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- PDB-8r4b: Roco protein from C. tepidum in the GTP state bound to the activa... -

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Basic information

Entry
Database: PDB / ID: 8r4b
TitleRoco protein from C. tepidum in the GTP state bound to the activating Nanobodies NbRoco1 and NbRoco2
Components
  • NbRoco1
  • NbRoco2
  • Rab family protein
KeywordsHYDROLASE / GTPase / Nanobody / Parkinson's Disease / Allostery activator
Function / homology
Function and homology information


GTP binding / identical protein binding
Similarity search - Function
: / C-terminal of Roc (COR) domain / C-terminal of Roc, COR, domain / Ras of Complex, Roc, domain of DAPkinase / Leucine rich repeat 4 / Leucine Rich repeats (2 copies) / Roc domain profile. / Roc domain / Leucine rich repeat, ribonuclease inhibitor type / Leucine Rich repeat ...: / C-terminal of Roc (COR) domain / C-terminal of Roc, COR, domain / Ras of Complex, Roc, domain of DAPkinase / Leucine rich repeat 4 / Leucine Rich repeats (2 copies) / Roc domain profile. / Roc domain / Leucine rich repeat, ribonuclease inhibitor type / Leucine Rich repeat / Leucine-rich repeats, bacterial type / Leucine-rich repeat, SDS22-like subfamily / 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 / Small GTP-binding protein domain / Winged helix-like DNA-binding domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
5'-GUANOSINE-DIPHOSPHATE-MONOTHIOPHOSPHATE / Rab family protein
Similarity search - Component
Biological speciesChlorobaculum tepidum (bacteria)
Lama glama (llama)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.88 Å
AuthorsGalicia, C. / Versees, W.
Funding support Belgium, 3items
OrganizationGrant numberCountry
Research Foundation - Flanders (FWO)G005219N Belgium
Research Foundation - Flanders (FWO)G003322N Belgium
Vrije Universiteit BrusselSRP50 Belgium
CitationJournal: Elife / Year: 2024
Title: Structural insights into the GTP-driven monomerization and activation of a bacterial LRRK2 homolog using allosteric nanobodies.
Authors: Christian Galicia / Giambattista Guaitoli / Marcus Fislage / Christian Johannes Gloeckner / Wim Versées /
Abstract: Roco proteins entered the limelight after mutations in human LRRK2 were identified as a major cause of familial Parkinson's disease. LRRK2 is a large and complex protein combining a GTPase and ...Roco proteins entered the limelight after mutations in human LRRK2 were identified as a major cause of familial Parkinson's disease. LRRK2 is a large and complex protein combining a GTPase and protein kinase activity, and disease mutations increase the kinase activity, while presumably decreasing the GTPase activity. Although a cross-communication between both catalytic activities has been suggested, the underlying mechanisms and the regulatory role of the GTPase domain remain unknown. Several structures of LRRK2 have been reported, but structures of Roco proteins in their activated GTP-bound state are lacking. Here, we use single-particle cryo-electron microscopy to solve the structure of a bacterial Roco protein (CtRoco) in its GTP-bound state, aided by two conformation-specific nanobodies: Nb and Nb. This structure presents CtRoco in an active monomeric state, featuring a very large GTP-induced conformational change using the LRR-Roc linker as a hinge. Furthermore, this structure shows how Nb and Nb collaborate to activate CtRoco in an allosteric way. Altogether, our data provide important new insights into the activation mechanism of Roco proteins, with relevance to LRRK2 regulation, and suggest new routes for the allosteric modulation of their GTPase activity.
History
DepositionNov 13, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 1, 2024Provider: repository / Type: Initial release
Revision 1.1May 8, 2024Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.pdbx_database_id_PubMed ..._citation.journal_volume / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID
Revision 1.2Nov 20, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Rab family protein
B: NbRoco1
C: NbRoco2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)154,2404
Polymers153,7013
Non-polymers5391
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Rab family protein


Mass: 124313.453 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: CtRoco bound to GTPgammaS / Source: (gene. exp.) Chlorobaculum tepidum (bacteria) / Gene: CT1526 / Production host: Escherichia coli (E. coli) / References: UniProt: Q8KC98
#2: Protein NbRoco1


Mass: 15206.718 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Production host: Escherichia coli (E. coli)
#3: Antibody NbRoco2


Mass: 14180.744 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Production host: Escherichia coli (E. coli)
#4: Chemical ChemComp-GSP / 5'-GUANOSINE-DIPHOSPHATE-MONOTHIOPHOSPHATE


Mass: 539.246 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O13P3S / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
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: CtRoco in the active GTP state bound to the two activating Nanobodies NbRoco1 and NbRoco2
Type: COMPLEX / Entity ID: #1-#3 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.15 MDa / Experimental value: NO
Source (natural)Organism: Chlorobaculum tepidum (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenConc.: 0.08 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE / Humidity: 90 %

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Electron microscopy imaging

MicroscopyModel: JEOL CRYO ARM 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 60000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm / Cs: 2.55 mm / C2 aperture diameter: 100 µm
Image recordingElectron dose: 63 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: cryoSPARC / Category: 3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.88 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 193185 / Symmetry type: POINT

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