- EMDB-45433: Human Mitochondrial LONP1 Stall State + casein -
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Open data
ID or keywords:
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Basic information
Entry
Database: EMDB / ID: EMD-45433
Title
Human Mitochondrial LONP1 Stall State + casein
Map data
Sharpened Map
Sample
Complex: LONP1 closed state in complex with casein
Protein or peptide: Endogenous Co-purified substrate modeled as unknown residues
Protein or peptide: Lon protease homolog, mitochondrial
Ligand: ADENOSINE-5'-DIPHOSPHATE
Ligand: MAGNESIUM ION
Keywords
ATPase / protease / HYDROLASE
Function / homology
Function and homology information
oxidation-dependent protein catabolic process / PH domain binding / mitochondrial protein catabolic process / G-quadruplex DNA binding / endopeptidase La / mitochondrial DNA metabolic process / mitochondrial genome maintenance / ATP-dependent peptidase activity / protein quality control for misfolded or incompletely synthesized proteins / mitochondrial nucleoid ...oxidation-dependent protein catabolic process / PH domain binding / mitochondrial protein catabolic process / G-quadruplex DNA binding / endopeptidase La / mitochondrial DNA metabolic process / mitochondrial genome maintenance / ATP-dependent peptidase activity / protein quality control for misfolded or incompletely synthesized proteins / mitochondrial nucleoid / regulation of peptidyl-tyrosine phosphorylation / insulin receptor substrate binding / chaperone-mediated protein complex assembly / DNA polymerase binding / negative regulation of insulin receptor signaling pathway / mitochondrion organization / proteolysis involved in protein catabolic process / Mitochondrial protein degradation / protein catabolic process / ADP binding / single-stranded DNA binding / cellular response to oxidative stress / sequence-specific DNA binding / single-stranded RNA binding / response to hypoxia / mitochondrial matrix / serine-type endopeptidase activity / ATP hydrolysis activity / mitochondrion / nucleoplasm / ATP binding / identical protein binding / membrane / cytosol Similarity search - Function
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
F32GM145143
United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)
NS095892
United States
Citation
Journal: bioRxiv / Year: 2024 Title: Structural and mechanistic studies on human LONP1 redefine the hand-over-hand translocation mechanism. Authors: Jeffrey T Mindrebo / Gabriel C Lander / Abstract: AAA+ enzymes use energy from ATP hydrolysis to remodel diverse cellular targets. Structures of substrate-bound AAA+ complexes suggest that these enzymes employ a conserved hand-over-hand mechanism to ...AAA+ enzymes use energy from ATP hydrolysis to remodel diverse cellular targets. Structures of substrate-bound AAA+ complexes suggest that these enzymes employ a conserved hand-over-hand mechanism to thread substrates through their central pore. However, the fundamental aspects of the mechanisms governing motor function and substrate processing within specific AAA+ families remain unresolved. We used cryo-electron microscopy to structurally interrogate reaction intermediates from in vitro biochemical assays to inform the underlying regulatory mechanisms of the human mitochondrial AAA+ protease, LONP1. Our results demonstrate that substrate binding allosterically regulates proteolytic activity, and that LONP1 can adopt a configuration conducive to substrate translocation even when the ATPases are bound to ADP. These results challenge the conventional understanding of the hand-over-hand translocation mechanism, giving rise to an alternative model that aligns more closely with biochemical and biophysical data on related enzymes like ClpX, ClpA, the 26S proteasome, and Lon protease.
Name: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 4 / Formula: MG
Molecular weight
Theoretical: 24.305 Da
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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
Concentration
3 mg/mL
Buffer
pH: 8 Component:
Concentration
Formula
Name
75.0 mM
KCl
potassium chloride
50.0 mM
C4H11NO3
Tris
Grid
Model: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 25 sec. / Pretreatment - Atmosphere: OTHER
Vitrification
Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV / Details: 5 second blot blot force 2.
Details
Sample was monodisperse with ~350 particles per image
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Electron microscopy
Microscope
FEI TITAN KRIOS
Specialist optics
Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recording
Film or detector model: GATAN K3 (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 2931 / Average exposure time: 1.0 sec. / Average electron dose: 54.0 e/Å2 Details: Images were collected in movie mode at 50 frames per second.
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
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