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- PDB-7b8r: Doxycycline bound structure of bacterial efflux pump. -

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Basic information

Entry
Database: PDB / ID: 7b8r
TitleDoxycycline bound structure of bacterial efflux pump.
Components
  • DARPin
  • Multidrug efflux pump subunit AcrB,Multidrug efflux pump subunit AcrB
KeywordsTRANSPORT PROTEIN / RND transporter / multidrug resistance / efflux pump / antibiotics
Function / homology
Function and homology information


xenobiotic detoxification by transmembrane export across the cell outer membrane / efflux pump complex / periplasmic side of plasma membrane / efflux transmembrane transporter activity / xenobiotic transmembrane transporter activity / outer membrane-bounded periplasmic space / identical protein binding / membrane / plasma membrane
Similarity search - Function
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
Chem-DXT / DI(HYDROXYETHYL)ETHER / Multidrug efflux pump subunit AcrB
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.1 Å
AuthorsWilhelm, J. / Sjuts, H. / Pos, K.M.
Funding support Germany, European Union, 3items
OrganizationGrant numberCountry
German Research Foundation (DFG)SFB807 Germany
German Research Foundation (DFG)EXC115 Germany
Innovative Medicines Initiative115525European Union
CitationJournal: Nat Commun / Year: 2021
Title: Structural and functional analysis of the promiscuous AcrB and AdeB efflux pumps suggests different drug binding mechanisms.
Authors: Alina Ornik-Cha / Julia Wilhelm / Jessica Kobylka / Hanno Sjuts / Attilio V Vargiu / Giuliano Malloci / Julian Reitz / Anja Seybert / Achilleas S Frangakis / Klaas M Pos /
Abstract: Upon antibiotic stress Gram-negative pathogens deploy resistance-nodulation-cell division-type tripartite efflux pumps. These include a H/drug antiporter module that recognizes structurally diverse ...Upon antibiotic stress Gram-negative pathogens deploy resistance-nodulation-cell division-type tripartite efflux pumps. These include a H/drug antiporter module that recognizes structurally diverse substances, including antibiotics. Here, we show the 3.5 Å structure of subunit AdeB from the Acinetobacter baumannii AdeABC efflux pump solved by single-particle cryo-electron microscopy. The AdeB trimer adopts mainly a resting state with all protomers in a conformation devoid of transport channels or antibiotic binding sites. However, 10% of the protomers adopt a state where three transport channels lead to the closed substrate (deep) binding pocket. A comparison between drug binding of AdeB and Escherichia coli AcrB is made via activity analysis of 20 AdeB variants, selected on basis of side chain interactions with antibiotics observed in the AcrB periplasmic domain X-ray co-structures with fusidic acid (2.3 Å), doxycycline (2.1 Å) and levofloxacin (2.7 Å). AdeABC, compared to AcrAB-TolC, confers higher resistance to E. coli towards polyaromatic compounds and lower resistance towards antibiotic compounds.
History
DepositionDec 13, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 20, 2021Provider: repository / Type: Initial release
Revision 1.1Dec 8, 2021Group: 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.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Jan 31, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Multidrug efflux pump subunit AcrB,Multidrug efflux pump subunit AcrB
B: Multidrug efflux pump subunit AcrB,Multidrug efflux pump subunit AcrB
C: Multidrug efflux pump subunit AcrB,Multidrug efflux pump subunit AcrB
D: DARPin
E: DARPin
F: DARPin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)254,66913
Polymers253,2506
Non-polymers1,4197
Water12,412689
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area25850 Å2
ΔGint-45 kcal/mol
Surface area84380 Å2
MethodPISA
Unit cell
Length a, b, c (Å)108.515, 145.422, 174.173
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121
Symmetry operation#1: x,y,z
#2: x+1/2,-y+1/2,-z
#3: -x,y+1/2,-z+1/2
#4: -x+1/2,-y,z+1/2

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Components

#1: Protein Multidrug efflux pump subunit AcrB,Multidrug efflux pump subunit AcrB / AcrAB-TolC multidrug efflux pump subunit AcrB / Acridine resistance protein B


Mass: 66099.047 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: acrB, acrE, b0462, JW0451 / Production host: Escherichia coli (E. coli) / References: UniProt: P31224
#2: Protein DARPin


Mass: 18317.566 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)
#3: Chemical
ChemComp-PEG / DI(HYDROXYETHYL)ETHER


Mass: 106.120 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C4H10O3
#4: Chemical ChemComp-DXT / (4S,4AR,5S,5AR,6R,12AS)-4-(DIMETHYLAMINO)-3,5,10,12,12A-PENTAHYDROXY-6-METHYL-1,11-DIOXO-1,4,4A,5,5A,6,11,12A-OCTAHYDROTETRACENE-2-CARBOXAMIDE / DOXYTETRACYCLINE / DOXYCYCLINE


Mass: 444.435 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C22H24N2O8 / Feature type: SUBJECT OF INVESTIGATION / Comment: antibiotic*YM
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 689 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.71 Å3/Da / Density % sol: 54.67 %
Crystal growTemperature: 291 K / Method: vapor diffusion, hanging drop / Details: 0.1 M MES pH 6.5, 0.21 M NaCl, 11.5% PEG4000

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: PETRA III, EMBL c/o DESY / Beamline: P13 (MX1) / Wavelength: 0.976253 Å
DetectorType: DECTRIS PILATUS 6M-F / Detector: PIXEL / Date: Sep 12, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.976253 Å / Relative weight: 1
ReflectionResolution: 2.1→48.799 Å / Num. obs: 158834 / % possible obs: 98.81 % / Redundancy: 13.7 % / Biso Wilson estimate: 41.76 Å2 / CC1/2: 0.999 / Net I/σ(I): 14.87
Reflection shellResolution: 2.1→2.175 Å / Redundancy: 14.3 % / Mean I/σ(I) obs: 1.46 / Num. unique obs: 15593 / CC1/2: 0.65 / % possible all: 98.01

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Processing

Software
NameVersionClassification
PHENIX1.13_2998refinement
XDSdata reduction
Aimlessdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5EN5

5en5


Resolution: 2.1→48.799 Å / SU ML: 0.26 / Cross valid method: THROUGHOUT / σ(F): 1.34 / Phase error: 26.25 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2391 7810 4.92 %
Rwork0.2029 150967 -
obs0.2047 158777 98.82 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 111.01 Å2 / Biso mean: 51.4338 Å2 / Biso min: 23.45 Å2
Refinement stepCycle: final / Resolution: 2.1→48.799 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms16885 0 99 689 17673
Biso mean--59.24 45.72 -
Num. residues----2220
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection Rwork% reflection obs (%)
2.1-2.12390.33372370.2988499098
2.1239-2.14890.32722510.2996490598
2.1489-2.17510.33322560.2857494998
2.1751-2.20260.3522650.279495398
2.2026-2.23160.30892580.2722494298
2.2316-2.26220.2812860.2595494798
2.2622-2.29450.30072570.255493098
2.2945-2.32870.31692550.2541499398
2.3287-2.36510.25812490.2382495398
2.3651-2.40390.24362600.2306494998
2.4039-2.44530.2752440.24503399
2.4453-2.48980.28112660.2401498199
2.4898-2.53770.29522560.2415499299
2.5377-2.58950.29332670.2345499499
2.5895-2.64580.27482550.2338500099
2.6458-2.70730.29282740.2336500699
2.7073-2.7750.29262420.2296502599
2.775-2.850.29462680.2352502399
2.85-2.93390.26012740.2258502499
2.9339-3.02860.29452190.2333506899
3.0286-3.13680.27662540.2341504199
3.1368-3.26240.2662650.2281505699
3.2624-3.41080.25552610.2125505899
3.4108-3.59060.24572790.2063506899
3.5906-3.81550.23712770.1925508199
3.8155-4.10990.21652640.1724510799
4.1099-4.52330.19192530.16045147100
4.5233-5.17710.17882740.1545160100
5.1771-6.52020.20352540.18375222100
6.5202-48.81170.18042900.1644537099

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