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- PDB-6vp7: Cryo-EM structure of the C-terminal half of the Parkinson's Disea... -

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Basic information

Entry
Database: PDB / ID: 6vp7
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 / : / beta-catenin destruction complex binding / regulation of branching morphogenesis of a nerve / Wnt signalosome assembly / negative regulation of motile cilium assembly / regulation of neuron maturation / regulation of kidney size / regulation of cell projection organization / regulation of dopamine receptor signaling pathway ...caveola neck / : / beta-catenin destruction complex binding / regulation of branching morphogenesis of a nerve / Wnt signalosome assembly / negative regulation of motile cilium assembly / regulation of neuron maturation / regulation of kidney size / regulation of cell projection organization / regulation of dopamine receptor signaling pathway / tangential migration from the subventricular zone to the olfactory bulb / GTP-dependent protein kinase activity / regulation of SNARE complex assembly / regulation of neuroblast proliferation / regulation of ER to Golgi vesicle-mediated transport / protein localization to endoplasmic reticulum exit site / negative regulation of late endosome to lysosome transport / regulation of mitochondrial depolarization / : / peroxidase inhibitor activity / positive regulation of dopamine receptor signaling pathway / amphisome / regulation of synaptic vesicle transport / : / regulation of CAMKK-AMPK signaling cascade / negative regulation of autophagosome assembly / co-receptor binding / positive regulation of microglial cell activation / olfactory bulb development / regulation of retrograde transport, endosome to Golgi / negative regulation of GTPase activity / cellular response to curcumin / striatum development / regulation of locomotion / positive regulation of synaptic vesicle endocytosis / cytoplasmic side of mitochondrial outer membrane / JUN kinase kinase kinase activity / regulation of cAMP/PKA signal transduction / multivesicular body, internal vesicle / negative regulation of excitatory postsynaptic potential / endoplasmic reticulum organization / neuron projection arborization / mitochondrion localization / regulation of dendritic spine morphogenesis / protein localization to mitochondrion / positive regulation of mitochondrial outer membrane permeabilization involved in apoptotic signaling pathway / cellular response to dopamine / negative regulation of protein processing / positive regulation of protein autoubiquitination / Wnt signalosome / exploration behavior / GTP metabolic process / regulation of reactive oxygen species metabolic process / regulation of canonical Wnt signaling pathway / lysosome organization / syntaxin-1 binding / positive regulation of programmed cell death / negative regulation of macroautophagy / Golgi-associated vesicle / PTK6 promotes HIF1A stabilization / clathrin binding / Golgi organization / regulation of synaptic vesicle exocytosis / regulation of mitochondrial fission / Lewy body / intracellular distribution of mitochondria / protein kinase A binding / microvillus / canonical Wnt signaling pathway / neuromuscular junction development / autolysosome / locomotory exploration behavior / endoplasmic reticulum exit site / MAP kinase kinase kinase activity / JNK cascade / regulation of synaptic vesicle endocytosis / negative regulation of endoplasmic reticulum stress-induced intrinsic apoptotic signaling pathway / Rho protein signal transduction / regulation of synaptic transmission, glutamatergic / presynaptic cytosol / determination of adult lifespan / cellular response to manganese ion / neuron projection morphogenesis / phagocytic vesicle / positive regulation of autophagy / dendrite cytoplasm / excitatory postsynaptic potential / positive regulation of protein ubiquitination / cellular response to starvation / regulation of autophagy / GTPase activator activity / mitochondrion organization / calcium-mediated signaling / SNARE binding / cellular response to reactive oxygen species / trans-Golgi network / regulation of protein stability / regulation of membrane potential / tubulin binding / mitochondrial membrane
Similarity search - Function
: / : / : / LRRK2 ARM repeat / LRRK2 ANK repeat / LRRK2 beta propeller / : / : / C-terminal of Roc, COR-B domain / C-terminal of Roc (COR) domain ...: / : / : / LRRK2 ARM repeat / LRRK2 ANK repeat / LRRK2 beta propeller / : / : / C-terminal of Roc, COR-B domain / 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 / 7 Propeller / Methylamine Dehydrogenase; Chain H / Leucine-rich repeat / Leucine-rich repeat domain superfamily / Rab subfamily of small GTPases / 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. / Protein kinase domain / Serine/Threonine protein kinases, catalytic domain / WD40-repeat-containing domain superfamily / 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
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
DepositionFeb 1, 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 1.3Oct 9, 2024Group: Data collection / Database references / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_admin / em_author_list / pdbx_entry_details / pdbx_modification_feature
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_entry_details.has_protein_modification

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)136,9441
Polymers136,9441
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area0 Å2
ΔGint0 kcal/mol
Surface area51830 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 1327-2527 / 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
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: all / 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 / Calibrated defocus min: 1000 nm / Calibrated defocus max: 1800 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 (eV)
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 particles went into the final map that achieved 3.8A resolution
Symmetry type: POINT

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