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- PDB-7sj4: Human Trio residues 1284-1959 in complex with Rac1 -

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Basic information

Entry
Database: PDB / ID: 7sj4
TitleHuman Trio residues 1284-1959 in complex with Rac1
Components
  • Ras-related C3 botulinum toxin substrate 1
  • Triple functional domain protein
KeywordsSIGNALING PROTEIN / guanine nucleotide exchange factor / GTPase / dbl homology / pleckstrin homology
Function / homology
Function and homology information


transmembrane receptor protein tyrosine phosphatase signaling pathway / regulation of respiratory burst / regulation of neutrophil migration / negative regulation of interleukin-23 production / localization within membrane / Activated NTRK2 signals through CDK5 / NADPH oxidase complex / negative regulation of receptor-mediated endocytosis / engulfment of apoptotic cell / regulation of hydrogen peroxide metabolic process ...transmembrane receptor protein tyrosine phosphatase signaling pathway / regulation of respiratory burst / regulation of neutrophil migration / negative regulation of interleukin-23 production / localization within membrane / Activated NTRK2 signals through CDK5 / NADPH oxidase complex / negative regulation of receptor-mediated endocytosis / engulfment of apoptotic cell / regulation of hydrogen peroxide metabolic process / ruffle assembly / NTRK2 activates RAC1 / Rho GDP-dissociation inhibitor binding / Inactivation of CDC42 and RAC1 / WNT5:FZD7-mediated leishmania damping / respiratory burst / SEMA3A-Plexin repulsion signaling by inhibiting Integrin adhesion / PCP/CE pathway / cell projection assembly / RHO GTPases activate CIT / RHO GTPases activate KTN1 / cortical cytoskeleton organization / ruffle organization / hepatocyte growth factor receptor signaling pathway / positive regulation of neutrophil chemotaxis / Azathioprine ADME / regulation of stress fiber assembly / negative regulation of fibroblast migration / sphingosine-1-phosphate receptor signaling pathway / thioesterase binding / regulation of nitric oxide biosynthetic process / Wnt signaling pathway, planar cell polarity pathway / regulation of lamellipodium assembly / Nef and signal transduction / motor neuron axon guidance / Sema4D mediated inhibition of cell attachment and migration / Activation of RAC1 / regulation of small GTPase mediated signal transduction / positive regulation of Rho protein signal transduction / Ephrin signaling / positive regulation of cell-substrate adhesion / MET activates RAP1 and RAC1 / DCC mediated attractive signaling / CD28 dependent Vav1 pathway / lamellipodium assembly / Activation of RAC1 downstream of NMDARs / semaphorin-plexin signaling pathway / NRAGE signals death through JNK / presynaptic active zone / negative regulation of fat cell differentiation / regulation of cell size / Rac protein signal transduction / DSCAM interactions / RHOJ GTPase cycle / positive regulation of lamellipodium assembly / establishment or maintenance of cell polarity / small GTPase mediated signal transduction / RHO GTPases activate PAKs / postsynaptic modulation of chemical synaptic transmission / ficolin-1-rich granule membrane / RHOA GTPase cycle / positive regulation of focal adhesion assembly / CDC42 GTPase cycle / RHOG GTPase cycle / Sema3A PAK dependent Axon repulsion / EPH-ephrin mediated repulsion of cells / RHO GTPases activate IQGAPs / anatomical structure morphogenesis / RAC3 GTPase cycle / RAC2 GTPase cycle / PTK6 Regulates RHO GTPases, RAS GTPase and MAP kinases / RHO GTPases Activate WASPs and WAVEs / RHO GTPases Activate NADPH Oxidases / extrinsic component of membrane / RHO GTPases activate PKNs / localization / regulation of actin cytoskeleton organization / neuron projection morphogenesis / GPVI-mediated activation cascade / EPHB-mediated forward signaling / positive regulation of microtubule polymerization / G protein activity / positive regulation of stress fiber assembly / positive regulation of substrate adhesion-dependent cell spreading / regulation of cell migration / RAC1 GTPase cycle / neuron migration / actin filament polymerization / small monomeric GTPase / guanyl-nucleotide exchange factor activity / cell motility / actin filament organization / cell-matrix adhesion / substrate adhesion-dependent cell spreading / secretory granule membrane / RHO GTPases Activate Formins / VEGFR2 mediated vascular permeability / Translocation of SLC2A4 (GLUT4) to the plasma membrane / positive regulation of endothelial cell migration / Signal transduction by L1
Similarity search - Function
Rho GDP/GTP exchange factor Kalirin/TRIO / CRAL/TRIO domain / CRAL-TRIO lipid binding domain profile. / Domain in homologues of a S. cerevisiae phosphatidylinositol transfer protein (Sec14p) / CRAL-TRIO lipid binding domain / CRAL-TRIO lipid binding domain superfamily / Spectrin repeat / Spectrin repeat / small GTPase Rho family profile. / Spectrin/alpha-actinin ...Rho GDP/GTP exchange factor Kalirin/TRIO / CRAL/TRIO domain / CRAL-TRIO lipid binding domain profile. / Domain in homologues of a S. cerevisiae phosphatidylinositol transfer protein (Sec14p) / CRAL-TRIO lipid binding domain / CRAL-TRIO lipid binding domain superfamily / Spectrin repeat / Spectrin repeat / small GTPase Rho family profile. / Spectrin/alpha-actinin / Spectrin repeats / Small GTPase Rho / Dbl homology (DH) domain superfamily / RhoGEF domain / Guanine nucleotide exchange factor for Rho/Rac/Cdc42-like GTPases / Dbl homology (DH) domain / Dbl homology (DH) domain profile. / Immunoglobulin I-set / Immunoglobulin I-set domain / PH domain / PH domain profile. / Rho (Ras homology) subfamily of Ras-like small GTPases / Pleckstrin homology domain. / Pleckstrin homology domain / Small GTPase / Ras family / Immunoglobulin subtype 2 / Immunoglobulin C-2 Type / SH3 domain / Src homology 3 domains / SH3-like domain superfamily / Src homology 3 (SH3) domain profile. / SH3 domain / Small GTP-binding protein domain / PH-like domain superfamily / Immunoglobulin subtype / Immunoglobulin / Ig-like domain profile. / Immunoglobulin-like domain / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / Immunoglobulin-like domain superfamily / Protein kinase domain / Serine/Threonine protein kinases, catalytic domain / Immunoglobulin-like fold / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Triple functional domain protein / Ras-related C3 botulinum toxin substrate 1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.86 Å
AuthorsChen, C.-L. / Ravala, S.K. / Bandekar, S.J. / Cash, J. / Tesmer, J.J.G.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)R01CA221289 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)F31CA224804 United States
CitationJournal: J Biol Chem / Year: 2022
Title: Structural/functional studies of Trio provide insights into its configuration and show that conserved linker elements enhance its activity for Rac1.
Authors: Sumit J Bandekar / Chun-Liang Chen / Sandeep K Ravala / Jennifer N Cash / Larisa V Avramova / Mariya V Zhalnina / J Silvio Gutkind / Sheng Li / John J G Tesmer /
Abstract: Trio is a large and highly conserved metazoan signaling scaffold that contains two Dbl family guanine nucleotide exchange factor (GEF) modules, TrioN and TrioC, selective for Rac and RhoA GTPases, ...Trio is a large and highly conserved metazoan signaling scaffold that contains two Dbl family guanine nucleotide exchange factor (GEF) modules, TrioN and TrioC, selective for Rac and RhoA GTPases, respectively. The GEF activities of TrioN and TrioC are implicated in several cancers, especially uveal melanoma. However, little is known about how these modules operate in the context of larger fragments of Trio. Here we show via negative stain electron microscopy that the N-terminal region of Trio is extended and could thus serve as a rigid spacer between the N-terminal putative lipid-binding domain and TrioN, whereas the C-terminal half of Trio seems globular. We found that regions C-terminal to TrioN enhance its Rac1 GEF activity and thus could play a regulatory role. We went on to characterize a minimal, well-behaved Trio fragment with enhanced activity, Trio, in complex with Rac1 using cryo-electron microscopy and hydrogen-deuterium exchange mass spectrometry and found that the region conferring enhanced activity is disordered. Deletion of two different strongly conserved motifs in this region eliminated this enhancement, suggesting that they form transient intramolecular interactions that promote GEF activity. Because Dbl family RhoGEF modules have been challenging to directly target with small molecules, characterization of accessory Trio domains such as these may provide alternate routes for the development of therapeutics that inhibit Trio activity in human cancer.
History
DepositionOct 15, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 6, 2022Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2022Group: Database references / Category: citation / citation_author
Item: _citation.page_first / _citation.page_last ..._citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Aug 24, 2022Group: Database references / Category: citation / Item: _citation.journal_volume

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Triple functional domain protein
B: Ras-related C3 botulinum toxin substrate 1


Theoretical massNumber of molelcules
Total (without water)97,4042
Polymers97,4042
Non-polymers00
Water43224
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Triple functional domain protein / PTPRF-interacting protein


Mass: 75592.969 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TRIO / Production host: Escherichia coli (E. coli) / Strain (production host): Rosetta (DE3) pLysS
References: UniProt: O75962, non-specific serine/threonine protein kinase
#2: Protein Ras-related C3 botulinum toxin substrate 1 / Cell migration-inducing gene 5 protein / Ras-like protein TC25 / p21-Rac1


Mass: 21811.453 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RAC1, TC25, MIG5 / Production host: Escherichia coli (E. coli) / Strain (production host): Rosetta (DE3) pLysS / References: UniProt: P63000
#3: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 24 / Source method: isolated from a natural source / Formula: H2O

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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: Human Trio residues 1284-1959 in complex with Rac1 / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: Rosetta (DE3) pLysS
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
120 mM(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)C8H18N2O4S1
2200 mMSodium ChlorideNaClSodium chloride1
32 mMdithiothreitolC4H10O2S21
SpecimenConc.: 0.25 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: Blot force 10

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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 FIELDBright-field microscopy / Nominal magnification: 81000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Residual tilt: 0.01 mradians
Image recordingAverage exposure time: 3.12 sec. / Electron dose: 55 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of real images: 2817
Details: 3514 movies were initially collected. After motion correction and CTF estimation, 697 micrographs were rejected due to poor CTF fitting.
Image scansWidth: 11520 / Height: 8184 / Movie frames/image: 40

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Processing

SoftwareName: PHENIX / Version: 1.20_4459: / Classification: refinement
EM software
IDNameVersionCategory
1cryoSPARC3.3.1particle selection
2Leginonimage acquisition
4cryoSPARC3.3.1CTF correction
7UCSF Chimera1.15model fitting
8Coot9.6model fitting
10Coot9.6model refinement
11PHENIX1.20-4459model refinement
12cryoSPARC3.3.1initial Euler assignment
13cryoSPARC3.3.1final Euler assignment
14cryoSPARC3.3.1classification
15cryoSPARC3.3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.86 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 922202 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL / Target criteria: correlation coefficient
Details: The initial model was generated from the pdb structure (2NZ8) using the SWISS-MODEL server and rigid-body fitted using Chimera. Several runs of structure refinement were done using the coot ...Details: The initial model was generated from the pdb structure (2NZ8) using the SWISS-MODEL server and rigid-body fitted using Chimera. Several runs of structure refinement were done using the coot and phenix real-space refinement.
Atomic model building
IDPDB-IDPdb chain-ID 3D fitting-ID
12NZ8A1
22NZ8B1
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0023964
ELECTRON MICROSCOPYf_angle_d0.4145350
ELECTRON MICROSCOPYf_dihedral_angle_d9.5631502
ELECTRON MICROSCOPYf_chiral_restr0.041593
ELECTRON MICROSCOPYf_plane_restr0.003681

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