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- PDB-8f4r: Gentamicin bound aminoglycoside efflux pump AcrD -

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Basic information

Entry
Database: PDB / ID: 8f4r
TitleGentamicin bound aminoglycoside efflux pump AcrD
ComponentsEfflux pump membrane transporter
KeywordsMEMBRANE PROTEIN / aminoglycoside efflux pump / AcrD / E.coli
Function / homologyHydrophobe/amphiphile efflux-1 HAE1 / Acriflavin resistance protein / Multidrug efflux transporter AcrB TolC docking domain, DN/DC subdomains / AcrB/AcrD/AcrF family / xenobiotic transport / efflux transmembrane transporter activity / plasma membrane / Chem-LLL / Efflux pump membrane transporter
Function and homology information
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.06 Å
AuthorsZhang, Z.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01AI145069 United States
CitationJournal: mBio / Year: 2023
Title: Cryo-EM Structures of AcrD Illuminate a Mechanism for Capturing Aminoglycosides from Its Central Cavity.
Authors: Zhemin Zhang / Christopher E Morgan / Meng Cui / Edward W Yu /
Abstract: The Escherichia coli acriflavine resistance protein D (AcrD) is an efflux pump that belongs to the resistance-nodulation-cell division (RND) superfamily. Its primary function is to provide resistance ...The Escherichia coli acriflavine resistance protein D (AcrD) is an efflux pump that belongs to the resistance-nodulation-cell division (RND) superfamily. Its primary function is to provide resistance to aminoglycoside-based drugs by actively extruding these noxious compounds out of E. coli cells. AcrD can also mediate resistance to a limited range of other amphiphilic agents, including bile acids, novobiocin, and fusidic acids. As there is no structural information available for any aminoglycoside-specific RND pump, here we describe cryo-electron microscopy (cryo-EM) structures of AcrD in the absence and presence of bound gentamicin. These structures provide new information about the RND superfamily of efflux pumps, specifically, that three negatively charged residues central to the aminoglycoside-binding site are located within the ceiling of the central cavity of the AcrD trimer. Thus, it is likely that AcrD is capable of picking up aminoglycosides via this central cavity. Through the combination of cryo-EM structural determination, mutagenesis analysis, and molecular simulation, we show that charged residues are critically important for this pump to shuttle drugs directly from the central cavity to the funnel of the AcrD trimer for extrusion. Here, we report cryo-EM structures of the AcrD aminoglycoside efflux pump in the absence and presence of bound gentamicin, posing the possibility that this pump is capable of capturing aminoglycosides from the central cavity of the AcrD trimer. The results indicate that AcrD utilizes charged residues to bind and export drugs, mediating resistance to these antibiotics.
History
DepositionNov 11, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 28, 2022Provider: repository / Type: Initial release
Revision 1.1Jan 11, 2023Group: Database references / Category: citation
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.title / _citation.year
Revision 1.2Jan 25, 2023Group: Database references / Category: citation / citation_author
Item: _citation.page_first / _citation.page_last ..._citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.3Mar 8, 2023Group: Database references / Category: citation / Item: _citation.journal_volume
Revision 1.4Jun 19, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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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
hetero molecules


Theoretical massNumber of molelcules
Total (without water)339,8694
Polymers339,4203
Non-polymers4501
Water00
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: 113139.891 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli)
Gene: acrD, ACU57_04565, BANRA_03325, BJI68_01485, BJJ90_06945, BMT91_11990, BON93_11695, BUO55_002264, C0P57_000121, C5N07_10560, C5Y87_22760, C9160_05345, CTR35_000190, CX938_001812, D3Y67_13030, ...Gene: acrD, ACU57_04565, BANRA_03325, BJI68_01485, BJJ90_06945, BMT91_11990, BON93_11695, BUO55_002264, C0P57_000121, C5N07_10560, C5Y87_22760, C9160_05345, CTR35_000190, CX938_001812, D3Y67_13030, DAH22_02795, DAH27_02260, DAH28_02170, DAH32_09030, DAH37_20010, DAH41_00685, DAH50_04680, DIV22_31685, E2119_05090, E2122_05015, E2127_22975, E2128_11985, E2132_10115, E2134_09425, E4T14_01660, E5P26_05645, E5P27_06290, E5P28_04085, E5P29_08410, E5P31_03360, E5P32_02065, E5P40_06505, E5P51_11900, E5S36_11905, E5S45_03290, EC1094V2_1219, ECs3332, ED648_19550, EI021_07760, EIZ93_19695, EKI52_22415, EL79_1194, EL80_1199, ELT21_03480, ERS139208_00580, EYV17_00615, EYV18_00295, F2N20_03885, F2N31_04825, F9V24_02735, FFF58_09795, FOI11_000625, FOI11_19425, FQF29_08200, G3V95_05155, G4A38_06285, G4A47_09235, GJO56_19765, GKF89_16975, GOP25_13330, GP975_00825, GRW05_00965, HMV95_10675, HNC36_05080, HX136_06730, IH772_01085, J0541_000250, JNP96_20105, NCTC8960_04125, NCTC9037_01546, SAMEA3472044_02066, SAMEA3753106_01071
Production host: Escherichia coli (E. coli) / References: UniProt: C3T0H0
#2: Chemical ChemComp-LLL / (2R,3R,4R,5R)-2-((1S,2S,3R,4S,6R)-4,6-DIAMINO-3-((2R,3R,6S)-3-AMINO-6-(AMINOMETHYL)-TETRAHYDRO-2H-PYRAN-2-YLOXY)-2-HYDR OXYCYCLOHEXYLOXY)-5-METHYL-4-(METHYLAMINO)-TETRAHYDRO-2H-PYRAN-3,5-DIOL / GENTAMICIN C1A


Mass: 449.542 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C19H39N5O7 / 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: AcrD / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Escherichia coli (E. coli)
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 / Nominal magnification: 81000 X / Nominal defocus max: 1500 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 35.9 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: 3.06 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 36919 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00424130
ELECTRON MICROSCOPYf_angle_d0.4632794
ELECTRON MICROSCOPYf_dihedral_angle_d10.4768675
ELECTRON MICROSCOPYf_chiral_restr0.0373823
ELECTRON MICROSCOPYf_plane_restr0.0034224

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