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- EMDB-23463: NorA in complex with Fab25 -

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ID or keywords:

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Basic information

Entry
Database: EMDB / ID: EMD-23463
TitleNorA in complex with Fab25
Map data
Sample
  • Complex: NorA:Fab25 complex
    • Protein or peptide: Quinolone resistance protein NorA
    • Protein or peptide: Fab25 Heavy Chain
    • Protein or peptide: Fab25 Light Chain
Function / homology
Function and homology information


xenobiotic transmembrane transporter activity / membrane => GO:0016020 / plasma membrane
Similarity search - Function
Tetracycline resistance protein TetA/multidrug resistance protein MdtG / Multidrug resistance protein / Major facilitator superfamily / Major Facilitator Superfamily / Major facilitator superfamily domain / Major facilitator superfamily (MFS) profile. / MFS transporter superfamily
Similarity search - Domain/homology
Quinolone resistance protein NorA
Similarity search - Component
Biological speciesStaphylococcus aureus (bacteria) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.74 Å
AuthorsBrawley DN / Sauer DB / Song JM / Koide A / Koide S / Traaseth NJ / Wang DN
Funding support United States, 8 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01AI108889 United States
National Science Foundation (NSF, United States)MCB1506420 United States
American Cancer Society129844-PF-17-135-01-TBE United States
Department of Defense (DOD, United States)W81XWH-16-1-0153 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM093825 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)R01NS108151 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM121994 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)T32GM881188 United States
CitationJournal: Nat Chem Biol / Year: 2022
Title: Structural basis for inhibition of the drug efflux pump NorA from Staphylococcus aureus.
Authors: Douglas N Brawley / David B Sauer / Jianping Li / Xuhui Zheng / Akiko Koide / Ganesh S Jedhe / Tiffany Suwatthee / Jinmei Song / Zheng Liu / Paramjit S Arora / Shohei Koide / Victor J Torres ...Authors: Douglas N Brawley / David B Sauer / Jianping Li / Xuhui Zheng / Akiko Koide / Ganesh S Jedhe / Tiffany Suwatthee / Jinmei Song / Zheng Liu / Paramjit S Arora / Shohei Koide / Victor J Torres / Da-Neng Wang / Nathaniel J Traaseth /
Abstract: Membrane protein efflux pumps confer antibiotic resistance by extruding structurally distinct compounds and lowering their intracellular concentration. Yet, there are no clinically approved drugs to ...Membrane protein efflux pumps confer antibiotic resistance by extruding structurally distinct compounds and lowering their intracellular concentration. Yet, there are no clinically approved drugs to inhibit efflux pumps, which would potentiate the efficacy of existing antibiotics rendered ineffective by drug efflux. Here we identified synthetic antigen-binding fragments (Fabs) that inhibit the quinolone transporter NorA from methicillin-resistant Staphylococcus aureus (MRSA). Structures of two NorA-Fab complexes determined using cryo-electron microscopy reveal a Fab loop deeply inserted in the substrate-binding pocket of NorA. An arginine residue on this loop interacts with two neighboring aspartate and glutamate residues essential for NorA-mediated antibiotic resistance in MRSA. Peptide mimics of the Fab loop inhibit NorA with submicromolar potency and ablate MRSA growth in combination with the antibiotic norfloxacin. These findings establish a class of peptide inhibitors that block antibiotic efflux in MRSA by targeting indispensable residues in NorA without the need for membrane permeability.
History
DepositionFeb 9, 2021-
Header (metadata) releaseApr 20, 2022-
Map releaseApr 20, 2022-
UpdateJul 13, 2022-
Current statusJul 13, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_23463.png

Downloads & links

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Map

FileDownload / File: emd_23463.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.079 Å
Density
Contour LevelBy AUTHOR: 7.0
Minimum - Maximum-36.408455 - 55.29559
Average (Standard dev.)1.7102204e-12 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 276.224 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : NorA:Fab25 complex

EntireName: NorA:Fab25 complex
Components
  • Complex: NorA:Fab25 complex
    • Protein or peptide: Quinolone resistance protein NorA
    • Protein or peptide: Fab25 Heavy Chain
    • Protein or peptide: Fab25 Light Chain

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Supramolecule #1: NorA:Fab25 complex

SupramoleculeName: NorA:Fab25 complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Staphylococcus aureus (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #1: Quinolone resistance protein NorA

MacromoleculeName: Quinolone resistance protein NorA / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Staphylococcus aureus (bacteria)
Molecular weightTheoretical: 42.353238 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MNKQIFVLYF NIFLIFLGIG LVIPVLPVYL KDLGLTGSDL GLLVAAFALS QMIISPFGGT LADKLGKKLI ICIGLILFSV SEFMFAVGH NFSVLMLSRV IGGMSAGMVM PGVTGLIADI SPSHQKAKNF GYMSAIINSG FILGPGIGGF MAEVSHRMPF Y FAGALGIL ...String:
MNKQIFVLYF NIFLIFLGIG LVIPVLPVYL KDLGLTGSDL GLLVAAFALS QMIISPFGGT LADKLGKKLI ICIGLILFSV SEFMFAVGH NFSVLMLSRV IGGMSAGMVM PGVTGLIADI SPSHQKAKNF GYMSAIINSG FILGPGIGGF MAEVSHRMPF Y FAGALGIL AFIMSIVLIH DPKKSTTSGF QKLEPQLLTK INWKVFITPV ILTLVLSFGL SAFETLYSLY TADKVNYSPK DI SIAITGG GIFGALFQIY FFDKFMKYFS ELTFIAWSLL YSVVVLILLV FANDYWSIML ISFVVFIGFD MIRPAITNYF SNI AGERQG FAGGLNSTFT SMGNFIGPLI AGALFDVHIE APIYMAIGVS LAGVVIVLIE KQHRAKLKEQ NM

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Macromolecule #2: Fab25 Heavy Chain

MacromoleculeName: Fab25 Heavy Chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 28.215697 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKKNIAFLLA SMFVFSIATN AYAEISEVQL VESGGGLVQP GGSLRLSCAA SGFTFSSSSI HWVRQAPGKG LEWVASISSS SGSTSYADS VKGRFTISAD TSKNTAYLQM NSLRAEDTAV YYCARWETGY YPYWRMYGFY WALDYWGQGT LVTVFNQIKG P SVFPLAPS ...String:
MKKNIAFLLA SMFVFSIATN AYAEISEVQL VESGGGLVQP GGSLRLSCAA SGFTFSSSSI HWVRQAPGKG LEWVASISSS SGSTSYADS VKGRFTISAD TSKNTAYLQM NSLRAEDTAV YYCARWETGY YPYWRMYGFY WALDYWGQGT LVTVFNQIKG P SVFPLAPS SKSTSGGTAA LGCLVKDYFP EPVTVSWNSG ALTSGVHTFP AVLQSSGLYS LSSVVTVPSS SLGTQTYICN VN HKPSNTK VDKKVEPKSC DKTHT

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Macromolecule #3: Fab25 Light Chain

MacromoleculeName: Fab25 Light Chain / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 25.794859 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKKNIAFLLA SMFVFSIATN AYASDIQMTQ SPSSLSASVG DRVTITCRAS QSVSSAVAWY QQKPGKAPKL LIYSASSLYS GVPSRFSGS RSGTDFTLTI SSLQPEDFAT YYCQQSSSSL ITFGQGTKVE IKRTVAAPSV FIFPPSDSQL KSGTASVVCL L NNFYPREA ...String:
MKKNIAFLLA SMFVFSIATN AYASDIQMTQ SPSSLSASVG DRVTITCRAS QSVSSAVAWY QQKPGKAPKL LIYSASSLYS GVPSRFSGS RSGTDFTLTI SSLQPEDFAT YYCQQSSSSL ITFGQGTKVE IKRTVAAPSV FIFPPSDSQL KSGTASVVCL L NNFYPREA KVQWKVDNAL QSGNSQESVT EQDSKDSTYS LSSTLTLSKA DYEKHKVYAC EVTHQGLSSP VTKSFNRGEC

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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

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 49.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.74 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 231085

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