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- PDB-8rlz: NMR solution structure of the N-terminal cytoplasmic domain, DdvA... -

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Basic information

Entry
Database: PDB / ID: 8rlz
TitleNMR solution structure of the N-terminal cytoplasmic domain, DdvANt, of the membrane antisigma factor DdvA
ComponentsAntisigma factor DdvA
KeywordsTRANSCRIPTION / Membrane antisigma / Zinc-associated antisigma domain / ECF sigma binding domain / Three-helix bundle
Function / homology:
Function and homology information
Biological speciesMyxococcus xanthus (bacteria)
MethodSOLUTION NMR / simulated annealing
AuthorsPantoja-Uceda, D. / Padmanabhan, S.
Funding support Spain, 4items
OrganizationGrant numberCountry
Ministerio de Ciencia e Innovacion (MCIN)PID2021-123336NB-C22 Spain
Ministerio de Ciencia e Innovacion (MCIN)PID2021-123336NB-C21 Spain
Ministerio de Ciencia e Innovacion (MCIN)PID2020-112821GB-I00 Spain
Other governmentICTS R-LRB
CitationJournal: Sci Adv / Year: 2024
Title: Structural basis for regulation of a CBASS-CRISPR-Cas defense island by a transmembrane anti-σ factor and its ECF σ partner.
Authors: Diego Bernal-Bernal / David Pantoja-Uceda / Jorge Pedro López-Alonso / Alfonso López-Rojo / José Antonio López-Ruiz / Marisa Galbis-Martínez / Borja Ochoa-Lizarralde / Igor Tascón / ...Authors: Diego Bernal-Bernal / David Pantoja-Uceda / Jorge Pedro López-Alonso / Alfonso López-Rojo / José Antonio López-Ruiz / Marisa Galbis-Martínez / Borja Ochoa-Lizarralde / Igor Tascón / Montserrat Elías-Arnanz / Iban Ubarretxena-Belandia / S Padmanabhan /
Abstract: How CRISPR-Cas and cyclic oligonucleotide-based antiphage signaling systems (CBASS) are coordinately deployed against invaders remains unclear. We show that a locus containing two CBASS and one type ...How CRISPR-Cas and cyclic oligonucleotide-based antiphage signaling systems (CBASS) are coordinately deployed against invaders remains unclear. We show that a locus containing two CBASS and one type III-B CRISPR-Cas system, regulated by the transmembrane anti-σ DdvA and its cognate extracytoplasmic function (ECF) σ DdvS, can defend against a phage. Cryo-electron microscopy reveals DdvA-DdvS pairs assemble as arrow-shaped transmembrane dimers. Each DdvA periplasmic domain adopts a separase/craspase-type tetratricopeptide repeat (TPR)-caspase HetF-associated with TPR (TPR-CHAT) architecture with an incomplete His-Cys active site, lacking three α-helices conserved among CHAT domains. Each active site faces the dimer interface, raising the possibility that signal-induced caspase-like DdvA autoproteolysis in trans precedes RseP-mediated intramembrane proteolysis and DdvS release. Nuclear magnetic resonance reveals a DdvA cytoplasmic CHCC-type zinc-bound three-helix bundle that binds to DdvS σ and σ domains, undergoing σ-induced helix extension to trap DdvS. Altogether, we provide structural-mechanistic insights into membrane anti-σ-ECF σ regulation of an antiviral CBASS-CRISPR-Cas defense island.
History
DepositionJan 4, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 6, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Antisigma factor DdvA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)10,1342
Polymers10,0681
Non-polymers651
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: NMR Distance Restraints, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)20 / 100target function
RepresentativeModel #1target function

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Components

#1: Protein Antisigma factor DdvA / Antisigma factor DdvA


Mass: 10068.254 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Myxococcus xanthus (bacteria) / Strain: DK1050 / Gene: I5Q59_35855 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A8E4SKQ1
#2: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: SOLUTION NMR
NMR experiment
Conditions-IDExperiment-IDSolution-IDSample stateSpectrometer-IDType
112isotropic12D 1H-15N HSQC
122isotropic12D 1H-13C HSQC
132isotropic13D HNCO
142isotropic13D HN(CA)CO
152isotropic13D HNCA
162isotropic13D HN(CA)CB
172isotropic13D CBCA(CO)NH
182isotropic13D H(CCO)NH
192isotropic13D (H)CC(CO)NH
1102isotropic13D HNHA
1112isotropic13D HBHA(CO)NH
1122isotropic13D (H)N(COCA)NH
1132isotropic13D H(MCOCA)NH
1142isotropic12D NOESY
1152isotropic12D TOCSY
1162isotropic13D 1H-15N NOESY
1172isotropic12D NOESY
1182isotropic12D TOCSY

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Sample preparation

Details
TypeSolution-IDContentsDetailsLabelSolvent system
solution10.7 mM [U-13C; U-15N] DdvANt, 100 mM sodium chloride, 20 mM Tris, 2 mM beta-mercaptoethanol, 0.05 % sodium azide, 90% H2O/10% D2ODdvANt sample for assignment13C_15N_sample90% H2O/10% D2O
solution20.7 mM DdvANt, 100 mM sodium chloride, 20 mM Tris, 2 mM beta-mercaptoethanol, 0.05 % sodium azide, 90% H2O/10% D2ODdvANt sample for assignment and structure calculation1H_sample90% H2O/10% D2O
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
0.7 mMDdvANt[U-13C; U-15N]1
100 mMsodium chloridenatural abundance1
20 mMTrisnatural abundance1
2 mMbeta-mercaptoethanolnatural abundance1
0.05 %sodium azidenatural abundance1
0.7 mMDdvANtnatural abundance2
100 mMsodium chloridenatural abundance2
20 mMTrisnatural abundance2
2 mMbeta-mercaptoethanolnatural abundance2
0.05 %sodium azidenatural abundance2
Sample conditionsDetails: DdvANt sample for assignment and structure calculation
Ionic strength: 100 mM / Label: condition_1 / pH: 7.0 Not defined / Pressure: 1 atm / Temperature: 298 K

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NMR measurement

NMR spectrometerType: Bruker AVANCE NEO / Manufacturer: Bruker / Model: AVANCE NEO / Field strength: 800 MHz

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Processing

NMR software
NameDeveloperClassification
TopSpinBruker Biospincollection
TopSpinBruker Biospinprocessing
NMRPipeDelaglio, Grzesiek, Vuister, Zhu, Pfeifer and Baxprocessing
NMRViewJohnson, One Moon Scientificdata analysis
CYANAGuntert, Mumenthaler and Wuthrichstructure calculation
AmberCase, Darden, Cheatham III, Simmerling, Wang, Duke, Luo, ... and Kollmanrefinement
NMRViewJohnson, One Moon Scientificchemical shift assignment
NMRViewJohnson, One Moon Scientificpeak picking
RefinementMethod: simulated annealing / Software ordinal: 6
NMR representativeSelection criteria: target function
NMR ensembleConformer selection criteria: target function / Conformers calculated total number: 100 / Conformers submitted total number: 20

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