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- PDB-7thz: Structure of Leucine Rich Repeat Kinase 2's ROC domain interactin... -

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Basic information

Entry
Database: PDB / ID: 7thz
TitleStructure of Leucine Rich Repeat Kinase 2's ROC domain interacting with the microtubule facing the plus end
ComponentsLeucine-rich repeat serine/threonine-protein kinase 2
KeywordsCYTOSOLIC PROTEIN / parkinson's disease / microtubule / kinase / gtpase
Function / homology
Function and homology information


caveola neck / negative regulation of protein processing involved in protein targeting to mitochondrion / beta-catenin destruction complex binding / regulation of branching morphogenesis of a nerve / regulation of kidney size / Wnt signalosome assembly / regulation of cell projection organization / tangential migration from the subventricular zone to the olfactory bulb / GTP-dependent protein kinase activity / regulation of SNARE complex assembly ...caveola neck / negative regulation of protein processing involved in protein targeting to mitochondrion / beta-catenin destruction complex binding / regulation of branching morphogenesis of a nerve / regulation of kidney size / Wnt signalosome assembly / regulation of cell projection organization / 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 / 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 / amphisome / regulation of synaptic vesicle transport / regulation of lysosomal lumen pH / regulation of CAMKK-AMPK signaling cascade / co-receptor binding / negative regulation of GTPase activity / regulation of dopamine receptor signaling pathway / regulation of neuron maturation / positive regulation of microglial cell activation / regulation of retrograde transport, endosome to Golgi / positive regulation of synaptic vesicle endocytosis / cytoplasmic side of mitochondrial outer membrane / negative regulation of excitatory postsynaptic potential / negative regulation of autophagosome assembly / JUN kinase kinase kinase activity / olfactory bulb development / neuron projection arborization / striatum development / multivesicular body, internal vesicle / regulation of dendritic spine morphogenesis / mitochondrion localization / protein localization to mitochondrion / cellular response to dopamine / positive regulation of mitochondrial outer membrane permeabilization involved in apoptotic signaling pathway / endoplasmic reticulum organization / positive regulation of protein autoubiquitination / Wnt signalosome / negative regulation of protein processing / positive regulation of programmed cell death / GTP metabolic process / regulation of canonical Wnt signaling pathway / syntaxin-1 binding / regulation of reactive oxygen species metabolic process / lysosome organization / Golgi-associated vesicle / clathrin binding / PTK6 promotes HIF1A stabilization / negative regulation of macroautophagy / protein kinase A binding / neuromuscular junction development / regulation of cAMP/PKA signal transduction / regulation of mitochondrial fission / regulation of locomotion / regulation of synaptic vesicle exocytosis / microvillus / Golgi organization / intracellular distribution of mitochondria / exploration behavior / endoplasmic reticulum exit site / autolysosome / locomotory exploration behavior / negative regulation of Notch signaling pathway / MAP kinase kinase kinase activity / regulation of synaptic vesicle endocytosis / canonical Wnt signaling pathway / regulation of synaptic transmission, glutamatergic / negative regulation of endoplasmic reticulum stress-induced intrinsic apoptotic signaling pathway / presynaptic cytosol / Rho protein signal transduction / neuron projection morphogenesis / phagocytic vesicle / cellular response to manganese ion / JNK cascade / positive regulation of autophagy / dendrite cytoplasm / tubulin binding / GTPase activator activity / cellular response to starvation / positive regulation of protein ubiquitination / SNARE binding / determination of adult lifespan / cellular response to reactive oxygen species / regulation of membrane potential / excitatory postsynaptic potential / mitochondrion organization / trans-Golgi network / calcium-mediated signaling / regulation of protein stability / autophagy / small GTPase binding / mitochondrial membrane / endocytosis
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 / C-terminal of Roc, COR-A 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. / Leucine-rich repeat / 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 / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / Protein kinase domain / WD40-repeat-containing domain superfamily / 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
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: 5 Å
AuthorsMatyszewski, M. / Leschziner, A.E.
Funding support United States, 4items
OrganizationGrant numberCountry
Other privateASAP-000519 United States
Michael J. Fox Foundation18321 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM121772 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM107214 United States
CitationJournal: Nat Struct Mol Biol / Year: 2022
Title: Structural basis for Parkinson's disease-linked LRRK2's binding to microtubules.
Authors: David M Snead / Mariusz Matyszewski / Andrea M Dickey / Yu Xuan Lin / Andres E Leschziner / Samara L Reck-Peterson /
Abstract: Leucine-rich repeat kinase 2 (LRRK2) is one of the most commonly mutated genes in familial Parkinson's disease (PD). Under some circumstances, LRRK2 co-localizes with microtubules in cells, an ...Leucine-rich repeat kinase 2 (LRRK2) is one of the most commonly mutated genes in familial Parkinson's disease (PD). Under some circumstances, LRRK2 co-localizes with microtubules in cells, an association enhanced by PD mutations. We report a cryo-EM structure of the catalytic half of LRRK2, containing its kinase, in a closed conformation, and GTPase domains, bound to microtubules. We also report a structure of the catalytic half of LRRK1, which is closely related to LRRK2 but is not linked to PD. Although LRRK1's structure is similar to that of LRRK2, we find that LRRK1 does not interact with microtubules. Guided by these structures, we identify amino acids in LRRK2's GTPase that mediate microtubule binding; mutating them disrupts microtubule binding in vitro and in cells, without affecting LRRK2's kinase activity. Our results have implications for the design of therapeutic LRRK2 kinase inhibitors.
History
DepositionJan 12, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 28, 2022Provider: repository / Type: Initial release
Revision 1.1Jun 5, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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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)22,6352
Polymers22,1921
Non-polymers4431
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Number of models5

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Components

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


Mass: 22191.762 Da / Num. of mol.: 1 / Fragment: ROC domain
Source method: isolated from a genetically manipulated source
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
Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: LRRK2RCKW filament bound to a 11-pf microtubule with MLi-2 present
Type: COMPLEX / Entity ID: #1 / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Buffer solutionpH: 7.4
Details: This is the final dilution buffer. The incubation buffer consisted of 1x BRB80, 10% glycerol, 1mM DTT, 1mM GTP, 1mM MgCl2, 10 uM taxol, and 5 uM MLi-2. Sample was diluted 3-fold right before ...Details: This is the final dilution buffer. The incubation buffer consisted of 1x BRB80, 10% glycerol, 1mM DTT, 1mM GTP, 1mM MgCl2, 10 uM taxol, and 5 uM MLi-2. Sample was diluted 3-fold right before freezing with the final buffer.
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPES1
280 mMSodium ChlorideNaCl1
30.5 mMTCEP1
42.5 mMMagnesium ChlorideMgCl21
520 uMGDP1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: 4.5 uM of LRRK2RCKW (I2020T) was allowed to incubate with 2.25 uM of tubulin dimer, causing both to co-polymerize. 5 uM of MLi-2 was present as well. The sample was diluted 3-fold right ...Details: 4.5 uM of LRRK2RCKW (I2020T) was allowed to incubate with 2.25 uM of tubulin dimer, causing both to co-polymerize. 5 uM of MLi-2 was present as well. The sample was diluted 3-fold right before freezing (1.5 uM LRRK2RCKW concentration final).
Specimen supportDetails: EMS LC-300 lacey grid used (not homemade, but can't choose EMS as the manufacturer)
Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Homemade
VitrificationCryogen 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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 36000 X / Nominal defocus max: 1500 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 10 sec. / Electron dose: 55 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2 / Details: 250 ms frames

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Processing

EM software
IDNameVersionCategoryDetails
1RELIONparticle selection
2Leginonimage acquisitionCustom Hole picker
4CTFFIND4CTF correction
7Rosetta3.13model fittingUsing Frank DiMaio's scripts
9ISOLDE1.2.2model refinementUsed to fix outliers by hand
10Rosetta3.13model refinementRelaxation in cryo-EM density to resolve leftover outliers
14cryoSPARC3.23D reconstructionlocal refinement, with non-uniform refinement turned off
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 557577
Details: Filament Autopicker with templates created by manual picking. This is before symmetry expansion.
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 99854 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT
Details: Used AlphaFold model as initial model (Q5S007) using only the ROC domain. TUB1 was added to the initial refinement to prevent ROC model from entering density reserved for the microtubule. ...Details: Used AlphaFold model as initial model (Q5S007) using only the ROC domain. TUB1 was added to the initial refinement to prevent ROC model from entering density reserved for the microtubule. TUB1 was discarded after the initial refinement.

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