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- PDB-7ry3: Multidrug Efflux pump AdeJ with TP-6076 bound -

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Basic information

Entry
Database: PDB / ID: 7ry3
TitleMultidrug Efflux pump AdeJ with TP-6076 bound
ComponentsEfflux pump membrane transporter
KeywordsMEMBRANE PROTEIN/ANTIBIOTIC / multidrug efflux pump / MEMBRANE PROTEIN-ANTIBIOTIC complex
Function / homologyHydrophobe/amphiphile efflux-1 HAE1 / Acriflavin resistance protein / Multidrug efflux transporter AcrB TolC docking domain, DN/DC subdomains / AcrB/AcrD/AcrF family / efflux transmembrane transporter activity / xenobiotic transmembrane transporter activity / plasma membrane / Chem-80P / Efflux pump membrane transporter
Function and homology information
Biological speciesAcinetobacter baumannii (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.91 Å
AuthorsZhang, Z.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID) United States
CitationJournal: mBio / Year: 2021
Title: An Analysis of the Novel Fluorocycline TP-6076 Bound to Both the Ribosome and Multidrug Efflux Pump AdeJ from Acinetobacter baumannii.
Authors: Christopher E Morgan / Zhemin Zhang / Robert A Bonomo / Edward W Yu /
Abstract: Antibiotic resistance among bacterial pathogens continues to pose a serious global health threat. Multidrug-resistant (MDR) strains of the Gram-negative organism Acinetobacter baumannii utilize a ...Antibiotic resistance among bacterial pathogens continues to pose a serious global health threat. Multidrug-resistant (MDR) strains of the Gram-negative organism Acinetobacter baumannii utilize a number of resistance determinants to evade current antibiotics. One of the major resistance mechanisms employed by these pathogens is the use of multidrug efflux pumps. These pumps extrude xenobiotics directly out of bacterial cells, resulting in treatment failures when common antibiotics are administered. Here, the structure of the novel tetracycline antibiotic TP-6076, bound to both the cinetobacter rug fflux pump AdeJ and the ribosome from Acinetobacter baumannii, using single-particle cryo-electron microscopy (cryo-EM), is elucidated. In this work, the structure of the AdeJ-TP-6076 complex is solved, and we show that AdeJ utilizes a network of hydrophobic interactions to recognize this fluorocycline. Concomitant with this, we elucidate three structures of TP-6076 bound to the A. baumannii ribosome and determine that its binding is stabilized largely by electrostatic interactions. We then compare the differences in binding modes between TP-6076 and the related tetracycline antibiotic eravacycline in both targets. These differences suggest that modifications to the tetracycline core may be able to alter AdeJ binding while maintaining interactions with the ribosome. Together, this work highlights how different mechanisms are used to stabilize the binding of tetracycline-based compounds to unique bacterial targets and provides guidance for the future clinical development of tetracycline antibiotics. Treatment of antibiotic-resistant organisms such as A. baumannii represents an ongoing issue for modern medicine. The multidrug efflux pump AdeJ serves as a major resistance determinant in A. baumannii through its action of extruding antibiotics from the cell. In this work, we use cryo-EM to show how AdeJ recognizes the experimental tetracycline antibiotic TP-6076 and prevents this drug from interacting with the A. baumannii ribosome. Since AdeJ and the ribosome use different binding modes to stabilize interactions with TP-6076, exploiting these differences may guide future drug development for combating antibiotic-resistant A. baumannii and potentially other strains of MDR bacteria.
History
DepositionAug 24, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 2, 2022Provider: repository / Type: Initial release
Revision 1.1Feb 16, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Dec 7, 2022Group: Database references / Category: citation / Item: _citation.journal_volume
Revision 1.3Jun 5, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Assembly

Deposited unit
A: Efflux pump membrane transporter
B: Efflux pump membrane transporter
C: Efflux pump membrane transporter
hetero molecules


Theoretical massNumber of molelcules
Total (without water)344,4934
Polymers343,9133
Non-polymers5801
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area17080 Å2
ΔGint-78 kcal/mol
Surface area119830 Å2

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Components

#1: Protein Efflux pump membrane transporter


Mass: 114637.781 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Acinetobacter baumannii (bacteria) / Gene: adeJ, mexB_1, mexB_2 / Production host: Escherichia coli (E. coli) / References: UniProt: Q2FD94
#2: Chemical ChemComp-80P / (4S,4aS,5aR,12aS)-4-(diethylamino)-3,10,12,12a-tetrahydroxy-1,11-dioxo-8-[(2S)-pyrrolidin-2-yl]-7-(trifluoromethyl)-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide / TP-6076


Mass: 579.565 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C28H32F3N3O7 / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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: multidrug efflux pump AdeJ / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Acinetobacter baumannii (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 FIELD
Image recordingElectron dose: 36 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.19.2_4158refinement
PHENIX1.19.2_4158refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.91 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 68346 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 62.46 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.010924443
ELECTRON MICROSCOPYf_angle_d1.548133249
ELECTRON MICROSCOPYf_chiral_restr0.11263913
ELECTRON MICROSCOPYf_plane_restr0.00874262
ELECTRON MICROSCOPYf_dihedral_angle_d12.47838789

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