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- PDB-8deu: Cryo-electron microscopy structure of Neisseria gonorrhoeae multi... -

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Basic information

Entry
Database: PDB / ID: 8deu
TitleCryo-electron microscopy structure of Neisseria gonorrhoeae multidrug efflux pump MtrD with CASP peptide complex
Components
  • CASP peptide
  • Efflux pump membrane transporter
KeywordsMEMBRANE PROTEIN / MtrD complex / multidrug efflux pump / Neisseria gonorrhoeae
Function / homology
Function and homology information


efflux transmembrane transporter activity / xenobiotic 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
PHOSPHATIDYLETHANOLAMINE / Efflux pump membrane transporter
Similarity search - Component
Biological speciesNeisseria gonorrhoeae (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.95 Å
AuthorsLyu, M. / Yu, E.W.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01 AI145069 United States
CitationJournal: Microbiol Spectr / Year: 2022
Title: Structural Basis of Peptide-Based Antimicrobial Inhibition of a Resistance-Nodulation-Cell Division Multidrug Efflux Pump.
Authors: Meinan Lyu / Julio C Ayala / Isabella Chirakos / Chih-Chia Su / William M Shafer / Edward W Yu /
Abstract: Bacterial efflux pumps in the resistance-nodulation-cell division (RND) family of Gram-negative bacteria contribute significantly to the development of antimicrobial resistance by many pathogens. In ...Bacterial efflux pumps in the resistance-nodulation-cell division (RND) family of Gram-negative bacteria contribute significantly to the development of antimicrobial resistance by many pathogens. In this study, we selected the MtrD transporter protein of Neisseria gonorrhoeae as it is the sole RND pump possessed by this strictly human pathogen and can export multiple antimicrobials, including antibiotics, bile salts, detergents, dyes, and antimicrobial peptides. Using knowledge from our previously published structures of MtrD in the presence or absence of bound antibiotics as a model and the known ability of MtrCDE to export cationic antimicrobial peptides, we hypothesized that cationic peptides could be accommodated within MtrD binding sites. Furthermore, we thought that MtrD-bound peptides lacking antibacterial action could sensitize bacteria to an antibiotic normally exported by the MtrCDE efflux pump or other similar RND-type pumps possessed by different Gram-negative bacteria. We now report the identification of a novel nonantimicrobial cyclic cationic antimicrobial peptide, which we termed CASP (ationic ntibiotic-ensitizing eptide). By single-particle cryo-electron microscopy, we found that CASP binds within the periplasmic cleft region of MtrD using overlapping and distinct amino acid contact sites that interact with another cyclic peptide (colistin) or a linear human cationic antimicrobial peptide derived from human LL-37. While CASP could not sensitize Neisseria gonorrhoeae to an antibiotic (novobiocin) that is a substrate for RND pumps, it could do so against multiple Gram-negative, rod-shaped bacteria. We propose that CASP (or future derivatives) could serve as an adjuvant for the antibiotic treatment of certain Gram-negative infections previously thwarted by RND transporters. RND efflux pumps can export numerous antimicrobials that enter Gram-negative bacteria, and their action can reduce the efficacy of antibiotics and provide decreased susceptibility to various host antimicrobials. Here, we identified a ationic ntibiotic-ensitizing eptide (CASP) that binds within the periplasmic cleft of an RND transporter protein (MtrD) produced by Neisseria gonorrhoeae. Surprisingly, CASP was able to render rod-shaped Gram-negative bacteria, but not gonococci, susceptible to an antibiotic that is a substrate for the gonococcal MtrCDE efflux pump. CASP (or its future derivatives) could be used as an adjuvant to treat infections for which RND efflux contributes to multidrug resistance.
History
DepositionJun 21, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 14, 2022Provider: repository / Type: Initial release
Revision 1.1Oct 5, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_abbrev / _citation.journal_id_CSD ..._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 1.2Nov 9, 2022Group: Database references / Category: citation / Item: _citation.journal_volume

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)362,58431
Polymers342,7644
Non-polymers19,81927
Water00
1


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

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Components

#1: Protein Efflux pump membrane transporter


Mass: 113932.469 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Neisseria gonorrhoeae (bacteria) / Gene: mtrD / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A6V7GUB3
#2: Protein/peptide CASP peptide


Mass: 967.055 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Neisseria gonorrhoeae (bacteria) / Production host: Escherichia coli BL21(DE3) (bacteria)
#3: Chemical...
ChemComp-PTY / PHOSPHATIDYLETHANOLAMINE


Mass: 734.039 Da / Num. of mol.: 27 / Source method: obtained synthetically / Formula: C40H80NO8P / Comment: phospholipid*YM
Has ligand of interestN

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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 MtrD with CASP peptide complex / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Source (natural)Organism: Neisseria gonorrhoeae (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
SpecimenConc.: 0.7 mg/ml / Embedding 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 / Nominal defocus max: 1750 nm / Nominal defocus min: 750 nm / C2 aperture diameter: 100 µm
Image recordingAverage exposure time: 2.6 sec. / Electron dose: 40 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5188

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.95 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 129244 / Symmetry type: POINT

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