Journal: J Biol Chem / Year: 2022 Title: Cryo-electron microscopy reveals how acetogenins inhibit mitochondrial respiratory complex I. Authors: Daniel N Grba / James N Blaza / Hannah R Bridges / Ahmed-Noor A Agip / Zhan Yin / Masatoshi Murai / Hideto Miyoshi / Judy Hirst / Abstract: Mitochondrial complex I (NADH:ubiquinone oxidoreductase), a crucial enzyme in energy metabolism, captures the redox potential energy from NADH oxidation/ubiquinone reduction to create the proton ...Mitochondrial complex I (NADH:ubiquinone oxidoreductase), a crucial enzyme in energy metabolism, captures the redox potential energy from NADH oxidation/ubiquinone reduction to create the proton motive force used to drive ATP synthesis in oxidative phosphorylation. High-resolution single-particle electron cryo-EM analyses have provided detailed structural knowledge of the catalytic machinery of complex I, but not of the molecular principles of its energy transduction mechanism. Although ubiquinone is considered to bind in a long channel at the interface of the membrane-embedded and hydrophilic domains, with channel residues likely involved in coupling substrate reduction to proton translocation, no structures with the channel fully occupied have yet been described. Here, we report the structure (determined by cryo-EM) of mouse complex I with a tight-binding natural product acetogenin inhibitor, which resembles the native substrate, bound along the full length of the expected ubiquinone-binding channel. Our structure reveals the mode of acetogenin binding and the molecular basis for structure-activity relationships within the acetogenin family. It also shows that acetogenins are such potent inhibitors because they are highly hydrophobic molecules that contain two specific hydrophilic moieties spaced to lock into two hydrophilic regions of the otherwise hydrophobic channel. The central hydrophilic section of the channel does not favor binding of the isoprenoid chain when the native substrate is fully bound but stabilizes the ubiquinone/ubiquinol headgroup as it transits to/from the active site. Therefore, the amphipathic nature of the channel supports both tight binding of the amphipathic inhibitor and rapid exchange of the ubiquinone/ubiquinol substrate and product.
EMPIAR-10927 (Title: Single particle cryo-EM dataset of Mus musculus mitochondrial complex I bound with an acetogenin inhibitor Data size: 3.3 TB Data #1: Single-particle cryo-EM dataset of Mus musculus mitochondrial complex I co-purified with the inhibitor acetogenin [micrographs - multiframe])
Model: UltrAuFoil R0.6/1 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Support film - Film thickness: 50.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR Details: 20 mA glow discharge. Followed by a 2-day PEGylation with a PEG-thiol reagent.
Vitrification
Cryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK III
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Electron microscopy
Microscope
FEI TITAN KRIOS
Specialist optics
Energy filter - Slit width: 20 eV
Image recording
Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 1286 / Average exposure time: 10.0 sec. / Average electron dose: 50.0 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Type: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1) Details: RELION solvent-flattened 3D refinement with a PDB-generated mask
Final 3D classification
Number classes: 2 / Software - Name: RELION (ver. 3.1) Details: Used a mask classification without alignment on a small region to isolated a compound-bound state.
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Mus musculus (house mouse) / 
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United Kingdom, 2 items 
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emd_13611.png
http://ftp.pdbj.org/pub/emdb/structures/EMD-13611
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Electron microscopy
FIELD EMISSION GUN
