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- PDB-8uyh: Structure of AMP-PNP-bound Pediculus humanus (Ph) PINK1 dimer -

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Basic information

Entry
Database: PDB / ID: 8uyh
TitleStructure of AMP-PNP-bound Pediculus humanus (Ph) PINK1 dimer
ComponentsSerine/threonine-protein kinase Pink1, mitochondrial
KeywordsTRANSFERASE / PINK1 / Kinase / Mitophagy / Parkinson's Disease / Ubiquitin / Phosphorylation / Phospho-ubiquitin
Function / homology
Function and homology information


autophagy of mitochondrion / positive regulation of mitochondrial fission / regulation of apoptotic process / mitochondrial outer membrane / mitochondrial inner membrane / non-specific serine/threonine protein kinase / protein kinase activity / protein serine/threonine kinase activity / ATP binding / metal ion binding / cytosol
Similarity search - Function
: / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / Protein kinase domain / Serine/Threonine protein kinases, catalytic domain / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily
Similarity search - Domain/homology
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / Serine/threonine-protein kinase Pink1, mitochondrial
Similarity search - Component
Biological speciesPediculus humanus corporis (human body louse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.84 Å
AuthorsGan, Z.Y. / Kirk, N.S. / Leis, A. / Komander, D.
Funding support Australia, United States, 2items
OrganizationGrant numberCountry
National Health and Medical Research Council (NHMRC, Australia) Australia
Michael J. Fox Foundation United States
CitationJournal: Sci Adv / Year: 2024
Title: Interaction of PINK1 with nucleotides and kinetin.
Authors: Zhong Yan Gan / Sylvie Callegari / Thanh N Nguyen / Nicholas S Kirk / Andrew Leis / Michael Lazarou / Grant Dewson / David Komander /
Abstract: The ubiquitin kinase PINK1 accumulates on damaged mitochondria to trigger mitophagy, and PINK1 loss-of-function mutations cause early onset Parkinson's disease. Nucleotide analogs such as kinetin ...The ubiquitin kinase PINK1 accumulates on damaged mitochondria to trigger mitophagy, and PINK1 loss-of-function mutations cause early onset Parkinson's disease. Nucleotide analogs such as kinetin triphosphate (KTP) were reported to enhance PINK1 activity and may represent a therapeutic strategy for the treatment of Parkinson's disease. Here, we investigate the interaction of PINK1 with nucleotides, including KTP. We establish a cryo-EM platform exploiting the dodecamer assembly of () PINK1 and determine PINK1 structures bound to AMP-PNP and ADP, revealing conformational changes in the kinase N-lobe that help establish PINK1's ubiquitin binding site. Notably, we find that KTP is unable to bind PINK1 or human () PINK1 due to a steric clash with the kinase "gatekeeper" methionine residue, and mutation to Ala or Gly is required for PINK1 to bind and use KTP as a phosphate donor in ubiquitin phosphorylation and mitophagy. PINK1 M318G can be used to conditionally uncouple PINK1 stabilization and activity on mitochondria.
History
DepositionNov 13, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 31, 2024Provider: repository / Type: Initial release
Revision 1.1Oct 30, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Serine/threonine-protein kinase Pink1, mitochondrial
B: Serine/threonine-protein kinase Pink1, mitochondrial
hetero molecules


Theoretical massNumber of molelcules
Total (without water)107,1937
Polymers106,1072
Non-polymers1,0855
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Serine/threonine-protein kinase Pink1, mitochondrial


Mass: 53053.602 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pediculus humanus corporis (human body louse)
Gene: Pink1 / Plasmid: pOPINK / Production host: Escherichia coli (E. coli) / References: UniProt: E0W1I1
#2: Chemical ChemComp-ANP / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER


Mass: 506.196 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H17N6O12P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: AMP-PNP, energy-carrying molecule analogue*YM
#3: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationY

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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: AMP-PNP-bound Pediculus humanus (Ph) PINK1 dodecamer / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Pediculus humanus corporis (human body louse)
Source (recombinant)Organism: Escherichia coli (E. coli) / Plasmid: pOPINK
Buffer solutionpH: 8.5
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMtrisaminomethane1
2150 mMsodium chlorideNaCl1
310 mMdithiothreitol1
410 mMmagnesium chlorideMgCl21
510 mMadenylyl-imidodiphosphate1
SpecimenConc.: 1.9 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
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: 277 K

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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: OTHER
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1500 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)
EM imaging opticsEnergyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
7Coot0.9.8.7model fitting
9PHENIX1.20.1-4487model refinement
10cryoSPARC4.2.1initial Euler assignment
11cryoSPARC4.2.1final Euler assignment
13cryoSPARC4.2.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.84 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 114790 / Symmetry type: POINT
Atomic model buildingSpace: REAL
Atomic model buildingPDB-ID: 7T4N

7t4n


Accession code: 7T4N / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0026152
ELECTRON MICROSCOPYf_angle_d0.4828342
ELECTRON MICROSCOPYf_dihedral_angle_d11.6222293
ELECTRON MICROSCOPYf_chiral_restr0.04947
ELECTRON MICROSCOPYf_plane_restr0.0051038

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