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- PDB-7usd: Cryo-EM structure of D-site Rac1-bound WAVE Regulatory Complex -

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Basic information

Entry
Database: PDB / ID: 7usd
TitleCryo-EM structure of D-site Rac1-bound WAVE Regulatory Complex
Components
  • Abl interactor 2
  • Cytoplasmic FMR1-interacting protein 1
  • Nck-associated protein 1
  • Protein BRICK1
  • Ras-related C3 botulinum toxin substrate 1
  • Wiskott-Aldrich syndrome protein family member 1
KeywordsCELL INVASION / actin regulator / GTPase binding protein / cytoskeletal regulator
Function / homology
Function and homology information


peripheral region of growth cone / negative regulation of synaptic vesicle recycling / SCAR complex / positive regulation of neurotrophin TRK receptor signaling pathway / lamellipodium morphogenesis / positive regulation of Arp2/3 complex-mediated actin nucleation / Arp2/3 complex binding / modification of synaptic structure / regulation of actin polymerization or depolymerization / central region of growth cone ...peripheral region of growth cone / negative regulation of synaptic vesicle recycling / SCAR complex / positive regulation of neurotrophin TRK receptor signaling pathway / lamellipodium morphogenesis / positive regulation of Arp2/3 complex-mediated actin nucleation / Arp2/3 complex binding / modification of synaptic structure / regulation of actin polymerization or depolymerization / central region of growth cone / modification of postsynaptic actin cytoskeleton / regulation of respiratory burst / dendrite extension / regulation of neutrophil migration / regulation of translation at postsynapse, modulating synaptic transmission / localization within membrane / negative regulation of interleukin-23 production / Activated NTRK2 signals through CDK5 / negative regulation of receptor-mediated endocytosis / filopodium tip / regulation of hydrogen peroxide metabolic process / ruffle assembly / NTRK2 activates RAC1 / engulfment of apoptotic cell / Inactivation of CDC42 and RAC1 / NADPH oxidase complex / respiratory burst / regulation of modification of postsynaptic actin cytoskeleton / cortical cytoskeleton organization / WNT5:FZD7-mediated leishmania damping / regulation of actin filament polymerization / SEMA3A-Plexin repulsion signaling by inhibiting Integrin adhesion / hepatocyte growth factor receptor signaling pathway / RNA 7-methylguanosine cap binding / ruffle organization / Nef and signal transduction / regulation of stress fiber assembly / thioesterase binding / cell projection assembly / negative regulation of fibroblast migration / RHO GTPases activate CIT / sphingosine-1-phosphate receptor signaling pathway / axon extension / RHO GTPases activate KTN1 / regulation of nitric oxide biosynthetic process / motor neuron axon guidance / PCP/CE pathway / Activation of RAC1 / positive regulation of neutrophil chemotaxis / regulation of lamellipodium assembly / positive regulation of ruffle assembly / Azathioprine ADME / MET activates RAP1 and RAC1 / DCC mediated attractive signaling / positive regulation of cell-substrate adhesion / positive regulation of dendrite development / Sema4D mediated inhibition of cell attachment and migration / CD28 dependent Vav1 pathway / Ephrin signaling / Wnt signaling pathway, planar cell polarity pathway / lamellipodium assembly / regulation of myelination / cortical actin cytoskeleton organization / protein kinase A binding / positive regulation of actin filament polymerization / NRAGE signals death through JNK / positive regulation of Rho protein signal transduction / small GTPase-mediated signal transduction / regulation of cell size / Rho GDP-dissociation inhibitor binding / Activation of RAC1 downstream of NMDARs / establishment or maintenance of cell polarity / Rac protein signal transduction / protein kinase A regulatory subunit binding / dendritic growth cone / filamentous actin / RHO GTPases activate PAKs / positive regulation of focal adhesion assembly / lamellipodium membrane / Sema3A PAK dependent Axon repulsion / semaphorin-plexin signaling pathway / excitatory synapse / ficolin-1-rich granule membrane / RHOG GTPase cycle / RHO GTPases Activate NADPH Oxidases / EPH-ephrin mediated repulsion of cells / anatomical structure morphogenesis / RAC2 GTPase cycle / RHO GTPases Activate WASPs and WAVEs / RAC3 GTPase cycle / RHO GTPases activate IQGAPs / positive regulation of axon extension / response to electrical stimulus / positive regulation of lamellipodium assembly / axonal growth cone / PTK6 Regulates RHO GTPases, RAS GTPase and MAP kinases / positive regulation of substrate adhesion-dependent cell spreading / RHO GTPases activate PKNs / translation repressor activity / 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 / CYRIA/CYRIB, Rac1 binding domain / Abl-interactor, homeo-domain homologous domain / SCAR/WAVE family / ABI family ...Cytoplasmic FMR1-interacting / Nck-associated protein 1 / Cytoplasmic Fragile-X interacting family / Nck-associated protein 1 / Protein BRICK1 / Abl interactor 2, SH3 domain / 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 family profile. / SH3 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 / Small GTP-binding protein domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
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
hetero molecules


Theoretical massNumber of molelcules
Total (without water)359,6708
Polymers359,1236
Non-polymers5472
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 , 6 types, 6 molecules ABCDEF

#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
#6: 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 , 2 types, 2 molecules

#7: 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
#8: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION

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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 D-site / Type: COMPLEX / Entity ID: #1-#6 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.36 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.: 0.41 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 30 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: 1000 nm / Nominal defocus min: 500 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: 45.27 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2512
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
4GctfCTF correction
7NAMDmodel fitting
8UCSF Chimeramodel fitting
10PHENIXmodel refinement
12RELION3.1 betafinal Euler assignment
13RELION3.1 betaclassification
14RELION3.1 beta3D reconstruction
CTF correctionDetails: Particles were CTF-corrected during projection matching and back projection
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1765193
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 87810 / 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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