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- PDB-7lo8: NorA in complex with Fab36 -

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Basic information

Entry
Database: PDB / ID: 7lo8
TitleNorA in complex with Fab36
Components
  • Fab36 Heavy Chain
  • Fab36 Light Chain
  • Quinolone resistance protein NorA
KeywordsTRANSPORT PROTEIN / efflux pump / antibiotic resistance
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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.16 Å
AuthorsBrawley, D.N. / Sauer, D.B. / Song, J.M. / Koide, A. / Koide, S. / Traaseth, N.J. / Wang, D.N.
Funding support United States, 8items
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, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 20, 2022Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
Z: Quinolone resistance protein NorA
H: Fab36 Heavy Chain
L: Fab36 Light Chain


Theoretical massNumber of molelcules
Total (without water)96,1933
Polymers96,1933
Non-polymers00
Water0
1


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

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Components

#1: Protein Quinolone resistance protein NorA


Mass: 42353.238 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Staphylococcus aureus (bacteria)
Gene: norA, tetA, tetA_2, BTN44_09105, DD547_00670, DQV53_04080, EP54_04170, EQ90_03440, FA040_06730, G0V24_03995, G0X12_10865, G0Z18_04370, G6Y10_04720, GO746_14090, GO803_07545, GO805_01365, GO821_ ...Gene: norA, tetA, tetA_2, BTN44_09105, DD547_00670, DQV53_04080, EP54_04170, EQ90_03440, FA040_06730, G0V24_03995, G0X12_10865, G0Z18_04370, G6Y10_04720, GO746_14090, GO803_07545, GO805_01365, GO821_08105, GO894_15090, GO942_03500, HMPREF2819_04810, HMPREF3211_00604, NCTC10654_00801, NCTC10702_01167, NCTC10988_00893, NCTC9944_00776, RK64_04180, SAMEA1029528_00668, SAMEA1029547_01044, SAMEA1029553_00751, SAMEA1964876_01266, SAMEA1965205_01031, SAMEA1966505_00819, SAMEA1969349_01052, SAMEA1969845_01011, SAMEA1971706_01410, SAMEA1972827_00559, SAMEA2076212_00937, SAMEA2076218_00794, SAMEA2076220_00416, SAMEA2076226_00094, SAMEA2076463_00402, SAMEA2076464_00402, SAMEA2076470_00989, SAMEA2076472_00906, SAMEA2076478_00948, SAMEA2076480_00403, SAMEA2076481_00754, SAMEA2076743_00851, SAMEA2076745_01198, SAMEA2076746_00709, SAMEA2076747_00402, SAMEA2076749_01092, SAMEA2076751_00767, SAMEA2076752_01693, SAMEA2076755_01477, SAMEA2076756_01730, SAMEA2076758_01750, SAMEA2076759_01476, SAMEA2076761_01673, SAMEA2076762_01568, SAMEA2076763_01005, SAMEA2076764_01192, SAMEA2076765_00813, SAMEA2077023_01193, SAMEA2077025_00963, SAMEA2077027_00992, SAMEA2077029_00946, SAMEA2077031_00749, SAMEA2077034_01532, SAMEA2077035_00441, SAMEA2077039_00933, SAMEA2077040_00402, SAMEA2077041_01231, SAMEA2077044_01041, SAMEA2077045_01087, SAMEA2077046_01075, SAMEA2077293_01085, SAMEA2077294_01001, SAMEA2077295_01088, SAMEA2077297_00893, SAMEA2077300_01388, SAMEA2077301_01079, SAMEA2077302_01294, SAMEA2077303_01165, SAMEA2077307_01110, SAMEA2077832_01179, SAMEA2078252_00991, SAMEA2078256_00442, SAMEA2078307_00767, SAMEA2078308_00437, SAMEA2078553_00705, SAMEA2078558_00440, SAMEA2078560_00441, SAMEA2078569_01033, SAMEA2078570_00726, SAMEA2078572_00766, SAMEA2078824_00440, SAMEA2078837_00724, SAMEA2079048_01019, SAMEA2079051_00890, SAMEA2079277_00570, SAMEA2079291_00132, SAMEA2079503_00716, SAMEA2079507_01569, SAMEA2079512_00399, SAMEA2079517_00401, SAMEA2079724_00193, SAMEA2079727_00743, SAMEA2079728_01205, SAMEA2079732_00659, SAMEA2079742_00929, SAMEA2079946_00528, SAMEA2079949_00512, SAMEA2079951_00193, SAMEA2079952_00660, SAMEA2079957_00881, SAMEA2079958_00559, SAMEA2079960_00657, SAMEA2079961_00193, SAMEA2079968_00614, SAMEA2080329_00094, SAMEA2080330_00193, SAMEA2080334_00929, SAMEA2080433_00981, SAMEA2080812_00715, SAMEA2080898_00727, SAMEA2080900_00662, SAMEA2080904_00710, SAMEA2080913_00729, SAMEA2081043_01808, SAMEA2081053_01006, SAMEA2081054_00654, SAMEA2081055_00993, SAMEA2081060_00553, SAMEA2081211_01079, SAMEA2081213_00399, SAMEA2081218_00473, SAMEA2081341_00094, SAMEA2081342_00612, SAMEA2081349_00936, SAMEA2081359_00094, SAMEA2081362_00860, SAMEA2081468_00628, SAMEA2081474_00441, SAMEA2081475_00651, SAMEA2081476_01465, SAMEA2081479_01707, SAMEA2081480_01854, SAMEA2081560_01571, SAMEA2081561_01009, SAMEA2081564_00963, SAMEA2081567_00976, SAMEA2081568_01004, SAMEA2081569_00706, SAMEA2081570_00962, SAMEA2081571_00711, SAMEA2081572_00947, SAMEA2081573_00955, SAMEA2081575_01082, SAMEA2081577_00891, SAMEA2081578_00766, SAMEA2081579_01168, SAMEA2081581_01073, SAMEA2081582_01166, SAMEA2081673_00935, SAMEA2081674_00934, SAMEA958766_00618, SAMEA958770_01028, SAMEA958772_00649, SAMEA958778_01022, SAMEA958779_00094, SAMEA958785_00651, SAMEA958793_00683, SAMEA958798_00328, SAMEA958804_01063, SAMEA958810_00661, SAMEA958836_01276, SAMEA958838_00069, SAMEA958845_00683, SAMEA958846_00694, SAMEA958848_01177, SAMEA958855_00578, SAMEA958858_00913, SAMEA958870_01324, SAMEA958898_01209, SAMEA958906_00445, SAMEA958925_01036, SAMEA958951_01582, SAMEA958953_00694, SAMEA958961_00691, SAMEA958979_00607, SAMEA958987_00401, SAMEA958995_00511
Plasmid: pET28a / Details (production host): C-term 10 His tag / Cell (production host): E. coli / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): C43 / References: UniProt: Q53459
#2: Antibody Fab36 Heavy Chain


Mass: 28044.465 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)
#3: Antibody Fab36 Light Chain


Mass: 25794.859 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)

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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: NorA:Fab36 complex / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Staphylococcus aureus (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenEmbedding 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 FIELDBright-field microscopy
Image recordingElectron dose: 55.54 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM software
IDNameCategory
2SerialEMimage acquisition
4cryoSPARCCTF correction
9cryoSPARCinitial Euler assignment
10cryoSPARCfinal Euler assignment
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.16 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 311335 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0066356
ELECTRON MICROSCOPYf_angle_d0.9388627
ELECTRON MICROSCOPYf_dihedral_angle_d9.442869
ELECTRON MICROSCOPYf_chiral_restr0.051989
ELECTRON MICROSCOPYf_plane_restr0.0081074

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