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- EMDB-28660: Cryo-EM structure of S. aureus BlaR1 TM and zinc metalloprotease ... -

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Basic information

Entry
Database: EMDB / ID: EMD-28660
TitleCryo-EM structure of S. aureus BlaR1 TM and zinc metalloprotease domain
Map dataSharpened map from cryosparc local refinement
Sample
  • Complex: Dimeric complex of S. aureus BlaR1
    • Protein or peptide: Beta-lactam sensor/signal transducer BlaR1
  • Ligand: ZINC ION
  • Ligand: (2S)-3-{[{[(2S)-2,3-DIHYDROXYPROPYL]OXY}(HYDROXY)PHOSPHORYL]OXY}-2-[(6E)-HEXADEC-6-ENOYLOXY]PROPYL (8E)-OCTADEC-8-ENOATE
  • Ligand: PHOSPHATE ION
KeywordsAntibiotic resistance / beta-lactam antibiotics / MRSA / BlaR1 / MecR1 / cryo-EM / transmembrane signalling / SIGNALING PROTEIN
Function / homology: / Peptidase M56 / BlaR1 peptidase M56 / Penicillin-binding protein, transpeptidase / Penicillin binding protein transpeptidase domain / penicillin binding / Beta-lactamase/transpeptidase-like / membrane / BlaR1
Function and homology information
Biological speciesStaphylococcus aureus (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsWorrall LJ / Alexander JAN / Vuckovic M / Strynadka NCJ
Funding support Canada, 1 items
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, 2022-
Header (metadata) releaseJan 11, 2023-
Map releaseJan 11, 2023-
UpdateJun 19, 2024-
Current statusJun 19, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_28660.png

Downloads & links

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Map

FileDownload / File: emd_28660.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map from cryosparc local refinement
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.77 Å/pix.
x 300 pix.
= 231. Å		0.77 Å/pix.
x 300 pix.
= 231. Å		0.77 Å/pix.
x 300 pix.
= 231. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.77 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.07
Minimum - Maximum-0.30921808 - 0.46021047
Average (Standard dev.)0.00027321183 (±0.013928027)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 231.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half map from cryosparc refinement. Maps have a...

Fileemd_28660_half_map_1.map
AnnotationHalf map from cryosparc refinement. Maps have a lot of high resolution noise. Lowpass filtering to 3.8 Ang. (FSC resolution of final map) reveals structure.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map from cryosparc refinement. Maps have a...

Fileemd_28660_half_map_2.map
AnnotationHalf map from cryosparc refinement. Maps have a lot of high resolution noise. Lowpass filtering to 3.8 Ang. (FSC resolution of final map) reveals structure.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Dimeric complex of S. aureus BlaR1

EntireName: Dimeric complex of S. aureus BlaR1
Components
  • Complex: Dimeric complex of S. aureus BlaR1
    • Protein or peptide: Beta-lactam sensor/signal transducer BlaR1
  • Ligand: ZINC ION
  • Ligand: (2S)-3-{[{[(2S)-2,3-DIHYDROXYPROPYL]OXY}(HYDROXY)PHOSPHORYL]OXY}-2-[(6E)-HEXADEC-6-ENOYLOXY]PROPYL (8E)-OCTADEC-8-ENOATE
  • Ligand: PHOSPHATE ION

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Supramolecule #1: Dimeric complex of S. aureus BlaR1

SupramoleculeName: Dimeric complex of S. aureus BlaR1 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Staphylococcus aureus (bacteria)
Molecular weightTheoretical: 142 KDa

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Macromolecule #1: Beta-lactam sensor/signal transducer BlaR1

MacromoleculeName: Beta-lactam sensor/signal transducer BlaR1 / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Staphylococcus aureus (bacteria)
Molecular weightTheoretical: 71.269703 KDa
Recombinant expressionOrganism: Lactobacillus delbrueckii subsp. lactis (bacteria)
SequenceString: MAKLLIMSIV SFCFIFLLLL FFRYILKRYF NYMLNYKVWY LTLLAGLIPF IPIKFSLFKF NNVNNQAPTV ESKSHDLNHN INTTKPIQE FATDIHKFNW DSIDNICTVI WIVLVIILSF KFLKALLYLK YLKKQSLYLN ENEKNKIDTI LFNHQYKKNI V IRKAETIQ ...String:
MAKLLIMSIV SFCFIFLLLL FFRYILKRYF NYMLNYKVWY LTLLAGLIPF IPIKFSLFKF NNVNNQAPTV ESKSHDLNHN INTTKPIQE FATDIHKFNW DSIDNICTVI WIVLVIILSF KFLKALLYLK YLKKQSLYLN ENEKNKIDTI LFNHQYKKNI V IRKAETIQ SPITFWYGKY IILIPSSYFK SVIDKRLKYI ILHEYAHAKN RDTLHLIIFN IFSIIMSYNP LVHIVKRKII HD NEVEADR FVLNNINKNE FKTYAESIMD SVLNVPFFNK NILSHSFNGK KSLLKRRLIN IKEANLKKQS KLILIFICIF TFL LMVIQS QFLMGQSITD YNYKKPLHND YQILDKSKIF GSNSGSFVMY SMKKDKYYIY NEKESRKRYS PNSTYKIYLA MFGL DRHII NDENSRMSWN HKHYPFDAWN KEQDLNTAMQ NSVNWYFERI SDQIPKNYTA TQLKQLNYGN KNLGSYKSYW MEDSL KISN LEQVIVFKNM MEQNNHFSKK AKNQLSSSLL IKKNEKYELY GKTGTGIVNG KYNNGWFVGY VITNHDKYYF ATHLSD GKP SGKNAELISE KILKEMGVLN GQELALVPRG SSAHHHHHH

UniProtKB: BlaR1

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Macromolecule #2: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 2 / Number of copies: 2 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #3: (2S)-3-{[{[(2S)-2,3-DIHYDROXYPROPYL]OXY}(HYDROXY)PHOSPHORYL]OXY}-...

MacromoleculeName: (2S)-3-{[{[(2S)-2,3-DIHYDROXYPROPYL]OXY}(HYDROXY)PHOSPHORYL]OXY}-2-[(6E)-HEXADEC-6-ENOYLOXY]PROPYL (8E)-OCTADEC-8-ENOATE
type: ligand / ID: 3 / Number of copies: 2 / Formula: P6L
Molecular weightTheoretical: 746.991 Da
Chemical component information

ChemComp-P6L:
(2S)-3-{[{[(2S)-2,3-DIHYDROXYPROPYL]OXY}(HYDROXY)PHOSPHORYL]OXY}-2-[(6E)-HEXADEC-6-ENOYLOXY]PROPYL (8E)-OCTADEC-8-ENOATE / phospholipid*YM

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Macromolecule #4: PHOSPHATE ION

MacromoleculeName: PHOSPHATE ION / type: ligand / ID: 4 / Number of copies: 3 / Formula: PO4
Molecular weightTheoretical: 94.971 Da
Chemical component information

ChemComp-PO4:
PHOSPHATE ION

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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

Concentration0.5 mg/mL
BufferpH: 7.5 / Details: 20 mM HEPES, pH 7.5, 150 mM sodium chloride
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 7508828
Startup modelType of model: INSILICO MODEL
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 66347
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
FSC plot (resolution estimation)

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