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- PDB-7t4l: Structure of dimeric phosphorylated Pediculus humanus (Ph) PINK1 ... -

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Basic information

Entry
Database: PDB / ID: 7t4l
TitleStructure of dimeric phosphorylated Pediculus humanus (Ph) PINK1 with extended alpha-C helix in chain B
ComponentsSerine/threonine-protein kinase PINK1, putative
KeywordsTRANSFERASE / PINK1 / Kinase / Mitophagy / Parkinson's Disease / Ubiquitin / Phosphorylation / Phospho-ubiquitin
Function / homology
Function and homology information


positive regulation of mitochondrial fission / autophagy / regulation of apoptotic process / mitochondrial outer membrane / mitochondrial inner membrane / non-specific serine/threonine protein kinase / protein kinase activity / phosphorylation / protein serine/threonine kinase activity / ATP binding ...positive regulation of mitochondrial fission / autophagy / regulation of apoptotic process / mitochondrial outer membrane / mitochondrial inner membrane / non-specific serine/threonine protein kinase / protein kinase activity / phosphorylation / 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
Serine/threonine-protein kinase Pink1, mitochondrial
Similarity search - Component
Biological speciesPediculus humanus corporis (human body louse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.28 Å
AuthorsGan, Z.Y. / Leis, A. / Dewson, G. / Glukhova, A. / Komander, D.
Funding support United States, 2items
OrganizationGrant numberCountry
National Health and Medical Research Council (NHMRC, Australia) United States
Michael J. Fox Foundation United States
CitationJournal: Nature / Year: 2022
Title: Activation mechanism of PINK1.
Authors: Zhong Yan Gan / Sylvie Callegari / Simon A Cobbold / Thomas R Cotton / Michael J Mlodzianoski / Alexander F Schubert / Niall D Geoghegan / Kelly L Rogers / Andrew Leis / Grant Dewson / Alisa ...Authors: Zhong Yan Gan / Sylvie Callegari / Simon A Cobbold / Thomas R Cotton / Michael J Mlodzianoski / Alexander F Schubert / Niall D Geoghegan / Kelly L Rogers / Andrew Leis / Grant Dewson / Alisa Glukhova / David Komander /
Abstract: Mutations in the protein kinase PINK1 lead to defects in mitophagy and cause autosomal recessive early onset Parkinson's disease. PINK1 has many unique features that enable it to phosphorylate ...Mutations in the protein kinase PINK1 lead to defects in mitophagy and cause autosomal recessive early onset Parkinson's disease. PINK1 has many unique features that enable it to phosphorylate ubiquitin and the ubiquitin-like domain of Parkin. Structural analysis of PINK1 from diverse insect species with and without ubiquitin provided snapshots of distinct structural states yet did not explain how PINK1 is activated. Here we elucidate the activation mechanism of PINK1 using crystallography and cryo-electron microscopy (cryo-EM). A crystal structure of unphosphorylated Pediculus humanus corporis (Ph; human body louse) PINK1 resolves an N-terminal helix, revealing the orientation of unphosphorylated yet active PINK1 on the mitochondria. We further provide a cryo-EM structure of a symmetric PhPINK1 dimer trapped during the process of trans-autophosphorylation, as well as a cryo-EM structure of phosphorylated PhPINK1 undergoing a conformational change to an active ubiquitin kinase state. Structures and phosphorylation studies further identify a role for regulatory PINK1 oxidation. Together, our research delineates the complete activation mechanism of PINK1, illuminates how PINK1 interacts with the mitochondrial outer membrane and reveals how PINK1 activity may be modulated by mitochondrial reactive oxygen species.
History
DepositionDec 10, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 12, 2022Provider: repository / Type: Initial release
Revision 1.1Feb 23, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year / _citation_author.identifier_ORCID

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Assembly

Deposited unit
A: Serine/threonine-protein kinase PINK1, putative
B: Serine/threonine-protein kinase PINK1, putative


Theoretical massNumber of molelcules
Total (without water)106,1072
Polymers106,1072
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Serine/threonine-protein kinase PINK1, putative


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: 8239562, Phum_PHUM577390 / Plasmid: pOPINK / Production host: Escherichia coli (E. coli)
References: UniProt: E0W1I1, non-specific serine/threonine protein kinase
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: Dodecameric phosphorylated Pediculus humanus (Ph) PINK1
Type: COMPLEX
Details: Dodecamer was stabilised by hydrogen peroxide treatment prior to phosphorylation and purification by SEC
Entity ID: all / 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 mMtrisaminomethaneTris1
2150 mMsodium chlorideNaCl1
SpecimenConc.: 1.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil 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: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Image recordingElectron dose: 1.25 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
EM imaging opticsEnergyfilter slit width: 10 eV

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.19.2_4158refinement
PHENIX1.19.2_4158refinement
EM software
IDNameVersionCategory
7Coot0.9model fitting
9PHENIX1.19.2-4158model refinement
10cryoSPARC3.2.0initial Euler assignment
11cryoSPARC3.2.0final Euler assignment
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.28 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 172764 / Symmetry type: POINT
Atomic model buildingSpace: REAL
Atomic model buildingPDB-ID: 6EQI

6eqi


Pdb chain-ID: C
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 79.38 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00496182
ELECTRON MICROSCOPYf_angle_d0.94778370
ELECTRON MICROSCOPYf_chiral_restr0.0532947
ELECTRON MICROSCOPYf_plane_restr0.00871056
ELECTRON MICROSCOPYf_dihedral_angle_d5.1605818

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