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- PDB-8gjl: multi-drug efflux pump RE-CmeB bound with Ciprofloxacin -

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Basic information

Entry
Database: PDB / ID: 8gjl
Titlemulti-drug efflux pump RE-CmeB bound with Ciprofloxacin
ComponentsEfflux pump membrane transporter
KeywordsMEMBRANE PROTEIN / efflux pump
Function / homology
Function and homology information


xenobiotic transport / efflux transmembrane transporter activity / plasma membrane
Similarity search - Function
Sterol-sensing domain (SSD) profile. / Sterol-sensing domain / Hydrophobe/amphiphile efflux-1 HAE1 / Acriflavin resistance protein / Multidrug efflux transporter AcrB TolC docking domain, DN/DC subdomains / AcrB/AcrD/AcrF family
Similarity search - Domain/homology
Biological speciesCampylobacter jejuni (Campylobacter)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.44 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: Microbiol Spectr / Year: 2023
Title: Cryo-Electron Microscopy Structures of a Campylobacter Multidrug Efflux Pump Reveal a Novel Mechanism of Drug Recognition and Resistance.
Authors: Zhemin Zhang / Nicholas Lizer / Zuowei Wu / Christopher E Morgan / Yuqi Yan / Qijing Zhang / Edward W Yu /
Abstract: Campylobacter jejuni is a bacterium that is commonly present in the intestinal tracts of animals. It is also a major foodborne pathogen that causes gastroenteritis in humans. The most predominant and ...Campylobacter jejuni is a bacterium that is commonly present in the intestinal tracts of animals. It is also a major foodborne pathogen that causes gastroenteritis in humans. The most predominant and clinically important multidrug efflux system in C. jejuni is the CmeABC (Campylobacter multidrug efflux) pump, a tripartite system that includes an inner membrane transporter (CmeB), a periplasmic fusion protein (CmeA), and an outer membrane channel protein (CmeC). This efflux protein machinery mediates resistance to a number of structurally diverse antimicrobial agents. A recently identified CmeB variant, termed resistance enhancing CmeB (RE-CmeB), can increase its multidrug efflux pump activity, likely by influencing antimicrobial recognition and extrusion. Here, we report structures of RE-CmeB in its apo form as well as in the presence of four different drugs by using single-particle cryo-electron microscopy (cryo-EM). Coupled with mutagenesis and functional studies, this structural information allows us to identify critical amino acids that are important for drug resistance. We also report that RE-CmeB utilizes a somewhat unique subset of residues to bind different drugs, thereby optimizing its ability to accommodate different compounds with distinct scaffolds. These findings provide insights into the structure-function relationship of this newly emerged antibiotic efflux transporter variant in Campylobacter. Campylobacter jejuni has emerged as one of the most problematic and highly antibiotic-resistant pathogens, worldwide. The Centers for Disease Control and Prevention have designated antibiotic-resistant C. jejuni as a serious antibiotic resistance threat in the United States. We recently identified a C. jejuni resistance enhancing CmeB (RE-CmeB) variant that can increase its multidrug efflux pump activity and confers an exceedingly high-level of resistance to fluoroquinolones. Here, we report the cryo-EM structures of this prevalent and clinically important C. jejuni RE-CmeB multidrug efflux pump in both the absence and presence of four antibiotics. These structures allow us to understand the action mechanism for multidrug recognition in this pump. Our studies will ultimately inform an era in structure-guided drug design to combat multidrug resistance in these Gram-negative pathogens.
History
DepositionMar 16, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 31, 2023Provider: repository / Type: Initial release
Revision 1.1Oct 4, 2023Group: Data collection / Database references
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / citation_author
Item: _citation.journal_abbrev / _citation.journal_id_CSD ..._citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)342,6994
Polymers342,3673
Non-polymers3311
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Efflux pump membrane transporter


Mass: 114122.477 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Campylobacter jejuni (Campylobacter) / Gene: cmeB / Production host: Escherichia coli (E. coli) / References: UniProt: A0A1C9A1J1
#2: Chemical ChemComp-CPF / 1-CYCLOPROPYL-6-FLUORO-4-OXO-7-PIPERAZIN-1-YL-1,4-DIHYDROQUINOLINE-3-CARBOXYLIC ACID / ciprofloxacin / Ciprofloxacin


Mass: 331.342 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C17H18FN3O3 / Feature type: SUBJECT OF INVESTIGATION / Comment: antibiotic*YM
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: RE-CmeB / Type: COMPLEX / Entity ID: #1 / Source: NATURAL
Source (natural)Organism: Campylobacter jejuni (Campylobacter)
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 / Nominal magnification: 81000 X / Nominal defocus max: 1500 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 37.9 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.44 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 43849 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00424379
ELECTRON MICROSCOPYf_angle_d0.60833125
ELECTRON MICROSCOPYf_dihedral_angle_d5.6043297
ELECTRON MICROSCOPYf_chiral_restr0.0413942
ELECTRON MICROSCOPYf_plane_restr0.0054197

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