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- PDB-7use: Cryo-EM structure of WAVE regulatory complex with Rac1 bound on b... -

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Basic information

Entry
Database: PDB / ID: 7use
TitleCryo-EM structure of WAVE regulatory complex with Rac1 bound on both A and D site
Components
  • (Ras-related C3 botulinum toxin substrate ...) x 2
  • Abl interactor 2
  • Cytoplasmic FMR1-interacting protein 1
  • Nck-associated protein 1
  • Protein BRICK1
  • Wiskott-Aldrich syndrome protein family member 1
KeywordsCELL INVASION / actin regulator / GTPase binding protein / cytoskeletal regulator
Function / homology
Function and homology information


dendritic transport of mitochondrion / SCAR complex / positive regulation of neurotrophin TRK receptor signaling pathway / lamellipodium morphogenesis / positive regulation of Arp2/3 complex-mediated actin nucleation / regulation of actin polymerization or depolymerization / Arp2/3 complex binding / dendrite extension / regulation of respiratory burst / positive regulation of ovarian follicle development ...dendritic transport of mitochondrion / SCAR complex / positive regulation of neurotrophin TRK receptor signaling pathway / lamellipodium morphogenesis / positive regulation of Arp2/3 complex-mediated actin nucleation / regulation of actin polymerization or depolymerization / Arp2/3 complex binding / dendrite extension / regulation of respiratory burst / positive regulation of ovarian follicle development / regulation of neutrophil migration / regulation of translation at postsynapse, modulating synaptic transmission / negative regulation of interleukin-23 production / Activated NTRK2 signals through CDK5 / negative regulation of fibroblast migration / ruffle assembly / localization within membrane / modification of postsynaptic actin cytoskeleton / kinocilium / regulation of cell adhesion involved in heart morphogenesis / NTRK2 activates RAC1 / NADPH oxidase complex / filopodium tip / Inactivation of CDC42 and RAC1 / regulation of hydrogen peroxide metabolic process / Rac protein signal transduction / engulfment of apoptotic cell / regulation of actin filament polymerization / axon extension / WNT5:FZD7-mediated leishmania damping / superoxide anion generation / cortical cytoskeleton organization / SEMA3A-Plexin repulsion signaling by inhibiting Integrin adhesion / cell projection assembly / regulation of modification of postsynaptic actin cytoskeleton / respiratory burst / motor neuron axon guidance / ruffle organization / midbrain dopaminergic neuron differentiation / positive regulation of bicellular tight junction assembly / GTP-dependent protein binding / RNA 7-methylguanosine cap binding / thioesterase binding / regulation of stress fiber assembly / regulation of lamellipodium assembly / RHO GTPases activate CIT / regulation of nitric oxide biosynthetic process / Nef and signal transduction / Activation of RAC1 / PCP/CE pathway / sphingosine-1-phosphate receptor signaling pathway / hepatocyte growth factor receptor signaling pathway / RHO GTPases activate KTN1 / forebrain development / cortical actin cytoskeleton organization / positive regulation of actin filament polymerization / DCC mediated attractive signaling / MET activates RAP1 and RAC1 / Azathioprine ADME / Sema4D mediated inhibition of cell attachment and migration / lamellipodium assembly / CD28 dependent Vav1 pathway / Ephrin signaling / positive regulation of neutrophil chemotaxis / positive regulation of ruffle assembly / Wnt signaling pathway, planar cell polarity pathway / regulation of receptor signaling pathway via JAK-STAT / NRAGE signals death through JNK / Rho GDP-dissociation inhibitor binding / Activation of RAC1 downstream of NMDARs / small GTPase-mediated signal transduction / protein kinase A regulatory subunit binding / positive regulation of Rho protein signal transduction / filamentous actin / pericentriolar material / protein kinase A binding / lamellipodium membrane / establishment or maintenance of cell polarity / RHO GTPases activate PAKs / semaphorin-plexin signaling pathway / RHOG GTPase cycle / Sema3A PAK dependent Axon repulsion / ficolin-1-rich granule membrane / EPH-ephrin mediated repulsion of cells / regulation of postsynapse assembly / RAC3 GTPase cycle / positive regulation of focal adhesion assembly / RAC2 GTPase cycle / RHO GTPases Activate NADPH Oxidases / positive regulation of substrate adhesion-dependent cell spreading / anatomical structure morphogenesis / positive regulation of stress fiber assembly / regulation of synaptic vesicle endocytosis / RHO GTPases Activate WASPs and WAVEs / substrate adhesion-dependent cell spreading / positive regulation of lamellipodium assembly / cell-matrix adhesion / cell morphogenesis / RHO GTPases activate IQGAPs / GPVI-mediated activation cascade
Similarity search - Function
Cytoplasmic FMR1-interacting / Nck-associated protein 1 / Cytoplasmic Fragile-X interacting family / Nck-associated protein 1 / Protein BRICK1 / Abl interactor 2, SH3 domain / : / SCAR helical domain / Abl interactor protein / CYRIA/CYRIB, Rac1 binding domain ...Cytoplasmic FMR1-interacting / Nck-associated protein 1 / Cytoplasmic Fragile-X interacting family / Nck-associated protein 1 / Protein BRICK1 / Abl interactor 2, SH3 domain / : / SCAR helical domain / Abl interactor protein / CYRIA/CYRIB, Rac1 binding domain / Abl-interactor, homeo-domain homologous domain / SCAR/WAVE family / ABI family / CYRIA/CYRIB Rac1 binding domain / Abl-interactor HHR / Wiskott Aldrich syndrome homology region 2 / WH2 motif / WH2 domain / WH2 domain profile. / t-SNARE coiled-coil homology domain profile. / Target SNARE coiled-coil homology domain / Small GTPase Rho / Small GTPase Rho domain profile. / SH3 domain / Rho (Ras homology) subfamily of Ras-like small GTPases / Ras subfamily of RAS small GTPases / Small GTPase / Ras family / Src homology 3 domains / Rab subfamily of small GTPases / SH3-like domain superfamily / Src homology 3 (SH3) domain profile. / SH3 domain / Small GTP-binding protein domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
PHOSPHOAMINOPHOSPHONIC ACID-GUANYLATE ESTER / GUANOSINE-5'-TRIPHOSPHATE / Abl interactor 2 / Ras-related C3 botulinum toxin substrate 1 / Cytoplasmic FMR1-interacting protein 1 / Protein BRICK1 / Actin-binding protein WASF1 / Nck-associated protein 1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsDing, B. / Yang, S. / Chen, B. / Chowdhury, S.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS) United States
CitationJournal: Nat Commun / Year: 2022
Title: Structures reveal a key mechanism of WAVE regulatory complex activation by Rac1 GTPase.
Authors: Bojian Ding / Sheng Yang / Matthias Schaks / Yijun Liu / Abbigale J Brown / Klemens Rottner / Saikat Chowdhury / Baoyu Chen /
Abstract: The Rho-family GTPase Rac1 activates the WAVE regulatory complex (WRC) to drive Arp2/3 complex-mediated actin polymerization in many essential processes. Rac1 binds to WRC at two distinct sites-the ...The Rho-family GTPase Rac1 activates the WAVE regulatory complex (WRC) to drive Arp2/3 complex-mediated actin polymerization in many essential processes. Rac1 binds to WRC at two distinct sites-the A and D sites. Precisely how Rac1 binds and how the binding triggers WRC activation remain unknown. Here we report WRC structures by itself, and when bound to single or double Rac1 molecules, at ~3 Å resolutions by cryogenic-electron microscopy. The structures reveal that Rac1 binds to the two sites by distinct mechanisms, and binding to the A site, but not the D site, drives WRC activation. Activation involves a series of unique conformational changes leading to the release of sequestered WCA (WH2-central-acidic) polypeptide, which stimulates the Arp2/3 complex to polymerize actin. Together with biochemical and cellular analyses, the structures provide a novel mechanistic understanding of how the Rac1-WRC-Arp2/3-actin signaling axis is regulated in diverse biological processes and diseases.
History
DepositionApr 25, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 21, 2022Provider: repository / Type: Initial release
Revision 1.1Nov 9, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Jun 12, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Cytoplasmic FMR1-interacting protein 1
B: Nck-associated protein 1
C: Wiskott-Aldrich syndrome protein family member 1
D: Protein BRICK1
E: Abl interactor 2
F: Ras-related C3 botulinum toxin substrate 1
G: Ras-related C3 botulinum toxin substrate 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)381,24211
Polymers380,1487
Non-polymers1,0944
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 5 types, 5 molecules ABCDE

#1: Protein Cytoplasmic FMR1-interacting protein 1 / Specifically Rac1-associated protein 1 / Sra-1 / p140sra-1


Mass: 145363.750 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: This construct contains two additional uncleaved residues "GA" in the N terminus from the construct design and purification procedure. Densities for these residues are not observed in the ...Details: This construct contains two additional uncleaved residues "GA" in the N terminus from the construct design and purification procedure. Densities for these residues are not observed in the map and were not included in the sample sequence to avoid numbering shifts.
Source: (gene. exp.) Homo sapiens (human) / Gene: CYFIP1, KIAA0068 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q7L576
#2: Protein Nck-associated protein 1 / NAP 1 / Membrane-associated protein HEM-2 / p125Nap1


Mass: 128940.727 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: This construct contains two additional uncleaved residues "GA" in the N terminus from the construct design and purification procedure. Densities for these residues are not observed in the ...Details: This construct contains two additional uncleaved residues "GA" in the N terminus from the construct design and purification procedure. Densities for these residues are not observed in the map and were not included in the sample sequence to avoid numbering shifts.
Source: (gene. exp.) Homo sapiens (human) / Gene: NCKAP1, HEM2, KIAA0587, NAP1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q9Y2A7
#3: Protein Wiskott-Aldrich syndrome protein family member 1 / WASP family protein member 1 / Protein WAVE-1 / Verprolin homology domain-containing protein 1


Mass: 37009.406 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Residues 231-248 are inserted as a flexible linker sequence. This construct contains two additional uncleaved residues "GA" in the N terminus from the construct design and purification ...Details: Residues 231-248 are inserted as a flexible linker sequence. This construct contains two additional uncleaved residues "GA" in the N terminus from the construct design and purification procedure. Densities for these residues are not observed in the map and were not included in the sample sequence to avoid numbering shifts.
Source: (gene. exp.) Homo sapiens (human) / Gene: WASF1, KIAA0269, SCAR1, WAVE1 / Production host: Escherichia coli (E. coli) / References: UniProt: Q92558
#4: Protein Protein BRICK1 / BRK1


Mass: 8756.915 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: This construct contains uncleaved residues "GHMGAA" in the N terminus from the construct design and purification procedure. Densities for the residues are not observed in the map and were ...Details: This construct contains uncleaved residues "GHMGAA" in the N terminus from the construct design and purification procedure. Densities for the residues are not observed in the map and were not included in the sample sequence to avoid numbering shifts.
Source: (gene. exp.) Homo sapiens (human) / Gene: BRK1, C3orf10, HSPC300, MDS027 / Production host: Escherichia coli (E. coli) / References: UniProt: Q8WUW1
#5: Protein Abl interactor 2


Mass: 18041.482 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: The sequence only contains residues 1-158. Also, there are two additional uncleaved residues "GH" in the N terminus from the construct design and purification procedure. Densities for these ...Details: The sequence only contains residues 1-158. Also, there are two additional uncleaved residues "GH" in the N terminus from the construct design and purification procedure. Densities for these residues are not observed in the map and were not included in the sample sequence to avoid numbering shifts.
Source: (gene. exp.) Homo sapiens (human) / Gene: ABI2 / Production host: Escherichia coli (E. coli) / References: UniProt: E9PEZ7

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Ras-related C3 botulinum toxin substrate ... , 2 types, 2 molecules FG

#6: Protein Ras-related C3 botulinum toxin substrate 1 / Cell migration-inducing gene 5 protein / Ras-like protein TC25 / p21-Rac1


Mass: 21025.459 Da / Num. of mol.: 1 / Mutation: P29S
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
#7: Protein Ras-related C3 botulinum toxin substrate 1 / Cell migration-inducing gene 5 protein / Ras-like protein TC25 / p21-Rac1


Mass: 21010.486 Da / Num. of mol.: 1 / Mutation: P29S, Q61L
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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Non-polymers , 3 types, 4 molecules

#8: Chemical ChemComp-GNP / PHOSPHOAMINOPHOSPHONIC ACID-GUANYLATE ESTER


Mass: 522.196 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H17N6O13P3 / Feature type: SUBJECT OF INVESTIGATION
Comment: GppNHp, GMPPNP, energy-carrying molecule analogue*YM
#9: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#10: 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

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Details

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: WAVE regulatory complex with Rac1 bound to A and D sites
Type: COMPLEX / Entity ID: #1-#7 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.38 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Trichoplusia ni (cabbage looper)7111
31Escherichia coli (E. coli)562
Buffer solutionpH: 7
SpecimenConc.: 0.15 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 15 mA / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 95 % / 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
Details: Data were collected by shifting the stage to target exposure positions.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 120000 X / Nominal defocus max: 1200 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 40 sec. / Electron dose: 41.34 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1285
Details: Each micrograph was acquired as dose-fractionated movies consisting of 62 frames per movie.
Image scansSampling size: 14 µm / Width: 4096 / Height: 4096

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Processing

EM software
IDNameVersionCategory
2EPU2.1image acquisition
4cryoSPARC2CTF correction
7NAMDmodel fitting
8UCSF Chimeramodel fitting
10PHENIXmodel refinement
12cryoSPARC2final Euler assignment
13cryoSPARC2classification
14cryoSPARC23D reconstruction
CTF correctionDetails: Particles were CTF-corrected during projection matching and back projection
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 666417
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 139296 / Algorithm: BACK PROJECTION / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
13P8C

3p8c

13P8C1PDBexperimental model
23SBD

3sbd

13SBD2PDBexperimental model

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