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- PDB-9cc3: Human Mitochondrial LONP1 Stall State + casein -

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Basic information

Entry
Database: PDB / ID: 9cc3
TitleHuman Mitochondrial LONP1 Stall State + casein
Components
  • Endogenous Co-purified substrate modeled as unknown residues
  • Lon protease homolog, mitochondrial
KeywordsHYDROLASE / ATPase / protease
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 / insulin receptor substrate binding / chaperone-mediated protein complex assembly / DNA polymerase binding / regulation of peptidyl-tyrosine phosphorylation / negative regulation of insulin receptor signaling pathway / Mitochondrial protein degradation / mitochondrion organization / proteolysis involved in protein catabolic process / 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
Lon protease homologue, chloroplastic/mitochondrial / Lon protease, bacterial/eukaryotic-type / Peptidase S16, active site / ATP-dependent serine proteases, lon family, serine active site. / Lon proteolytic domain profile. / Peptidase S16, Lon proteolytic domain / Lon protease / Lon protease (S16) C-terminal proteolytic domain / Lon protease, N-terminal domain superfamily / Lon N-terminal domain profile. ...Lon protease homologue, chloroplastic/mitochondrial / Lon protease, bacterial/eukaryotic-type / Peptidase S16, active site / ATP-dependent serine proteases, lon family, serine active site. / Lon proteolytic domain profile. / Peptidase S16, Lon proteolytic domain / Lon protease / Lon protease (S16) C-terminal proteolytic domain / Lon protease, N-terminal domain superfamily / Lon N-terminal domain profile. / Lon protease, N-terminal domain / ATP-dependent protease La (LON) substrate-binding domain / Found in ATP-dependent protease La (LON) / PUA-like superfamily / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / Ribosomal protein S5 domain 2-type fold, subgroup / Ribosomal protein S5 domain 2-type fold / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / Lon protease homolog, mitochondrial
Similarity search - Component
Biological speciesHomo sapiens (human)
Escherichia coli 'BL21-GoldpLysS AG'
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.23 Å
AuthorsMindrebo, J.T. / Lander, G.C.
Funding support United States, 2items
OrganizationGrant numberCountry
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
CitationJournal: 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.
History
DepositionJun 20, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 7, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
G: Endogenous Co-purified substrate modeled as unknown residues
A: Lon protease homolog, mitochondrial
B: Lon protease homolog, mitochondrial
C: Lon protease homolog, mitochondrial
D: Lon protease homolog, mitochondrial
E: Lon protease homolog, mitochondrial
F: Lon protease homolog, mitochondrial
hetero molecules


Theoretical massNumber of molelcules
Total (without water)589,18615
Polymers587,3807
Non-polymers1,8068
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein/peptide Endogenous Co-purified substrate modeled as unknown residues


Mass: 2571.161 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
#2: Protein
Lon protease homolog, mitochondrial / LONHs / Lon protease-like protein / LONP / Mitochondrial ATP-dependent protease Lon / Serine protease 15


Mass: 97468.156 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: LONP1, PRSS15 / Plasmid: pET-20a
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: P36776, endopeptidase La
#3: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
#4: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg / 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: LONP1 closed state in complex with casein / Type: COMPLEX / Details: LONP1 incubated with casein before vitrification / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: .583950 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Plasmid: pET-20a
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
175 mMpotassium chlorideKCl1
250 mMTrisC4H11NO31
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Sample was monodisperse with ~350 particles per image
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 K / Details: 5 second blot blot force 2

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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: 105000 X / Calibrated magnification: 60024 X / Nominal defocus max: 3000 nm / Nominal defocus min: 400 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1 sec. / Electron dose: 54 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2931
Details: Images were collected in movie mode at 50 frames per second.
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansWidth: 5760 / Height: 4092

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Processing

EM softwareName: PHENIX / Version: 1.19.2_4158: / Category: model refinement
Image processingDetails: movies were processed using motion cor 2 version 1.5.0
CTF correctionType: PHASE FLIPPING ONLY
Particle selectionNum. of particles selected: 1100000
Details: Template picking using templates generated from blob picker
3D reconstructionResolution: 3.23 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 59170 / Algorithm: BACK PROJECTION / Num. of class averages: 1 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00226229
ELECTRON MICROSCOPYf_angle_d0.49835550
ELECTRON MICROSCOPYf_dihedral_angle_d5.1293654
ELECTRON MICROSCOPYf_chiral_restr0.0394118
ELECTRON MICROSCOPYf_plane_restr0.0044584

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