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- PDB-7t3x: Structure of unphosphorylated Pediculus humanus (Ph) PINK1 D334A ... -

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Entry
Database: PDB / ID: 7t3x
TitleStructure of unphosphorylated Pediculus humanus (Ph) PINK1 D334A mutant
ComponentsSerine/threonine-protein kinase PINK1
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 / non-specific serine/threonine protein kinase / protein kinase activity / mitochondrial inner membrane / 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)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.53 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 9, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 22, 2021Provider: repository / Type: Initial release
Revision 1.1Jan 12, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID
Revision 1.2Feb 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
Revision 1.3Oct 18, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
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Assembly

Deposited unit
A: Serine/threonine-protein kinase PINK1


Theoretical massNumber of molelcules
Total (without water)52,9301
Polymers52,9301
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)116.118, 116.118, 70.565
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number170
Space group name H-MP65
Space group name HallP65
Symmetry operation#1: x,y,z
#2: x-y,x,z+5/6
#3: y,-x+y,z+1/6
#4: -y,x-y,z+2/3
#5: -x+y,-x,z+1/3
#6: -x,-y,z+1/2

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Components

#1: Protein Serine/threonine-protein kinase PINK1


Mass: 52929.613 Da / Num. of mol.: 1 / Mutation: D334A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pediculus humanus corporis (human body louse)
Gene: 8239562, Phum_PHUM577390 / Plasmid: pOPIN-K / Production host: Escherichia coli (E. coli)
References: UniProt: E0W1I1, non-specific serine/threonine protein kinase

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.59 Å3/Da / Density % sol: 52.59 %
Crystal growTemperature: 281 K / Method: vapor diffusion, sitting drop / pH: 7
Details: 20% polyethylene glycol 3350, 0.2 M triammonium citrate, 0.1 M ammonium sulphate, 0.01 M magnesium chloride

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Australian Synchrotron / Beamline: MX2 / Wavelength: 0.9537 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Nov 27, 2020
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9537 Å / Relative weight: 1
ReflectionResolution: 3.53→44.83 Å / Num. obs: 6769 / % possible obs: 99.9 % / Redundancy: 5.7 % / Biso Wilson estimate: 80.2 Å2 / Rmerge(I) obs: 0.41 / Rpim(I) all: 0.188 / Net I/σ(I): 4.2
Reflection shellResolution: 3.53→3.87 Å / Redundancy: 5.8 % / Rmerge(I) obs: 0.953 / Mean I/σ(I) obs: 2 / Num. unique obs: 1609 / % possible all: 99.7

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Processing

Software
NameVersionClassification
Coot0.9model building
PHENIX1.19.2_4158refinement
XDSNov 1, 2016data reduction
Aimless0.5.21data scaling
PHASER2.8.3phasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 6EQI

6eqi


Resolution: 3.53→44.83 Å / SU ML: 0.3182 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 26.8819
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2495 362 5.36 %
Rwork0.2162 6389 -
obs0.2181 6751 99.87 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 58.17 Å2
Refinement stepCycle: LAST / Resolution: 3.53→44.83 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3071 0 0 0 3071
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0023130
X-RAY DIFFRACTIONf_angle_d0.50164256
X-RAY DIFFRACTIONf_chiral_restr0.0396498
X-RAY DIFFRACTIONf_plane_restr0.0035540
X-RAY DIFFRACTIONf_dihedral_angle_d3.8646425
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.53-4.040.28931200.25632101X-RAY DIFFRACTION99.78
4.05-5.090.22611290.20242114X-RAY DIFFRACTION100
5.1-44.830.24611130.20152174X-RAY DIFFRACTION99.83

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