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- EMDB-23013: Human mitochondrial LONP1 in complex with Bortezomib -

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Entry
Database: EMDB / ID: EMD-23013
TitleHuman mitochondrial LONP1 in complex with Bortezomib
Map dataComposite stitched map of human LONP1 in complex with Bortezomib
Sample
  • Complex: Human mitochondrial LONP1 in complex with Bortezomib
    • Protein or peptide: Lon protease homolog, mitochondrial
    • Protein or peptide: Endogenous co-purified substrate
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: N-[(1R)-1-(DIHYDROXYBORYL)-3-METHYLBUTYL]-N-(PYRAZIN-2-YLCARBONYL)-L-PHENYLALANINAMIDE
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
KeywordsAAA+ / ATPase / protease / mitochondrial / LONP1 / LON / 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 / 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 / proteolysis involved in protein catabolic process / mitochondrion organization / ADP binding / protein catabolic process / 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 / Lon protease AAA+ ATPase lid domain / 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 N-terminal domain profile. ...Lon protease homologue, chloroplastic/mitochondrial / Lon protease, bacterial/eukaryotic-type / Lon protease AAA+ ATPase lid domain / 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 N-terminal domain profile. / Lon protease, N-terminal domain / Lon protease, N-terminal domain superfamily / 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
Lon protease homolog, mitochondrial
Similarity search - Component
Biological speciesHomo sapiens (human) / Escherichia coli BL21(DE3) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsShin M / Watson ER
Funding support United States, 4 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute on Aging (NIH/NIA)AG067594 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)NS095892 United States
National Institutes of Health/National Institute on Aging (NIH/NIA)AG061697 United States
National Institutes of Health/Office of the DirectorS10OD021634 United States
CitationJournal: Nat Commun / Year: 2021
Title: Structures of the human LONP1 protease reveal regulatory steps involved in protease activation.
Authors: Mia Shin / Edmond R Watson / Albert S Song / Jeffrey T Mindrebo / Scott J Novick / Patrick R Griffin / R Luke Wiseman / Gabriel C Lander /
Abstract: The human mitochondrial AAA+ protein LONP1 is a critical quality control protease involved in regulating diverse aspects of mitochondrial biology including proteostasis, electron transport chain ...The human mitochondrial AAA+ protein LONP1 is a critical quality control protease involved in regulating diverse aspects of mitochondrial biology including proteostasis, electron transport chain activity, and mitochondrial transcription. As such, genetic or aging-associated imbalances in LONP1 activity are implicated in pathologic mitochondrial dysfunction associated with numerous human diseases. Despite this importance, the molecular basis for LONP1-dependent proteolytic activity remains poorly defined. Here, we solved cryo-electron microscopy structures of human LONP1 to reveal the underlying molecular mechanisms governing substrate proteolysis. We show that, like bacterial Lon, human LONP1 adopts both an open and closed spiral staircase orientation dictated by the presence of substrate and nucleotide. Unlike bacterial Lon, human LONP1 contains a second spiral staircase within its ATPase domain that engages substrate as it is translocated toward the proteolytic chamber. Intriguingly, and in contrast to its bacterial ortholog, substrate binding within the central ATPase channel of LONP1 alone is insufficient to induce the activated conformation of the protease domains. To successfully induce the active protease conformation in substrate-bound LONP1, substrate binding within the protease active site is necessary, which we demonstrate by adding bortezomib, a peptidomimetic active site inhibitor of LONP1. These results suggest LONP1 can decouple ATPase and protease activities depending on whether AAA+ or both AAA+ and protease domains bind substrate. Importantly, our structures provide a molecular framework to define the critical importance of LONP1 in regulating mitochondrial proteostasis in health and disease.
History
DepositionNov 20, 2020-
Header (metadata) releaseFeb 24, 2021-
Map releaseFeb 24, 2021-
UpdateOct 23, 2024-
Current statusOct 23, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7krz
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23013.png

Downloads & links

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Map

FileDownload / File: emd_23013.map.gz / Format: CCP4 / Size: 15.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationComposite stitched map of human LONP1 in complex with Bortezomib
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.15 Å/pix.
x 288 pix.
= 331.2 Å		1.15 Å/pix.
x 288 pix.
= 331.2 Å		1.15 Å/pix.
x 288 pix.
= 331.2 Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.15 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.02 / Movie #1: 0.02
Minimum - Maximum-5.2704945 - 18.124131999999999
Average (Standard dev.)0.31875432 (±0.84339076)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions160160160
Spacing160160160
CellA=B=C: 184.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.151.151.15
M x/y/z288288288
origin x/y/z0.0000.0000.000
length x/y/z331.200331.200331.200
α/β/γ90.00090.00090.000
start NX/NY/NZ158149174
NX/NY/NZ205194166
MAP C/R/S123
start NC/NR/NS-64-64-64
NC/NR/NS288288288
D min/max/mean-0.0650.1210.001

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Supplemental data

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Mask #1

Fileemd_23013_msk_1.map
Projections & Slices
AxesZYX

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Mask #2

Fileemd_23013_msk_2.map
Projections & Slices
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Additional map: Half-map of protease reconstruction used in composite stitched...

Fileemd_23013_additional_1.map
AnnotationHalf-map of protease reconstruction used in composite stitched map of human LONP1 in complex with Bortezomib
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Half-map of protease reconstruction used in composite stitched...

Fileemd_23013_additional_2.map
AnnotationHalf-map of protease reconstruction used in composite stitched map of human LONP1 in complex with Bortezomib
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map of ATPase reconstruction used in composite stitched...

Fileemd_23013_half_map_1.map
AnnotationHalf-map of ATPase reconstruction used in composite stitched map of human LONP1 in complex with Bortezomib
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map of ATPase reconstruction used in composite stitched...

Fileemd_23013_half_map_2.map
AnnotationHalf-map of ATPase reconstruction used in composite stitched map of human LONP1 in complex with Bortezomib
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Human mitochondrial LONP1 in complex with Bortezomib

EntireName: Human mitochondrial LONP1 in complex with Bortezomib
Components
  • Complex: Human mitochondrial LONP1 in complex with Bortezomib
    • Protein or peptide: Lon protease homolog, mitochondrial
    • Protein or peptide: Endogenous co-purified substrate
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: N-[(1R)-1-(DIHYDROXYBORYL)-3-METHYLBUTYL]-N-(PYRAZIN-2-YLCARBONYL)-L-PHENYLALANINAMIDE
  • Ligand: ADENOSINE-5'-DIPHOSPHATE

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Supramolecule #1: Human mitochondrial LONP1 in complex with Bortezomib

SupramoleculeName: Human mitochondrial LONP1 in complex with Bortezomib / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Details: Complexes consisting of homohexameric LONP1 protease from Homo sapiens bound to endogenous co-purified substrate and Bortezomib.
Source (natural)Organism: Homo sapiens (human) / Organelle: Mitochondria / Location in cell: Matrix
Molecular weightTheoretical: 462 KDa

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Macromolecule #1: Lon protease homolog, mitochondrial

MacromoleculeName: Lon protease homolog, mitochondrial / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO / EC number: endopeptidase La
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 59.314016 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: KDAIEEKFRE RLKELVVPKH VMDVVDEELS KLGLLDNHSS EFNVTRNYLD WLTSIPWGKY SNENLDLARA QAVLEEDHYG MEDVKKRIL EFIAVSQLRG STQGKILCFY GPPGVGKTSI ARSIARALNR EYFRFSVGGM TDVAEIKGHR RTYVGAMPGK I IQCLKKTK ...String:
KDAIEEKFRE RLKELVVPKH VMDVVDEELS KLGLLDNHSS EFNVTRNYLD WLTSIPWGKY SNENLDLARA QAVLEEDHYG MEDVKKRIL EFIAVSQLRG STQGKILCFY GPPGVGKTSI ARSIARALNR EYFRFSVGGM TDVAEIKGHR RTYVGAMPGK I IQCLKKTK TENPLILIDE VDKIGRGYQG DPSSALLELL DPEQNANFLD HYLDVPVDLS KVLFICTANV TDTIPEPLRD RM EMINVSG YVAQEKLAIA ERYLVPQARA LCGLDESKAK LSSDVLTLLI KQYCRESGVR NLQKQVEKVL RKSAYKIVSG EAE SVEVTP ENLQDFVGKP VFTVERMYDV TPPGVVMGLA WTAMGGSTLF VETSLRRPQD KDAKGDKDGS LEVTGQLGEV MKES ARIAY TFARAFLMQH APANDYLVTS HIHLHVPEGA TPKDGPSAGC TIVTALLSLA MGRPVRQNLA MTGEVSLTGK ILPVG GIKE KTIAAKRAGV TCIVLPAENK KDFYDLAAFI TEGLEVHFVE HYREIFDIAF

UniProtKB: Lon protease homolog, mitochondrial

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Macromolecule #2: Endogenous co-purified substrate

MacromoleculeName: Endogenous co-purified substrate / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: Rosetta 2(DE3)pLysS
Molecular weightTheoretical: 1.039273 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)

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Macromolecule #3: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 4 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 4 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #5: N-[(1R)-1-(DIHYDROXYBORYL)-3-METHYLBUTYL]-N-(PYRAZIN-2-YLCARBONYL...

MacromoleculeName: N-[(1R)-1-(DIHYDROXYBORYL)-3-METHYLBUTYL]-N-(PYRAZIN-2-YLCARBONYL)-L-PHENYLALANINAMIDE
type: ligand / ID: 5 / Number of copies: 6 / Formula: BO2
Molecular weightTheoretical: 384.237 Da
Chemical component information

ChemComp-BO2:
N-[(1R)-1-(DIHYDROXYBORYL)-3-METHYLBUTYL]-N-(PYRAZIN-2-YLCARBONYL)-L-PHENYLALANINAMIDE / medication, anticancer*YM

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Macromolecule #6: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 6 / Number of copies: 2 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration2.5 mg/mL
BufferpH: 8
Component:
ConcentrationNameFormula
50.0 mMTris Base
75.0 mMPotassium ChlorideKCl
10.0 mMMagnesium ChlorideMgCl2
1.0 mMTCEP
1.0 mMAdenosine Triphosphate
0.263 mMBortezomib

Details: Solutions were made fresh from concentrated and filtered using a 0.1 um syringe filter to avoid microbial contamination. Samples were mixed on ice and incubated at 37 degrees C for 30 ...Details: Solutions were made fresh from concentrated and filtered using a 0.1 um syringe filter to avoid microbial contamination. Samples were mixed on ice and incubated at 37 degrees C for 30 minutes to ensure Bortezomib binding. Additional ATP was added to the sample mix on ice 5 minutes prior to vitrification.
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 7 sec. / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: HOMEMADE PLUNGER
Details: 4 uL of sample was applied per grid and manually blotted for 4 seconds followed by immediately plunge-freezing in liquid ethane cooled by liquid nitrogen..
DetailsThis sample was monodisperse.

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
TemperatureMin: 80.0 K / Max: 90.0 K
Alignment procedureComa free - Residual tilt: 0.14 mrad
DetailsComa-free alignment procedure from Herzik & Wu, Nature Methods (2017). Preliminary grid screening was performed manually prior to data collection.
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3710 pixel / Digitization - Dimensions - Height: 3838 pixel / Digitization - Frames/image: 0-51 / Number grids imaged: 1 / Number real images: 4774 / Average exposure time: 10.4 sec. / Average electron dose: 50.0 e/Å2
Details: Images were collected in counting mode at 5 frames per second.
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated defocus max: 1.5 µm / Calibrated defocus min: 0.5 µm / Calibrated magnification: 43478 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.2 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 36000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2736565 / Details: Template-based particle selection in RELION
Startup modelType of model: OTHER
Details: A low-resolution negative stain reconstruction of Human mitochondrial LONP1 was used as an initial model.
Final reconstructionNumber classes used: 1 / Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1)
Software - details: RELION 3.1 was used to perform final reconstruction
Number images used: 532298
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Software - details: RELION 3.1 was used to assign initial euler angles
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Software - details: RELION 3.1 was used to assign final euler angles
Details: RELION 3.1 was used to assign initial angles
Final 3D classificationNumber classes: 2 / Avg.num./class: 500000 / Software - Name: RELION (ver. 3.1)
Software - details: RELION 3.1 was used to perform final classification
Details: The final 3D classification had an somewhat even distribution owing to preferred specimen orientation due to differential interactions with the air-water interface
FSC plot (resolution estimation)

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Atomic model buiding 1

DetailsInitial homology model was built using SWISS-MODEL and initial rigid body docking was done using UCSF Chimera.
RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 52 / Target criteria: Correlation coefficient
Output model

PDB-7krz:
Human mitochondrial LONP1 in complex with Bortezomib

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