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- PDB-28vj: NorA bound to miniprotein I-23 -

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Basic information

Entry
Database: PDB / ID: 28vj
TitleNorA bound to miniprotein I-23
Components
  • I23 miniprotein
  • Quinolone resistance protein NorA,Soluble cytochrome b562
KeywordsTRANSPORT PROTEIN / Multidrug efflux transporter / efflux pump inhibitor
Function / homology
Function and homology information


xenobiotic transmembrane transporter activity / electron transport chain / periplasmic space / electron transfer activity / iron ion binding / heme binding / plasma membrane
Similarity search - Function
: / Tetracycline resistance protein TetA/multidrug resistance protein MdtG / Major facilitator superfamily / Major Facilitator Superfamily / Major facilitator superfamily domain / Major facilitator superfamily (MFS) profile. / Cytochrome b562 / Cytochrome b562 / Cytochrome c/b562 / MFS transporter superfamily
Similarity search - Domain/homology
Soluble cytochrome b562 / Quinolone resistance protein NorA
Similarity search - Component
Biological speciesSynthetic construct (others)
Staphylococcus aureus (bacteria)
Escherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.79 Å
AuthorsLamon, G. / Mishra, P. / Chazin-Gray, A. / Baker, D. / Traaseth, N.J.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI165782 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI108889 United States
CitationJournal: bioRxiv / Year: 2026
Title: Miniprotein inhibitors of the efflux transporter NorA.
Authors: Priyanka Mishra / Adam Chazin-Gray / Gaëlle Lamon / David Kim / David Baker / Nathaniel J Traaseth /
Abstract: Multidrug efflux pumps transport antibiotics across the cellular membrane resulting in resistance conferred to the host organism. Efflux pump inhibitors (EPIs) potentiate the efficacy of antibiotics ...Multidrug efflux pumps transport antibiotics across the cellular membrane resulting in resistance conferred to the host organism. Efflux pump inhibitors (EPIs) potentiate the efficacy of antibiotics by blocking drug efflux and hold promise as adjuvant therapeutics in the fight against multidrug resistant pathogenic bacteria. A hurdle in the field has been the lack of selectivity of small molecule EPIs which often display off-target toxicity due to non-specific binding. To tackle this specificity challenge, we aimed to maximize an inhibitor's binding surface area to efflux pumps by designing miniprotein EPIs using computational protein design and an co-expression assay to screen inhibition in cells. We used NorA as a model efflux transporter since it confers drug resistance to fluoroquinolones, puromycin, and other cytotoxic compounds. Starting from a focused miniprotein library of only 86 members, we identified inhibitors in the screen that blocked NorA transport under active efflux conditions . Our most promising inhibitor I-23 was validated by solving a cryo-EM structure of the miniprotein in complex with NorA, which stabilized the transporter in the outward-open conformation. I-23 has a ferredoxin-like fold with one of its β-hairpins inserted into the substrate binding pocket of NorA and other parts of the globular fold occupying the shallow pocket and making extensive intermolecular contacts with NorA. An arginine residue on the tip of the hairpin loop was positioned near an anionic patch required for NorA antibiotic efflux. The identified structural motifs in this work could be employed to explore the molecular properties of peptidoglycan penetration; full realization of the therapeutic potential of the designed miniprotein inhibitors will require determining the principles for facilitating passage of ~7 to 8 kDa miniproteins across the peptidoglycan bacterial cell wall.
History
DepositionFeb 21, 2026Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 8, 2026Provider: repository / Type: Initial release
Revision 1.0Apr 8, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Apr 8, 2026Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Apr 8, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Apr 8, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Apr 8, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Apr 8, 2026Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: I23 miniprotein
B: Quinolone resistance protein NorA,Soluble cytochrome b562


Theoretical massNumber of molelcules
Total (without water)61,3882
Polymers61,3882
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein I23 miniprotein


Mass: 7999.065 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Synthetic construct (others) / Production host: Escherichia coli BL21(DE3) (bacteria)
#2: Protein Quinolone resistance protein NorA,Soluble cytochrome b562 / Cytochrome b-562


Mass: 53388.516 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Staphylococcus aureus (bacteria), (gene. exp.) Escherichia coli (E. coli)
Gene: norA, SACOL0754, cybC / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q5HHX4, UniProt: P0ABE7
Has protein modificationN

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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 bound to miniprotein I-23 / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Staphylococcus aureus (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
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: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2400 nm / Nominal defocus min: 700 nm
Image recordingElectron dose: 49.59 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameCategory
1cryoSPARCparticle selection
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.79 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 302069 / Symmetry type: POINT

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