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- PDB-7zbq: Structure of the ADP-ribosyltransferase TccC3HVR from Photorhabdu... -

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Basic information

Entry
Database: PDB / ID: 7zbq
TitleStructure of the ADP-ribosyltransferase TccC3HVR from Photorhabdus Luminescens
ComponentsTccC3
KeywordsTOXIN / ADP-ribosylation / R-S-E motif / actin modification
Function / homologyToxin complex C-like repeat / Tripartite Tc toxins repeat / Rhs repeat-associated core / TccC3
Function and homology information
Biological speciesPhotorhabdus luminescens (bacteria)
MethodSOLUTION NMR / simulated annealing
AuthorsLindemann, F. / Belyy, A. / Friedrich, D. / Schmieder, P. / Bardiaux, B. / Roderer, D. / Funk, J. / Protze, J. / Bieling, P. / Oschkinat, H. / Raunser, S.
Funding support Germany, 1items
OrganizationGrant numberCountry
German Research Foundation (DFG)Os 106/17-1 Germany
CitationJournal: Nat Commun / Year: 2022
Title: Mechanism of threonine ADP-ribosylation of F-actin by a Tc toxin.
Authors: Alexander Belyy / Florian Lindemann / Daniel Roderer / Johanna Funk / Benjamin Bardiaux / Jonas Protze / Peter Bieling / Hartmut Oschkinat / Stefan Raunser /
Abstract: Tc toxins deliver toxic enzymes into host cells by a unique injection mechanism. One of these enzymes is the actin ADP-ribosyltransferase TccC3, whose activity leads to the clustering of the cellular ...Tc toxins deliver toxic enzymes into host cells by a unique injection mechanism. One of these enzymes is the actin ADP-ribosyltransferase TccC3, whose activity leads to the clustering of the cellular cytoskeleton and ultimately cell death. Here, we show in atomic detail how TccC3 modifies actin. We find that the ADP-ribosyltransferase does not bind to G-actin but interacts with two consecutive actin subunits of F-actin. The binding of TccC3 to F-actin occurs via an induced-fit mechanism that facilitates access of NAD to the nucleotide binding pocket. The following nucleophilic substitution reaction results in the transfer of ADP-ribose to threonine-148 of F-actin. We demonstrate that this site-specific modification of F-actin prevents its interaction with depolymerization factors, such as cofilin, which impairs actin network turnover and leads to steady actin polymerization. Our findings reveal in atomic detail a mechanism of action of a bacterial toxin through specific targeting and modification of F-actin.
History
DepositionMar 24, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 29, 2022Provider: repository / Type: Initial release
Revision 1.1Aug 3, 2022Group: 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 / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: TccC3


Theoretical massNumber of molelcules
Total (without water)21,7241
Polymers21,7241
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)10 / 400structures with the lowest energy
RepresentativeModel #1closest to the average

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Components

#1: Protein TccC3


Mass: 21723.785 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Photorhabdus luminescens (bacteria) / Gene: TccC3 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21-CodonPlus(DE3)-RIPL / References: UniProt: Q8GF97

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Experimental details

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Experiment

ExperimentMethod: SOLUTION NMR
NMR experiment
Conditions-IDExperiment-IDSolution-IDSample stateSpectrometer-IDType
112isotropic23D (H)CCH-TOCSY
123isotropic23D HNCA
132isotropic22D 1H-1H NOESY
142isotropic23D 1H-13C NOESY aliphatic
153isotropic13D 1H-13C NOESY aliphatic
173isotropic13D 1H-13C NOESY aromatic
163isotropic13D 1H-15N NOESY

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Sample preparation

Details
TypeSolution-IDContentsLabelSolvent system
solution1500 uM non-exchangeable-D, U-13C, U-15N TcART, 20 mM HEPES, 150 mM sodium chloride, 90% H2O/10% D2OTcART_DCN90% H2O/10% D2O
solution2500 uM [U-13C; U-15N] TcART, 20 mM d11-Tris, 150 mM sodium chloride, 100% D2OTcART_CN_D2O100% D2O
solution3500 uM [U-13C; U-15N] TcART, 20 mM HEPES, 150 mM sodium chloride, 90% H2O/10% D2OTcART_CN90% H2O/10% D2O
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
500 uMTcARTnon-exchangeable-D, U-13C, U-15N1
20 mMHEPESnatural abundance1
150 mMsodium chloridenatural abundance1
500 uMTcART[U-13C; U-15N]2
20 mMd11-Trisnatural abundance2
150 mMsodium chloridenatural abundance2
500 uMTcART[U-13C; U-15N]3
20 mMHEPESnatural abundance3
150 mMsodium chloridenatural abundance3
Sample conditionsIonic strength: 150 mM / Label: TcART_cond / pH: 7.8 / Pressure: 1 atm / Temperature: 280 K

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NMR measurement

NMR spectrometer
TypeManufacturerModelField strength (MHz)Spectrometer-ID
Bruker AVANCE IIBrukerAVANCE II7501
Bruker AVANCE IIIBrukerAVANCE III6002

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Processing

NMR software
NameVersionDeveloperClassification
TopSpin3.5pl6Bruker Biospincollection
TopSpin3.5pl6Bruker Biospinprocessing
CcpNmr Analysis2.4.2CCPNchemical shift assignment
CcpNmr Analysis2.4.2CCPNpeak picking
ARIA2.3.2Linge, O'Donoghue and Nilgesstructure calculation
CNS1.21Brunger A. T. et.al.refinement
RefinementMethod: simulated annealing / Software ordinal: 1 / Details: standard aria/cns water refinement protocol
NMR representativeSelection criteria: closest to the average
NMR ensembleConformer selection criteria: structures with the lowest energy
Conformers calculated total number: 400 / Conformers submitted total number: 10

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