Journal: Proc Natl Acad Sci U S A / Year: 2012 Title: Structural basis for TetM-mediated tetracycline resistance. Authors: Alexandra Dönhöfer / Sibylle Franckenberg / Stephan Wickles / Otto Berninghausen / Roland Beckmann / Daniel N Wilson / Abstract: Ribosome protection proteins (RPPs) confer tetracycline resistance by binding to the ribosome and chasing the drug from its binding site. The current model for the mechanism of action of RPPs ...Ribosome protection proteins (RPPs) confer tetracycline resistance by binding to the ribosome and chasing the drug from its binding site. The current model for the mechanism of action of RPPs proposes that drug release is indirect and achieved via conformational changes within the drug-binding site induced upon binding of the RPP to the ribosome. Here we report a cryo-EM structure of the RPP TetM in complex with the 70S ribosome at 7.2-Å resolution. The structure reveals the contacts of TetM with the ribosome, including interaction between the conserved and functionally critical C-terminal extension of TetM and the decoding center of the small subunit. Moreover, we observe direct interaction between domain IV of TetM and the tetracycline binding site and identify residues critical for conferring tetracycline resistance. A model is presented whereby TetM directly dislodges tetracycline to confer resistance.
History
Deposition
Aug 24, 2012
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Header (metadata) release
Sep 5, 2012
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Map release
Oct 10, 2012
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Update
May 20, 2015
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Current status
May 20, 2015
Processing site: PDBe / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Entire : Escherichia coli 70S ribosome in complex with ribosome protection...
Entire
Name: Escherichia coli 70S ribosome in complex with ribosome protection protein TetM
Components
Sample: Escherichia coli 70S ribosome in complex with ribosome protection protein TetM
Complex: Escherichia coli 70S ribosome
Protein or peptide: Tetracycline resistance protein TetM
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Supramolecule #1000: Escherichia coli 70S ribosome in complex with ribosome protection...
Supramolecule
Name: Escherichia coli 70S ribosome in complex with ribosome protection protein TetM type: sample / ID: 1000 / Number unique components: 2
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Supramolecule #1: Escherichia coli 70S ribosome
Supramolecule
Name: Escherichia coli 70S ribosome / type: complex / ID: 1 / Recombinant expression: No / Ribosome-details: ribosome-prokaryote: ALL
Source (natural)
Organism: Escherichia coli (E. coli)
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Macromolecule #1: Tetracycline resistance protein TetM
Macromolecule
Name: Tetracycline resistance protein TetM / type: protein_or_peptide / ID: 1 / Name.synonym: TetM / Recombinant expression: No
Source (natural)
Organism: Enterococcus faecalis (bacteria)
Sequence
UniProtKB: Tetracycline resistance protein TetM from transposon Tn916
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Experimental details
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Structure determination
Method
cryo EM
Processing
single particle reconstruction
Aggregation state
particle
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Sample preparation
Buffer
pH: 7.8 / Details: 20mM Hepes-KOH pH 7.8, 30 mM NH4Cl and 10 mM MgCl2
Grid
Details: Quantifoil grids (3/3) with 2 nm carbon on top
Vitrification
Cryogen name: ETHANE / Chamber humidity: 95 % / Instrument: FEI VITROBOT MARK IV Method: Blot for 10 seconds before plunging, used 2 layers of filter paper
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Electron microscopy
Microscope
FEI TITAN KRIOS
Date
Jul 1, 2011
Image recording
Category: CCD / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Average electron dose: 20 e/Å2
Electron beam
Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 7.2 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER Details: Sorting for ribosome conformation and ligand presence was performed Number images used: 52701
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