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- PDB-8u1b: C-terminal LRRK2 bound to E11 DARPin -

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Basic information

Entry
Database: PDB / ID: 8u1b
TitleC-terminal LRRK2 bound to E11 DARPin
Components
  • E11 DARPin
  • Leucine-rich repeat serine/threonine-protein kinase 2
KeywordsPROTEIN BINDING / Kinase / complex / DARPins
Function / homology
Function and homology information


caveola neck / : / beta-catenin destruction complex binding / regulation of branching morphogenesis of a nerve / Wnt signalosome assembly / regulation of kidney size / 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 / : / beta-catenin destruction complex binding / regulation of branching morphogenesis of a nerve / Wnt signalosome assembly / regulation of kidney size / 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 / : / 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 / positive regulation of microglial cell activation / regulation of neuron maturation / regulation of retrograde transport, endosome to Golgi / positive regulation of synaptic vesicle endocytosis / negative regulation of excitatory postsynaptic potential / cytoplasmic side of mitochondrial outer membrane / negative regulation of autophagosome assembly / olfactory bulb development / JUN kinase kinase kinase activity / 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 / regulation of cAMP/PKA signal transduction / regulation of mitochondrial fission / protein kinase A binding / neuromuscular junction development / regulation of locomotion / regulation of synaptic vesicle exocytosis / Golgi organization / intracellular distribution of mitochondria / microvillus / 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 / Rho protein signal transduction / presynaptic cytosol / 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 / regulation of membrane potential / cellular response to reactive oxygen species / mitochondrion organization / excitatory postsynaptic potential / trans-Golgi network / calcium-mediated signaling / regulation of protein stability / autophagy / small GTPase binding / mitochondrial membrane / regulation of autophagy
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
Leucine-rich repeat serine/threonine-protein kinase 2
Similarity search - Component
Biological speciesHomo sapiens (human)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsSanz-Murillo, M. / Mathea, S. / Dederer, V. / Knapp, S. / Leschziner, A.
Funding support United States, 1items
OrganizationGrant numberCountry
Other privateASAP-000519 United States
CitationJournal: J Biol Chem / Year: 2024
Title: A designed ankyrin-repeat protein that targets Parkinson's disease-associated LRRK2.
Authors: Verena Dederer / Marta Sanz Murillo / Eva P Karasmanis / Kathryn S Hatch / Deep Chatterjee / Franziska Preuss / Kamal R Abdul Azeez / Landon Vu Nguyen / Christian Galicia / Birgit Dreier / ...Authors: Verena Dederer / Marta Sanz Murillo / Eva P Karasmanis / Kathryn S Hatch / Deep Chatterjee / Franziska Preuss / Kamal R Abdul Azeez / Landon Vu Nguyen / Christian Galicia / Birgit Dreier / Andreas Plückthun / Wim Versees / Sebastian Mathea / Andres E Leschziner / Samara L Reck-Peterson / Stefan Knapp /
Abstract: Leucine rich repeat kinase 2 (LRRK2) is a large multidomain protein containing two catalytic domains, a kinase and a GTPase, as well as protein interactions domains, including a WD40 domain. The ...Leucine rich repeat kinase 2 (LRRK2) is a large multidomain protein containing two catalytic domains, a kinase and a GTPase, as well as protein interactions domains, including a WD40 domain. The association of increased LRRK2 kinase activity with both the familial and sporadic forms of Parkinson's disease has led to an intense interest in determining its cellular function. However, small molecule probes that can bind to LRRK2 and report on or affect its cellular activity are needed. Here, we report the identification and characterization of the first high-affinity LRRK2-binding designed ankyrin-repeat protein (DARPin), named E11. Using cryo-EM, we show that DARPin E11 binds to the LRRK2 WD40 domain. LRRK2 bound to DARPin E11 showed improved behavior on cryo-EM grids, resulting in higher resolution LRRK2 structures. DARPin E11 did not affect the catalytic activity of a truncated form of LRRK2 in vitro but decreased the phosphorylation of Rab8A, a LRRK2 substrate, in cells. We also found that DARPin E11 disrupts the formation of microtubule-associated LRRK2 filaments in cells, which are known to require WD40-based dimerization. Thus, DARPin E11 is a new tool to explore the function and dysfunction of LRRK2 and guide the development of LRRK2 kinase inhibitors that target the WD40 domain instead of the kinase.
History
DepositionAug 31, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 4, 2024Provider: repository / Type: Initial release
Revision 1.1Oct 23, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update
Revision 1.2Mar 26, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Leucine-rich repeat serine/threonine-protein kinase 2
C: E11 DARPin


Theoretical massNumber of molelcules
Total (without water)155,8282
Polymers155,8282
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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


Mass: 136060.656 Da / Num. of mol.: 1
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: Protein E11 DARPin


Mass: 19766.912 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Nterminal corresponds to HisTag. Some amino acids are not modeled due to the lack of density
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)
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: C-terminal LRRK2 bound to E11 DARPin / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.156 MDa / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPES1
2150 mMSodium ChlorideNaCl1
32.5 mMMagnesium ChlorideMgCl21
40.5 mMTCEP1
55 %Glycerol1
620 uMGDP1
SpecimenConc.: 0.68 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 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: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 52 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2468

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Processing

EM software
IDNameVersionCategory
1Topaz0.2.5particle selection
2Leginonimage acquisition
4CTFFIND4CTF correction
7UCSF ChimeraX1.5model fitting
9cryoSPARC4initial Euler assignment
10cryoSPARC4final Euler assignment
12cryoSPARC43D reconstruction
13PHENIX1.2model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 341041
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 185601 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Atomic model buildingPDB-ID: 6VP7

6vp7


Pdb chain-ID: A / Accession code: 6VP7 / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0044956
ELECTRON MICROSCOPYf_angle_d0.7746716
ELECTRON MICROSCOPYf_dihedral_angle_d4.919653
ELECTRON MICROSCOPYf_chiral_restr0.045802
ELECTRON MICROSCOPYf_plane_restr0.005844

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