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- PDB-8xm7: Cryo-EM structure of the RhoG/DOCK5/ELMO1/Rac1 complex: RhoG/DOCK... -

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Basic information

Entry
Database: PDB / ID: 8xm7
TitleCryo-EM structure of the RhoG/DOCK5/ELMO1/Rac1 complex: RhoG/DOCK5/ELMO1 focused map
Components
  • Dedicator of cytokinesis protein 5
  • Engulfment and cell motility protein 1
  • Rho-related GTP-binding protein RhoG
KeywordsSIGNALING PROTEIN / ELMO / DOCK / GEF / GTPASE / RHO / RAC
Function / homology
Function and homology information


negative regulation of vascular associated smooth muscle contraction / regulation of ruffle assembly / podosome assembly / engulfment of apoptotic cell / guanyl-nucleotide exchange factor complex / cortical cytoskeleton organization / bone remodeling / myoblast fusion / Nef and signal transduction / activation of GTPase activity ...negative regulation of vascular associated smooth muscle contraction / regulation of ruffle assembly / podosome assembly / engulfment of apoptotic cell / guanyl-nucleotide exchange factor complex / cortical cytoskeleton organization / bone remodeling / myoblast fusion / Nef and signal transduction / activation of GTPase activity / positive regulation of vascular associated smooth muscle cell migration / podosome / RHO GTPases activate KTN1 / motor neuron axon guidance / anchoring junction / establishment or maintenance of cell polarity / phagocytosis, engulfment / positive regulation of epithelial cell migration / Rac protein signal transduction / Rho protein signal transduction / RHOG GTPase cycle / PTK6 Regulates RHO GTPases, RAS GTPase and MAP kinases / positive regulation of substrate adhesion-dependent cell spreading / GPVI-mediated activation cascade / RAC1 GTPase cycle / GTPase activator activity / guanyl-nucleotide exchange factor activity / cell chemotaxis / secretory granule membrane / cell projection / cell motility / regulation of actin cytoskeleton organization / positive regulation of protein localization to plasma membrane / actin filament organization / FCGR3A-mediated phagocytosis / Regulation of actin dynamics for phagocytic cup formation / small GTPase binding / VEGFA-VEGFR2 Pathway / SH3 domain binding / Constitutive Signaling by Aberrant PI3K in Cancer / cell migration / PIP3 activates AKT signaling / Factors involved in megakaryocyte development and platelet production / regulation of cell shape / PI5P, PP2A and IER3 Regulate PI3K/AKT Signaling / cytoplasmic vesicle / actin cytoskeleton organization / cytoskeleton / focal adhesion / GTPase activity / apoptotic process / positive regulation of cell population proliferation / Neutrophil degranulation / endoplasmic reticulum membrane / GTP binding / protein kinase binding / positive regulation of DNA-templated transcription / extracellular exosome / membrane / plasma membrane / cytoplasm / cytosol
Similarity search - Function
Rho-related GTP-binding protein RhoG / : / Dedicator of cytokinesis protein 5 / Dedicator of cytokinesis, N-terminal domain / Dedicator of cytokinesis, N-terminal, subdomain 1 / DOCK N-terminus / ELMO domain / ELMO/CED-12 family / ELMO domain profile. / ELMO, armadillo-like helical domain ...Rho-related GTP-binding protein RhoG / : / Dedicator of cytokinesis protein 5 / Dedicator of cytokinesis, N-terminal domain / Dedicator of cytokinesis, N-terminal, subdomain 1 / DOCK N-terminus / ELMO domain / ELMO/CED-12 family / ELMO domain profile. / ELMO, armadillo-like helical domain / ELMO, armadillo-like helical domain / DOCKER, Lobe A / DOCKER, Lobe B / DOCKER, Lobe C / DHR-2, Lobe C / DHR-2, Lobe B / Dedicator of cytokinesis / C2 DOCK-type domain / DOCKER domain / Dedicator of cytokinesis, C-terminal, lobe A / Dedicator of cytokinesis, C-terminal, lobe C / DHR-2, Lobe A / C2 domain in Dock180 and Zizimin proteins / C2 DOCK-type domain profile. / DOCKER domain profile. / Pleckstrin homology domain / Small GTPase Rho / small GTPase Rho family profile. / Ran (Ras-related nuclear proteins) /TC4 subfamily of small GTPases / C2 domain superfamily / Pleckstrin homology domain / Rho (Ras homology) subfamily of Ras-like small GTPases / Ras subfamily of RAS small GTPases / SH3 domain / Small GTPase / Ras family / Rab subfamily of small GTPases / Src homology 3 domains / SH3-like domain superfamily / Src homology 3 (SH3) domain profile. / SH3 domain / Armadillo-like helical / Small GTP-binding protein domain / PH-like domain superfamily / Armadillo-type fold / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
GUANOSINE-5'-TRIPHOSPHATE / Rho-related GTP-binding protein RhoG / Engulfment and cell motility protein 1 / Dedicator of cytokinesis protein 5
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.91 Å
AuthorsKukimoto-Niino, M. / Katsura, K. / Ishizuka-Katsura, Y. / Mishima-Tsumagari, C. / Yonemochi, M. / Inoue, M. / Nakagawa, R. / Kaushik, R. / Zhang, K.Y.J. / Shirouzu, M.
Funding support Japan, 3items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)KAKENHI JP19K06575 Japan
Japan Society for the Promotion of Science (JSPS)KAKENHI JP22KH05551 Japan
Japan Science and TechnologyCREST JPMJCR22E3 Japan
CitationJournal: J Biol Chem / Year: 2024
Title: RhoG facilitates a conformational transition in the guanine nucleotide exchange factor complex DOCK5/ELMO1 to an open state.
Authors: Mutsuko Kukimoto-Niino / Kazushige Katsura / Yoshiko Ishizuka-Katsura / Chiemi Mishima-Tsumagari / Mayumi Yonemochi / Mio Inoue / Reiko Nakagawa / Rahul Kaushik / Kam Y J Zhang / Mikako Shirouzu /
Abstract: The dedicator of cytokinesis (DOCK)/engulfment and cell motility (ELMO) complex serves as a guanine nucleotide exchange factor (GEF) for the GTPase Rac. RhoG, another GTPase, activates the ELMO-DOCK- ...The dedicator of cytokinesis (DOCK)/engulfment and cell motility (ELMO) complex serves as a guanine nucleotide exchange factor (GEF) for the GTPase Rac. RhoG, another GTPase, activates the ELMO-DOCK-Rac pathway during engulfment and migration. Recent cryo-EM structures of the DOCK2/ELMO1 and DOCK2/ELMO1/Rac1 complexes have identified closed and open conformations that are key to understanding the autoinhibition mechanism. Nevertheless, the structural details of RhoG-mediated activation of the DOCK/ELMO complex remain elusive. Herein, we present cryo-EM structures of DOCK5/ELMO1 alone and in complex with RhoG and Rac1. The DOCK5/ELMO1 structure exhibits a closed conformation similar to that of DOCK2/ELMO1, suggesting a shared regulatory mechanism of the autoinhibitory state across DOCK-A/B subfamilies (DOCK1-5). Conversely, the RhoG/DOCK5/ELMO1/Rac1 complex adopts an open conformation that differs from that of the DOCK2/ELMO1/Rac1 complex, with RhoG binding to both ELMO1 and DOCK5. The alignment of the DOCK5 phosphatidylinositol (3,4,5)-trisphosphate binding site with the RhoG C-terminal lipidation site suggests simultaneous binding of RhoG and DOCK5/ELMO1 to the plasma membrane. Structural comparison of the apo and RhoG-bound states revealed that RhoG facilitates a closed-to-open state conformational change of DOCK5/ELMO1. Biochemical and surface plasmon resonance (SPR) assays confirm that RhoG enhances the Rac GEF activity of DOCK5/ELMO1 and increases its binding affinity for Rac1. Further analysis of structural variability underscored the conformational flexibility of the DOCK5/ELMO1/Rac1 complex core, potentially facilitating the proximity of the DOCK5 GEF domain to the plasma membrane. These findings elucidate the structural mechanism underlying the RhoG-induced allosteric activation and membrane binding of the DOCK/ELMO complex.
History
DepositionDec 27, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jun 26, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Engulfment and cell motility protein 1
B: Dedicator of cytokinesis protein 5
D: Rho-related GTP-binding protein RhoG
hetero molecules


Theoretical massNumber of molelcules
Total (without water)298,7405
Polymers298,1923
Non-polymers5472
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 Engulfment and cell motility protein 1 / Protein ced-12 homolog


Mass: 84337.719 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ELMO1, KIAA0281 / Production host: Homo sapiens (human) / References: UniProt: Q92556
#2: Protein Dedicator of cytokinesis protein 5


Mass: 191492.125 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: DOCK5 / Production host: Homo sapiens (human) / References: UniProt: Q9H7D0
#3: Protein Rho-related GTP-binding protein RhoG


Mass: 22362.359 Da / Num. of mol.: 1 / Mutation: Q61L
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RHOG, ARHG / Production host: Escherichia coli (E. coli) / References: UniProt: P84095
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE


Mass: 523.180 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: GTP, energy-carrying molecule*YM
Has ligand of interestY

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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: RhoG/DOCK5/ELMO1 complex / Type: COMPLEX / Entity ID: #1-#3 / 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
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / 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: 64000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 11976

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Processing

EM software
IDNameVersionCategory
1crYOLOparticle selection
2EPUimage acquisition
4CTFFIND4.1CTF correction
7UCSF Chimeramodel fitting
9RELION3.1initial Euler assignment
10RELION3.1final Euler assignment
11RELION3.1classification
12RELION3.13D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2483502
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.91 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 181978 / Symmetry type: POINT
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
17DPA

7dpa

17DPA1PDBexperimental model
26IE1

6ie1

16IE12PDBexperimental model
37Y4A

7y4a

17Y4A3PDBexperimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00717532
ELECTRON MICROSCOPYf_angle_d0.94823688
ELECTRON MICROSCOPYf_dihedral_angle_d11.7712300
ELECTRON MICROSCOPYf_chiral_restr0.0532658
ELECTRON MICROSCOPYf_plane_restr0.0063037

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