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- PDB-6vno: Cryo-EM structure of the C-terminal half of the Parkinson's Disea... -

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Basic information

Entry
Database: PDB / ID: 6vno
TitleCryo-EM structure of the C-terminal half of the Parkinson's Disease-linked protein Leucine Rich Repeat Kinase 2 (LRRK2)
ComponentsLeucine-rich repeat serine/threonine-protein kinase 2
KeywordsSIGNALING PROTEIN / Kinase / GTPase
Function / homology
Function and homology information


caveola neck / negative regulation of thioredoxin peroxidase activity by peptidyl-threonine phosphorylation / negative regulation of protein processing involved in protein targeting to mitochondrion / Wnt signalosome assembly / beta-catenin destruction complex binding / regulation of branching morphogenesis of a nerve / regulation of kidney size / regulation of neuron maturation / regulation of cell projection organization / tangential migration from the subventricular zone to the olfactory bulb ...caveola neck / negative regulation of thioredoxin peroxidase activity by peptidyl-threonine phosphorylation / negative regulation of protein processing involved in protein targeting to mitochondrion / Wnt signalosome assembly / beta-catenin destruction complex binding / regulation of branching morphogenesis of a nerve / regulation of kidney size / regulation of neuron maturation / regulation of cell projection organization / tangential migration from the subventricular zone to the olfactory bulb / protein localization to endoplasmic reticulum exit site / GTP-dependent protein kinase activity / regulation of neuroblast proliferation / regulation of ER to Golgi vesicle-mediated transport / peroxidase inhibitor activity / negative regulation of late endosome to lysosome transport / regulation of mitochondrial depolarization / negative regulation of protein targeting to mitochondrion / positive regulation of dopamine receptor signaling pathway / regulation of synaptic vesicle transport / regulation of CAMKK-AMPK signaling cascade / regulation of lysosomal lumen pH / amphisome / co-receptor binding / mitochondrion localization / negative regulation of excitatory postsynaptic potential / regulation of retrograde transport, endosome to Golgi / regulation of dopamine receptor signaling pathway / positive regulation of microglial cell activation / positive regulation of synaptic vesicle endocytosis / negative regulation of autophagosome assembly / cytoplasmic side of mitochondrial outer membrane / neuron projection arborization / regulation of cAMP/PKA signal transduction / olfactory bulb development / striatum development / multivesicular body, internal vesicle / regulation of dendritic spine morphogenesis / protein localization to mitochondrion / JUN kinase kinase kinase activity / cellular response to dopamine / endoplasmic reticulum organization / positive regulation of protein autoubiquitination / positive regulation of programmed cell death / Wnt signalosome / negative regulation of protein processing / GTP metabolic process / regulation of canonical Wnt signaling pathway / syntaxin-1 binding / negative regulation of GTPase activity / exploration behavior / regulation of reactive oxygen species metabolic process / lysosome organization / Golgi-associated vesicle / clathrin binding / regulation of locomotion / protein kinase A binding / phosphorylation / negative regulation of macroautophagy / neuromuscular junction development / regulation of synaptic vesicle exocytosis / PTK6 promotes HIF1A stabilization / regulation of mitochondrial fission / Golgi organization / intracellular distribution of mitochondria / locomotory exploration behavior / regulation of synaptic vesicle endocytosis / endoplasmic reticulum exit site / autolysosome / microvillus / MAP kinase kinase kinase activity / negative regulation of Notch signaling pathway / Rho protein signal transduction / positive regulation of protein kinase activity / cellular response to manganese ion / canonical Wnt signaling pathway / presynaptic cytosol / negative regulation of endoplasmic reticulum stress-induced intrinsic apoptotic signaling pathway / regulation of synaptic transmission, glutamatergic / phagocytic vesicle / JNK cascade / positive regulation of autophagy / dendrite cytoplasm / negative regulation of protein binding / positive regulation of MAP kinase activity / tubulin binding / GTPase activator activity / SNARE binding / neuron projection morphogenesis / cellular response to starvation / positive regulation of protein ubiquitination / regulation of membrane potential / excitatory postsynaptic potential / determination of adult lifespan / mitochondrion organization / cellular response to reactive oxygen species / peptidyl-threonine phosphorylation / trans-Golgi network / calcium-mediated signaling / mitochondrial membrane
Similarity search - Function
LRRK2 ARM repeat / LRRK2 ANK repeat / LRRK2 beta propeller / : / C-terminal of Roc (COR) domain / C-terminal of Roc, COR-A domain / Ras of Complex, Roc, domain of DAPkinase / Roc domain profile. / Roc domain / Leucine-rich repeats, bacterial type ...LRRK2 ARM repeat / LRRK2 ANK repeat / LRRK2 beta propeller / : / C-terminal of Roc (COR) domain / C-terminal of Roc, COR-A domain / Ras of Complex, Roc, domain of DAPkinase / Roc domain profile. / Roc domain / Leucine-rich repeats, bacterial type / Leucine rich repeat / Leucine-rich repeat, typical subtype / Leucine-rich repeats, typical (most populated) subfamily / Leucine-rich repeat profile. / YVTN repeat-like/Quinoprotein amine dehydrogenase / Leucine-rich repeat / 7 Propeller / Methylamine Dehydrogenase; Chain H / Rab subfamily of small GTPases / Leucine-rich repeat domain superfamily / Ankyrin repeat-containing domain superfamily / Armadillo-like helical / Small GTP-binding protein domain / Armadillo-type fold / Phosphorylase Kinase; domain 1 / Phosphorylase Kinase; domain 1 / Transferase(Phosphotransferase) domain 1 / Transferase(Phosphotransferase); domain 1 / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / WD40-repeat-containing domain superfamily / Protein kinase domain / Serine/Threonine protein kinases, catalytic domain / WD40/YVTN repeat-like-containing domain superfamily / Protein kinase, ATP binding site / Protein kinases ATP-binding region signature. / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily / P-loop containing nucleoside triphosphate hydrolase / 2-Layer Sandwich / Orthogonal Bundle / Mainly Beta / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
GUANOSINE-5'-DIPHOSPHATE / Leucine-rich repeat serine/threonine-protein kinase 2
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsLeschziner, A. / Deniston, C. / Lahiri, I.
Funding support United States, 3items
OrganizationGrant numberCountry
Michael J. Fox Foundation11425 United States
Michael J. Fox Foundation11425.02 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM107214 United States
CitationJournal: Nature / Year: 2020
Title: Structure of LRRK2 in Parkinson's disease and model for microtubule interaction.
Authors: C K Deniston / J Salogiannis / S Mathea / D M Snead / I Lahiri / M Matyszewski / O Donosa / R Watanabe / J Böhning / A K Shiau / S Knapp / E Villa / S L Reck-Peterson / A E Leschziner /
Abstract: Leucine-rich repeat kinase 2 (LRRK2) is the most commonly mutated gene in familial Parkinson's disease and is also linked to its idiopathic form. LRRK2 has been proposed to function in membrane ...Leucine-rich repeat kinase 2 (LRRK2) is the most commonly mutated gene in familial Parkinson's disease and is also linked to its idiopathic form. LRRK2 has been proposed to function in membrane trafficking and colocalizes with microtubules. Despite the fundamental importance of LRRK2 for understanding and treating Parkinson's disease, structural information on the enzyme is limited. Here we report the structure of the catalytic half of LRRK2, and an atomic model of microtubule-associated LRRK2 built using a reported cryo-electron tomography in situ structure. We propose that the conformation of the LRRK2 kinase domain regulates its interactions with microtubules, with a closed conformation favouring oligomerization on microtubules. We show that the catalytic half of LRRK2 is sufficient for filament formation and blocks the motility of the microtubule-based motors kinesin 1 and cytoplasmic dynein 1 in vitro. Kinase inhibitors that stabilize an open conformation relieve this interference and reduce the formation of LRRK2 filaments in cells, whereas inhibitors that stabilize a closed conformation do not. Our findings suggest that LRRK2 can act as a roadblock for microtubule-based motors and have implications for the design of therapeutic LRRK2 kinase inhibitors.
History
DepositionJan 29, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 26, 2020Provider: repository / Type: Initial release
Revision 1.1Sep 2, 2020Group: 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
Revision 1.2Dec 23, 2020Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Nov 20, 2024Group: Data collection / Database references / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

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Assembly

Deposited unit
A: Leucine-rich repeat serine/threonine-protein kinase 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)137,4113
Polymers136,9441
Non-polymers4682
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area770 Å2
ΔGint-16 kcal/mol
Surface area51880 Å2
Number of models10

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Components

#1: Protein Leucine-rich repeat serine/threonine-protein kinase 2 / Dardarin


Mass: 136943.609 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: C-terminal residues 1330-2527 / Source: (gene. exp.) Homo sapiens (human) / Gene: LRRK2, PARK8 / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: Q5S007, non-specific serine/threonine protein kinase, Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement
#2: Chemical ChemComp-GDP / GUANOSINE-5'-DIPHOSPHATE


Type: RNA linking / Mass: 443.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O11P2 / Comment: GDP, energy-carrying molecule*YM
#3: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
Has ligand of interestN
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: The C-terminal half of the Leucine Rich Repeat Kinase 2 (LRRK2) protein.
Type: COMPLEX / Details: C-terminal half runs from residue 1327-2527. / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.137 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPES1
280 mMNaCl1
30.5 mMTCEP1
45 %Glycerol1
52.5 mMMgCl21
620 uMGDP1
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: 4uM concentration
Specimen supportGrid material: GOLD / Grid type: Quantifoil, UltrAuFoil, R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK II / 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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 1800 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 8 sec. / Electron dose: 6.65 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3826
EM imaging opticsEnergyfilter name: GIF 2002
Image scansMovie frames/image: 40

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Processing

EM software
IDNameVersionCategoryDetails
2Leginon3image acquisition
4Gctf1CTF correction
7Rosetta3model fitting
9Rosetta3model refinement
10RELION3initial Euler assignment
11cryoSPARC2initial Euler assignment
12RELION3final Euler assignment
13cryoSPARC2final Euler assignment
15RELION33D reconstructionC1
16cryoSPARC23D reconstructionC3
CTF correctionDetails: Per-particle CTF values / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 836956
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 70953
Details: For the signal subtracted map, 105,787 (tripled) particles went into the final map that achieved 3.8A resolution.
Symmetry type: POINT

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