordsTOXIN / CSPG4 / TcdB / Clostridioides difficile infection
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 / angiogenesis / collagen-containing extracellular matrix / positive regulation of MAPK cascade / Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases / 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. / TcdA/TcdB toxin, N-terminal helical domain / TcdB toxin N-terminal helical domain / Dermonecrotic/RTX toxin, membrane localization domain / TcdA/TcdB toxin, catalytic glycosyltransferase domain / Membrane Localization Domain / TcdA/TcdB catalytic glycosyltransferase domain / TcdA/TcdB toxin, pore forming domain ...Cadherin-like / CSPG repeat / CSPG repeat profile. / TcdA/TcdB toxin, N-terminal helical domain / TcdB toxin N-terminal helical domain / Dermonecrotic/RTX toxin, membrane localization domain / TcdA/TcdB toxin, catalytic glycosyltransferase domain / Membrane Localization 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.17 Å
AuthorsChen, P. / Jin, R.
Funding support United States, 1items
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, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 9, 2021Provider: repository / Type: Initial release
Revision 1.1Jun 30, 2021Group: Database references / Category: 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

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Structure visualization

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  • Deposited structure unit
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Assembly

Deposited unit
A: Toxin B
B: Chondroitin sulfate proteoglycan 4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)311,1163
Polymers311,0512
Non-polymers651
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area2720 Å2
ΔGint-12 kcal/mol
Surface area57390 Å2

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Components

#1: Protein Toxin B


Mass: 228752.531 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Clostridioides difficile (bacteria) / Gene: tcdB, toxB / Production host: Escherichia coli (E. coli)
References: UniProt: P18177, Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases
#2: Protein Chondroitin sulfate proteoglycan 4 / Chondroitin sulfate proteoglycan NG2 / Melanoma chondroitin sulfate proteoglycan / Melanoma- ...Chondroitin sulfate proteoglycan NG2 / Melanoma chondroitin sulfate proteoglycan / Melanoma-associated chondroitin sulfate proteoglycan


Mass: 82298.305 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CSPG4, MCSP
Production host: Mammalian expression vector BsrGI-MCS-pcDNA3.1 (others)
References: UniProt: Q6UVK1
#3: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
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

Component
IDNameTypeEntity IDParent-IDSource
1EM map of TcdB in complex with CSPG4COMPLEX#1-#20MULTIPLE SOURCES
2TcdBCOMPLEX#11RECOMBINANT
3CSPG4COMPLEX#21RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Clostridioides difficile (bacteria)1496
23Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli (E. coli)562
23Mammalian expression vector BsrGI-MCS-pcDNA3.1 (others)2021187
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 46 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refinedev_3742refinement
PHENIXdev_3742refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.17 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 177995 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 63.27 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.004111109
ELECTRON MICROSCOPYf_angle_d0.725915027
ELECTRON MICROSCOPYf_chiral_restr0.05031685
ELECTRON MICROSCOPYf_plane_restr0.00511952
ELECTRON MICROSCOPYf_dihedral_angle_d15.13121455

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