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- PDB-6tgc: CryoEM structure of the ternary DOCK2-ELMO1-RAC1 complex. -

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Basic information

Entry
Database: PDB / ID: 6tgc
TitleCryoEM structure of the ternary DOCK2-ELMO1-RAC1 complex.
Components
  • Dedicator of cytokinesis protein 2
  • Engulfment and cell motility protein 1
  • Ras-related C3 botulinum toxin substrate 1
KeywordsSIGNALING PROTEIN / guanine nucleotide exchange factor / cytoskeleton / actin / cryoEM
Function / homology
Function and homology information


membrane raft polarization / alpha-beta T cell proliferation / myeloid dendritic cell activation involved in immune response / establishment of T cell polarity / macropinocytosis / regulation of respiratory burst / regulation of neutrophil migration / negative regulation of interleukin-23 production / localization within membrane / Activated NTRK2 signals through CDK5 ...membrane raft polarization / alpha-beta T cell proliferation / myeloid dendritic cell activation involved in immune response / establishment of T cell polarity / macropinocytosis / regulation of respiratory burst / regulation of neutrophil migration / negative regulation of interleukin-23 production / localization within membrane / Activated NTRK2 signals through CDK5 / immunological synapse formation / regulation of hydrogen peroxide metabolic process / negative regulation of receptor-mediated endocytosis / ruffle assembly / NTRK2 activates RAC1 / NADPH oxidase complex / Inactivation of CDC42 and RAC1 / cortical cytoskeleton organization / negative thymic T cell selection / respiratory burst / guanyl-nucleotide exchange factor complex / WNT5:FZD7-mediated leishmania damping / SEMA3A-Plexin repulsion signaling by inhibiting Integrin adhesion / hepatocyte growth factor receptor signaling pathway / myoblast fusion / ruffle organization / cell projection assembly / positive regulation of bicellular tight junction assembly / positive thymic T cell selection / thioesterase binding / negative regulation of fibroblast migration / regulation of stress fiber assembly / RHO GTPases activate CIT / regulation of nitric oxide biosynthetic process / regulation of lamellipodium assembly / Nef and signal transduction / sphingosine-1-phosphate receptor signaling pathway / motor neuron axon guidance / RHO GTPases activate KTN1 / PCP/CE pathway / Activation of RAC1 / MET activates RAP1 and RAC1 / DCC mediated attractive signaling / Azathioprine ADME / regulation of small GTPase mediated signal transduction / positive regulation of cell-substrate adhesion / positive regulation of neutrophil chemotaxis / Sema4D mediated inhibition of cell attachment and migration / Ephrin signaling / CD28 dependent Vav1 pathway / Wnt signaling pathway, planar cell polarity pathway / lamellipodium assembly / small GTPase-mediated signal transduction / Activation of RAC1 downstream of NMDARs / regulation of cell size / phagocytosis, engulfment / positive regulation of Rho protein signal transduction / NRAGE signals death through JNK / establishment or maintenance of cell polarity / Rho GDP-dissociation inhibitor binding / Rac protein signal transduction / RHO GTPases activate PAKs / semaphorin-plexin signaling pathway / regulation of postsynapse assembly / ficolin-1-rich granule membrane / RHOG GTPase cycle / Sema3A PAK dependent Axon repulsion / EPH-ephrin mediated repulsion of cells / RHO GTPases Activate NADPH Oxidases / anatomical structure morphogenesis / RHOA GTPase cycle / positive regulation of focal adhesion assembly / RHO GTPases Activate WASPs and WAVEs / RAC2 GTPase cycle / RHO GTPases activate IQGAPs / T cell receptor binding / membrane scission GTPase motor activity / positive regulation of phagocytosis / PTK6 Regulates RHO GTPases, RAS GTPase and MAP kinases / RHO GTPases activate PKNs / positive regulation of lamellipodium assembly / GPVI-mediated activation cascade / positive regulation of stress fiber assembly / positive regulation of microtubule polymerization / EPHB-mediated forward signaling / positive regulation of substrate adhesion-dependent cell spreading / RAC1 GTPase cycle / actin filament polymerization / positive regulation of endothelial cell migration / substrate adhesion-dependent cell spreading / GTPase activator activity / regulation of cell migration / actin filament organization / secretory granule membrane / guanyl-nucleotide exchange factor activity / small monomeric GTPase / cell-matrix adhesion / Signal transduction by L1 / VEGFR2 mediated vascular permeability / Translocation of SLC2A4 (GLUT4) to the plasma membrane
Similarity search - Function
Dedicator of cytokinesis protein 2 / 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 ...Dedicator of cytokinesis protein 2 / 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 / Dedicator of cytokinesis / C2 DOCK-type domain / DOCKER domain / Dedicator of cytokinesis, C-terminal, lobe A / Dedicator of cytokinesis, C-terminal, lobe C / DOCKER, Lobe A / DOCKER, Lobe B / DOCKER, Lobe C / DHR-2, Lobe A / C2 domain in Dock180 and Zizimin proteins / DHR-2, Lobe C / DHR-2, Lobe B / C2 DOCK-type domain profile. / DOCKER domain profile. / Pleckstrin homology domain / Variant SH3 domain / Small GTPase Rho / small GTPase Rho family profile. / C2 domain superfamily / Pleckstrin homology domain / Rho (Ras homology) subfamily of Ras-like small GTPases / Ras subfamily of RAS small GTPases / 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
Ras-related C3 botulinum toxin substrate 1 / Engulfment and cell motility protein 1 / Dedicator of cytokinesis protein 2
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsChang, L. / Yang, J. / Chang, J.H. / Zhang, Z. / Boland, A. / McLaughlin, S.H. / Abu-Thuraia, A. / Killoran, R.C. / Smith, M.J. / Cote, J.F. / Barford, D.
Funding support United Kingdom, European Union, 3items
OrganizationGrant numberCountry
Medical Research Council (United Kingdom)MC_UP_1201/6 United Kingdom
Cancer Research UKC576/A14109 United Kingdom
Marie Sklodowska-Curie Actions, FragNET ITN657725European Union
CitationJournal: Nat Commun / Year: 2020
Title: Structure of the DOCK2-ELMO1 complex provides insights into regulation of the auto-inhibited state.
Authors: Leifu Chang / Jing Yang / Chang Hwa Jo / Andreas Boland / Ziguo Zhang / Stephen H McLaughlin / Afnan Abu-Thuraia / Ryan C Killoran / Matthew J Smith / Jean-Francois Côté / David Barford /
Abstract: DOCK (dedicator of cytokinesis) proteins are multidomain guanine nucleotide exchange factors (GEFs) for RHO GTPases that regulate intracellular actin dynamics. DOCK proteins share catalytic (DOCK) ...DOCK (dedicator of cytokinesis) proteins are multidomain guanine nucleotide exchange factors (GEFs) for RHO GTPases that regulate intracellular actin dynamics. DOCK proteins share catalytic (DOCK) and membrane-associated (DOCK) domains. The structurally-related DOCK1 and DOCK2 GEFs are specific for RAC, and require ELMO (engulfment and cell motility) proteins for function. The N-terminal RAS-binding domain (RBD) of ELMO (ELMO) interacts with RHOG to modulate DOCK1/2 activity. Here, we determine the cryo-EM structures of DOCK2-ELMO1 alone, and as a ternary complex with RAC1, together with the crystal structure of a RHOG-ELMO2 complex. The binary DOCK2-ELMO1 complex adopts a closed, auto-inhibited conformation. Relief of auto-inhibition to an active, open state, due to a conformational change of the ELMO1 subunit, exposes binding sites for RAC1 on DOCK2, and RHOG and BAI GPCRs on ELMO1. Our structure explains how up-stream effectors, including DOCK2 and ELMO1 phosphorylation, destabilise the auto-inhibited state to promote an active GEF.
History
DepositionNov 15, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 29, 2020Provider: repository / Type: Initial release
Revision 1.1May 24, 2023Group: Database references / Structure summary / Category: database_2 / struct
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct.title
Revision 1.2Jul 26, 2023Group: Author supporting evidence / Other / Category: pdbx_audit_support / pdbx_database_status
Item: _pdbx_audit_support.country / _pdbx_database_status.pdb_format_compatible
Revision 1.3Jul 10, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond / em_admin / Item: _em_admin.last_update

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Assembly

Deposited unit
A: Dedicator of cytokinesis protein 2
B: Engulfment and cell motility protein 1
C: Ras-related C3 botulinum toxin substrate 1
D: Dedicator of cytokinesis protein 2
E: Engulfment and cell motility protein 1
F: Ras-related C3 botulinum toxin substrate 1


Theoretical massNumber of molelcules
Total (without water)602,5446
Polymers602,5446
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: cross-linking
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area18800 Å2
ΔGint-139 kcal/mol
Surface area210600 Å2
MethodPISA

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Components

#1: Protein Dedicator of cytokinesis protein 2


Mass: 195902.516 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: DOCK2, KIAA0209 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q92608
#2: Protein Engulfment and cell motility protein 1 / Protein ced-12 homolog


Mass: 83891.328 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ELMO1, KIAA0281 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q92556
#3: Protein Ras-related C3 botulinum toxin substrate 1 / Cell migration-inducing gene 5 protein / Ras-like protein TC25 / p21-Rac1


Mass: 21478.113 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RAC1, TC25, MIG5 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P63000, small monomeric GTPase

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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: Ternary complex of DOCK2-ELMO1-RAC1 / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.6 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHepesC8H18N2O4S1
2200 mMsodium chlorideNaCl1
SpecimenConc.: 0.2 mg/ml / Embedding 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 III / 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
Image recordingElectron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
EM imaging opticsEnergyfilter name: GIF Quantum ER / Energyfilter slit width: 20 eV
Image scansMovie frames/image: 20

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.16_3549refinement
PHENIX1.16_3549refinement
EM software
IDNameVersionCategory
1RELION1.4particle selection
2EPUimage acquisition
4GctfCTF correction
13RELION1.43D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 245763 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 538.5 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.015433882
ELECTRON MICROSCOPYf_angle_d1.789446026
ELECTRON MICROSCOPYf_chiral_restr0.07475416
ELECTRON MICROSCOPYf_plane_restr0.01065930
ELECTRON MICROSCOPYf_dihedral_angle_d15.86320486

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