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- PDB-8exr: Cryo-EM structure of S. aureus BlaR1 TM and zinc metalloprotease ... -

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Basic information

Entry
Database: PDB / ID: 8exr
TitleCryo-EM structure of S. aureus BlaR1 TM and zinc metalloprotease domain
ComponentsBeta-lactam sensor/signal transducer BlaR1
KeywordsSIGNALING PROTEIN / Antibiotic resistance / beta-lactam antibiotics / MRSA / BlaR1 / MecR1 / cryo-EM / transmembrane signalling
Function / homology
Function and homology information


penicillin binding / cell wall organization / membrane
Similarity search - Function
Peptidase M56 / BlaR1 peptidase M56 / : / Penicillin-binding protein, transpeptidase / Penicillin binding protein transpeptidase domain / Beta-lactamase/transpeptidase-like
Similarity search - Domain/homology
Chem-P6L / PHOSPHATE ION / BlaR1
Similarity search - Component
Biological speciesStaphylococcus aureus (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsWorrall, L.J. / Alexander, J.A.N. / Vuckovic, M. / Strynadka, N.C.J.
Funding support Canada, 1items
OrganizationGrant numberCountry
Canadian Institutes of Health Research (CIHR) Canada
CitationJournal: Nature / Year: 2023
Title: Structural basis of broad-spectrum β-lactam resistance in Staphylococcus aureus.
Authors: J Andrew N Alexander / Liam J Worrall / Jinhong Hu / Marija Vuckovic / Nidhi Satishkumar / Raymond Poon / Solmaz Sobhanifar / Federico I Rosell / Joshua Jenkins / Daniel Chiang / Wesley A ...Authors: J Andrew N Alexander / Liam J Worrall / Jinhong Hu / Marija Vuckovic / Nidhi Satishkumar / Raymond Poon / Solmaz Sobhanifar / Federico I Rosell / Joshua Jenkins / Daniel Chiang / Wesley A Mosimann / Henry F Chambers / Mark Paetzel / Som S Chatterjee / Natalie C J Strynadka /
Abstract: Broad-spectrum β-lactam antibiotic resistance in Staphylococcus aureus is a global healthcare burden. In clinical strains, resistance is largely controlled by BlaR1, a receptor that senses β- ...Broad-spectrum β-lactam antibiotic resistance in Staphylococcus aureus is a global healthcare burden. In clinical strains, resistance is largely controlled by BlaR1, a receptor that senses β-lactams through the acylation of its sensor domain, inducing transmembrane signalling and activation of the cytoplasmic-facing metalloprotease domain. The metalloprotease domain has a role in BlaI derepression, inducing blaZ (β-lactamase PC1) and mecA (β-lactam-resistant cell-wall transpeptidase PBP2a) expression. Here, overcoming hurdles in isolation, we show that BlaR1 cleaves BlaI directly, as necessary for inactivation, with no requirement for additional components as suggested previously. Cryo-electron microscopy structures of BlaR1-the wild type and an autocleavage-deficient F284A mutant, with or without β-lactam-reveal a domain-swapped dimer that we suggest is critical to the stabilization of the signalling loops within. BlaR1 undergoes spontaneous autocleavage in cis between Ser283 and Phe284 and we describe the catalytic mechanism and specificity underlying the self and BlaI cleavage. The structures suggest that allosteric signalling emanates from β-lactam-induced exclusion of the prominent extracellular loop bound competitively in the sensor-domain active site, driving subsequent dynamic motions, including a shift in the sensor towards the membrane and accompanying changes in the zinc metalloprotease domain. We propose that this enhances the expulsion of autocleaved products from the active site, shifting the equilibrium to a state that is permissive of efficient BlaI cleavage. Collectively, this study provides a structure of a two-component signalling receptor that mediates action-in this case, antibiotic resistance-through the direct cleavage of a repressor.
History
DepositionOct 25, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 11, 2023Provider: repository / Type: Initial release
Revision 1.1Jan 18, 2023Group: Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Jan 25, 2023Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Jun 19, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: Beta-lactam sensor/signal transducer BlaR1
A: Beta-lactam sensor/signal transducer BlaR1
C: Beta-lactam sensor/signal transducer BlaR1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)215,71910
Polymers213,8093
Non-polymers1,9107
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Beta-lactam sensor/signal transducer BlaR1 / Beta-lactamase regulatory sensor-transducer BlaR1 / Bla regulator protein blaR1 / BlaR1 / BlaR1 ...Beta-lactamase regulatory sensor-transducer BlaR1 / Bla regulator protein blaR1 / BlaR1 / BlaR1 family beta-lactam sensor/signal transducer / BlaR1 protein / Regulatory protein BlaR1


Mass: 71269.703 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Staphylococcus aureus (bacteria)
Gene: blaR1, blaR, BN1321_410015, BSZ10_11995, BTN44_15560, FA040_05105, GO941_06445, NCTC13131_00091, SAMEA2077334_00715, SAMEA2078260_01199, SAMEA2078588_01099, SAMEA2080344_00713, SAMEA2081063_ ...Gene: blaR1, blaR, BN1321_410015, BSZ10_11995, BTN44_15560, FA040_05105, GO941_06445, NCTC13131_00091, SAMEA2077334_00715, SAMEA2078260_01199, SAMEA2078588_01099, SAMEA2080344_00713, SAMEA2081063_00867, SAMEA2081470_00938, SAMEA70146418_00144, SAP084B_008, SAP086A_011
Production host: Lactobacillus delbrueckii subsp. lactis (bacteria)
References: UniProt: Q00419
#2: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
#3: Chemical ChemComp-P6L / (2S)-3-{[{[(2S)-2,3-DIHYDROXYPROPYL]OXY}(HYDROXY)PHOSPHORYL]OXY}-2-[(6E)-HEXADEC-6-ENOYLOXY]PROPYL (8E)-OCTADEC-8-ENOATE


Mass: 746.991 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C40H75O10P / Comment: phospholipid*YM
#4: Chemical ChemComp-PO4 / PHOSPHATE ION


Mass: 94.971 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: PO4
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

ComponentName: Dimeric complex of S. aureus BlaR1 / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.142 MDa / Experimental value: NO
Source (natural)Organism: Staphylococcus aureus (bacteria)
Source (recombinant)Organism: Lactobacillus delbrueckii subsp. lactis (bacteria)
Buffer solutionpH: 7.5 / Details: 20 mM HEPES, pH 7.5, 150 mM sodium chloride
SpecimenConc.: 0.5 mg/ml / Embedding 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 / Nominal defocus max: 3000 nm / Nominal defocus min: 500 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

EM software
IDNameCategoryDetails
1Topazparticle selection
4cryoSPARCCTF correction
9PHENIXmodel refinementReal space refine
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
12cryoSPARCclassification
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 7508828
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 66347 / Symmetry type: POINT

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