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- PDB-8vh5: Cryo-EM structure of Rab12-LRRK2 complex in the LRRK2 dimer state -

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Basic information

Entry
Database: PDB / ID: 8vh5
TitleCryo-EM structure of Rab12-LRRK2 complex in the LRRK2 dimer state
Components
  • Leucine-rich repeat serine/threonine-protein kinase 2
  • Ras-related protein Rab-12
KeywordsHYDROLASE / Cryo-EM / Parkinson's disease / Kinase / LRRK2 / Rab GTPases
Function / homology
Function and homology information


Rab protein signal transduction / peroxidase inhibitor activity / 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 ...Rab protein signal transduction / peroxidase inhibitor activity / 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 / 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 / negative regulation of late endosome to lysosome transport / regulation of mitochondrial depolarization / negative regulation of protein targeting to mitochondrion / regulation of synaptic vesicle transport / regulation of lysosomal lumen pH / positive regulation of dopamine receptor signaling pathway / amphisome / regulation of CAMKK-AMPK signaling cascade / cytoplasmic side of mitochondrial outer membrane / co-receptor binding / mitochondrion localization / regulation of retrograde transport, endosome to Golgi / negative regulation of excitatory postsynaptic potential / regulation of dopamine receptor signaling pathway / negative regulation of autophagosome assembly / positive regulation of microglial cell activation / neuron projection arborization / positive regulation of synaptic vesicle endocytosis / JUN kinase kinase kinase activity / olfactory bulb development / RAB geranylgeranylation / regulation of protein kinase A signaling / regulation of dendritic spine morphogenesis / striatum development / multivesicular body, internal vesicle / trans-Golgi network transport vesicle / protein localization to mitochondrion / RAB GEFs exchange GTP for GDP on RABs / cellular response to dopamine / endocytic recycling / endoplasmic reticulum organization / positive regulation of protein autoubiquitination / presynaptic cytosol / positive regulation of programmed cell death / Wnt signalosome / GTP metabolic process / negative regulation of protein processing / regulation of canonical Wnt signaling pathway / syntaxin-1 binding / negative regulation of GTPase activity / regulation of reactive oxygen species metabolic process / exploration behavior / protein kinase A binding / regulation of locomotion / regulation of synaptic vesicle exocytosis / PTK6 promotes HIF1A stabilization / Golgi-associated vesicle / negative regulation of macroautophagy / clathrin binding / neuromuscular junction development / lysosome organization / endosome to lysosome transport / regulation of mitochondrial fission / intracellular distribution of mitochondria / Golgi organization / autolysosome / : / locomotory exploration behavior / exocytosis / endoplasmic reticulum exit site / microvillus / Rho protein signal transduction / MAP kinase kinase kinase activity / protein secretion / positive regulation of protein kinase activity / cellular response to manganese ion / canonical Wnt signaling pathway / negative regulation of endoplasmic reticulum stress-induced intrinsic apoptotic signaling pathway / phosphorylation / positive regulation of autophagy / JNK cascade / regulation of synaptic transmission, glutamatergic / dendrite cytoplasm / autophagosome / tubulin binding / GTPase activator activity / cellular response to starvation / neuron projection morphogenesis / regulation of membrane potential / SNARE binding / excitatory postsynaptic potential / negative regulation of protein phosphorylation / negative regulation of protein binding / positive regulation of protein ubiquitination / regulation of autophagy
Similarity search - Function
Rab12 / : / : / C-terminal of Roc (COR) domain / C-terminal of Roc, COR, domain / Ras of Complex, Roc, domain of DAPkinase / Roc domain profile. / Roc domain / small GTPase Rab1 family profile. / Leucine-rich repeats, bacterial type ...Rab12 / : / : / C-terminal of Roc (COR) domain / C-terminal of Roc, COR, domain / Ras of Complex, Roc, domain of DAPkinase / Roc domain profile. / Roc domain / small GTPase Rab1 family profile. / Leucine-rich repeats, bacterial type / Leucine rich repeat / Ran (Ras-related nuclear proteins) /TC4 subfamily of small GTPases / Leucine-rich repeat, typical subtype / Leucine-rich repeats, typical (most populated) subfamily / Leucine-rich repeat profile. / Leucine-rich repeat / Rho (Ras homology) subfamily of Ras-like small GTPases / Ras subfamily of RAS small GTPases / Small GTPase / Ras family / 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 / WD40-repeat-containing domain superfamily / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / Protein kinase domain / WD40/YVTN repeat-like-containing domain superfamily / Serine/Threonine protein kinases, catalytic domain / 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
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / GUANOSINE-5'-DIPHOSPHATE / PHOSPHOAMINOPHOSPHONIC ACID-GUANYLATE ESTER / Leucine-rich repeat serine/threonine-protein kinase 2 / Ras-related protein Rab-12
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4 Å
AuthorsZhu, H. / Sun, J.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)R01NS129795 United States
CitationJournal: Nat Commun / Year: 2024
Title: RAB12-LRRK2 complex suppresses primary ciliogenesis and regulates centrosome homeostasis in astrocytes.
Authors: Xingjian Li / Hanwen Zhu / Bik Tzu Huang / Xianting Li / Heesoo Kim / Haiyan Tan / Yuanxi Zhang / Insup Choi / Junmin Peng / Pingyi Xu / Ji Sun / Zhenyu Yue /
Abstract: The leucine-rich repeat kinase 2 (LRRK2) phosphorylates a subset of RAB GTPases, and their phosphorylation levels are elevated by Parkinson's disease (PD)-linked mutations of LRRK2. However, the ...The leucine-rich repeat kinase 2 (LRRK2) phosphorylates a subset of RAB GTPases, and their phosphorylation levels are elevated by Parkinson's disease (PD)-linked mutations of LRRK2. However, the precise function of the LRRK2-regulated RAB GTPase in the brain remains to be elucidated. Here, we identify RAB12 as a robust LRRK2 substrate in the mouse brain through phosphoproteomics profiling and solve the structure of RAB12-LRRK2 protein complex through Cryo-EM analysis. Mechanistically, RAB12 cooperates with LRRK2 to inhibit primary ciliogenesis and regulate centrosome homeostasis in astrocytes through enhancing the phosphorylation of RAB10 and recruiting RILPL1, while the functions of RAB12 require a direct interaction with LRRK2 and LRRK2 activity. Furthermore, the ciliary and centrosome defects caused by the PD-linked LRRK2-G2019S mutation are prevented by Rab12 deletion in astrocytes. Thus, our study reveals a physiological function of the RAB12-LRRK2 complex in regulating ciliogenesis and centrosome homeostasis. The RAB12-LRRK2 structure offers a guidance in the therapeutic development of PD by targeting the RAB12-LRRK2 interaction.
History
DepositionDec 30, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 9, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Leucine-rich repeat serine/threonine-protein kinase 2
B: Ras-related protein Rab-12
C: Leucine-rich repeat serine/threonine-protein kinase 2
D: Ras-related protein Rab-12
hetero molecules


Theoretical massNumber of molelcules
Total (without water)616,26212
Polymers613,2704
Non-polymers2,9928
Water00
1


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

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Components

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Protein , 2 types, 4 molecules ACBD

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


Mass: 286427.656 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: LRRK2, PARK8 / Production host: Homo sapiens (human)
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 Ras-related protein Rab-12


Mass: 20207.365 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RAB12 / Production host: Escherichia coli (E. coli) / References: UniProt: Q6IQ22

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Non-polymers , 4 types, 8 molecules

#3: Chemical ChemComp-GDP / GUANOSINE-5'-DIPHOSPHATE


Type: RNA linking / Mass: 443.201 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H15N5O11P2 / Comment: GDP, energy-carrying molecule*YM
#4: Chemical ChemComp-ANP / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER


Mass: 506.196 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H17N6O12P3 / Comment: AMP-PNP, energy-carrying molecule analogue*YM
#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#6: Chemical ChemComp-GNP / PHOSPHOAMINOPHOSPHONIC ACID-GUANYLATE ESTER


Mass: 522.196 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H17N6O13P3 / Feature type: SUBJECT OF INVESTIGATION
Comment: GppNHp, GMPPNP, energy-carrying molecule analogue*YM

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Details

Has ligand of interestY
Has protein modificationN

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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: Rab12-LRRK2 / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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: OTHER
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 2600 nm / Nominal defocus min: 1200 nm
Image recordingElectron dose: 77.6 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameCategory
2EPUimage acquisition
4CTFFINDCTF correction
12cryoSPARCclassification
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 77265 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00634484
ELECTRON MICROSCOPYf_angle_d0.76947270
ELECTRON MICROSCOPYf_dihedral_angle_d7.2444982
ELECTRON MICROSCOPYf_chiral_restr0.0495964
ELECTRON MICROSCOPYf_plane_restr0.0065938

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