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- EMDB-27400: Cryo-electron microscopy structure of Neisseria gonorrhoeae multi... -

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Basic information

Entry
Database: EMDB / ID: EMD-27400
TitleCryo-electron microscopy structure of Neisseria gonorrhoeae multidrug efflux pump MtrD with colistin complex
Map data
Sample
  • Complex: Multidrug efflux pump MtrD with colistin complex
    • Protein or peptide: Efflux pump membrane transporter
  • Protein or peptide: Colistin
  • Ligand: PHOSPHATIDYLETHANOLAMINE
KeywordsMtrD complex / multidrug efflux pump / Neisseria gonorrhoeae / MEMBRANE PROTEIN
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 / Multidrug efflux transporter AcrB TolC docking domain, DN/DC subdomains / Acriflavin resistance protein / AcrB/AcrD/AcrF family
Similarity search - Domain/homology
Efflux pump membrane transporter
Similarity search - Component
Biological speciesNeisseria gonorrhoeae (bacteria) / synthethic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.08 Å
AuthorsLyu M / Yu EW
Funding support United States, 1 items
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, 2022-
Header (metadata) releaseSep 14, 2022-
Map releaseSep 14, 2022-
UpdateMay 14, 2025-
Current statusMay 14, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27400.png

Downloads & links

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Map

FileDownload / File: emd_27400.map.gz / Format: CCP4 / Size: 163.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.12 Å/pix.
x 350 pix.
= 392.7 Å		1.12 Å/pix.
x 350 pix.
= 392.7 Å		1.12 Å/pix.
x 350 pix.
= 392.7 Å

Surface

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.122 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.176
Minimum - Maximum-0.9061258 - 2.0424166
Average (Standard dev.)-0.0001985191 (±0.04427655)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions350350350
Spacing350350350
CellA=B=C: 392.69998 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_27400_msk_1.map
Projections & Slices
AxesZYX

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Half map: #2

Fileemd_27400_half_map_1.map
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Histogram

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Half map: #1

Fileemd_27400_half_map_2.map
Projections & Slices
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Sample components

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Entire : Multidrug efflux pump MtrD with colistin complex

EntireName: Multidrug efflux pump MtrD with colistin complex
Components
  • Complex: Multidrug efflux pump MtrD with colistin complex
    • Protein or peptide: Efflux pump membrane transporter
  • Protein or peptide: Colistin
  • Ligand: PHOSPHATIDYLETHANOLAMINE

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Supramolecule #1: Multidrug efflux pump MtrD with colistin complex

SupramoleculeName: Multidrug efflux pump MtrD with colistin complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Neisseria gonorrhoeae (bacteria)

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Macromolecule #1: Efflux pump membrane transporter

MacromoleculeName: Efflux pump membrane transporter / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Neisseria gonorrhoeae (bacteria)
Molecular weightTheoretical: 111.643969 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MAKFFIDRPI FAWVISIFII AAGIFGIKSL PVSQYPSVAA PTITLHAIYP GASAQVMEGS VLSVIERNMN GVEGLDYMST SADSSGSGS VSLTFTPDTD ENLAQVEVQN KLSEVLSTLP ATVQQYGVTV SKARSNFLMI VMLSSDVQST EEMNDYAQRN V VPELQRIE ...String:
MAKFFIDRPI FAWVISIFII AAGIFGIKSL PVSQYPSVAA PTITLHAIYP GASAQVMEGS VLSVIERNMN GVEGLDYMST SADSSGSGS VSLTFTPDTD ENLAQVEVQN KLSEVLSTLP ATVQQYGVTV SKARSNFLMI VMLSSDVQST EEMNDYAQRN V VPELQRIE GVGQVRLFGA QRAMRIWVDP KKLQNYNLSF ADVGSALSAQ NIQISAGSIG SLPAVRGQTV TATVTAQGQL GT AEEFGNV ILRANTDGSN IYLKDVAKVG LGMEDYSSST RLNGVNTTGM AVMLSNSGNA MATAKAVKER LAVLEKYFPQ GMS WKTPYD TSKFVEISIE KVIHTLIEAM VLVFVVMYLF LQNIRYTLIP TIVVPISLLG GFAFISYMGM SINVLTMFAM ILVI GIVVD DAIVVVENVE RIMAGEGLPP KEATKKAMGQ ISGAVIGITA VLISVFVPLA MFSGAAGNIY KQFALTMASS IAFSA FLAL TLTPALCATM LKTIPKGHHE EKKGFFGWFN KKFDSWTHGY EGRVAKVLRK TFRMMVVYIG LAVVGVFLFM RLPTSF LPT EDQGFVMVSV QLPAGATKER TDATLAQVTQ LAKSIPEIEN IITVSGFSFS GSGQNMAMGF AILKDWNERT ASGSDAV AV AGKLTGMMMG TLKDGFGIAV VPPPILELGN GSGLSINLQD RNNTGHTALL AKRNELIQKM RASGLFDPST VRAGGLED S PQLKIDINRA AAAAQGVSFA DIRTALASAL SSSYVSDFPN QGRLQRVMVQ ADGDARMQPA DILNLTVPNS SGIAVPLSS IATVSWQMGT EQSVRFNGYP AMELSGSPAT GVSTGQAMEA VQKMVDELGS GYSLEWGGQS REEAKGGSQT IALYALAAVA VFLVLAALY ESWSIPLAVL LVMPLGLAGA AAGVTGRNLF EGLLGSVPSF ANDIYFQVGF VTVMGLSAKN AILIIEFAKD L QAQGKSAV EAALEAARLR FRPIIMTSFA FILGVVPLYI AGGASSASQR AIGTTVFWGM LIGTLLSVFL VPLFYVVVRK FF KETAHE

UniProtKB: Efflux pump membrane transporter

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Macromolecule #2: Colistin

MacromoleculeName: Colistin / type: protein_or_peptide / ID: 2 / Details: polymyxin E / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthethic construct (others)
Molecular weightTheoretical: 1.173447 KDa
SequenceString:
(RE6)(DAB)T(DAB)(DAB)(DAB)(DLE)L(DAB)(DAB) T

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Macromolecule #3: PHOSPHATIDYLETHANOLAMINE

MacromoleculeName: PHOSPHATIDYLETHANOLAMINE / type: ligand / ID: 3 / Number of copies: 23 / Formula: PTY
Molecular weightTheoretical: 734.039 Da
Chemical component information

ChemComp-PTY:
PHOSPHATIDYLETHANOLAMINE / phospholipid*YM

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.7 mg/mL
BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 5647 / Average exposure time: 2.8 sec. / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.75 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.08 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 301061
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: OTHER
FSC plot (resolution estimation)

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