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- EMDB-25153: Human Trio residues 1284-1959 in complex with Rac1 -

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Basic information

Entry
Database: EMDB / ID: EMD-25153
TitleHuman Trio residues 1284-1959 in complex with Rac1
Map dataTrioN-Rac1 complex
Sample
  • Complex: Human Trio residues 1284-1959 in complex with Rac1
    • Protein or peptide: Triple functional domain protein
    • Protein or peptide: Ras-related C3 botulinum toxin substrate 1
  • Ligand: water
Keywordsguanine nucleotide exchange factor / GTPase / dbl homology / pleckstrin homology / signaling protein
Function / homology
Function and homology information


cell surface receptor protein tyrosine phosphatase signaling pathway / embryonic olfactory bulb interneuron precursor migration / anatomical structure arrangement / regulation of ERK5 cascade / angiotensin-activated signaling pathway involved in heart process / positive regulation of ovarian follicle development / cerebral cortex GABAergic interneuron development / regulation of respiratory burst / auditory receptor cell morphogenesis / cerebral cortex radially oriented cell migration ...cell surface receptor protein tyrosine phosphatase signaling pathway / embryonic olfactory bulb interneuron precursor migration / anatomical structure arrangement / regulation of ERK5 cascade / angiotensin-activated signaling pathway involved in heart process / positive regulation of ovarian follicle development / cerebral cortex GABAergic interneuron development / regulation of respiratory burst / auditory receptor cell morphogenesis / cerebral cortex radially oriented cell migration / erythrocyte enucleation / regulation of neutrophil migration / negative regulation of interleukin-23 production / localization within membrane / Activated NTRK2 signals through CDK5 / regulation of hydrogen peroxide metabolic process / interneuron migration / kinocilium / regulation of cell adhesion involved in heart morphogenesis / negative regulation of receptor-mediated endocytosis / engulfment of apoptotic cell / ruffle assembly / NTRK2 activates RAC1 / NADPH oxidase complex / Inactivation of CDC42 and RAC1 / cochlea morphogenesis / regulation of neuron maturation / respiratory burst / WNT5:FZD7-mediated leishmania damping / cortical cytoskeleton organization / SEMA3A-Plexin repulsion signaling by inhibiting Integrin adhesion / positive regulation of skeletal muscle acetylcholine-gated channel clustering / hepatocyte growth factor receptor signaling pathway / GTP-dependent protein binding / midbrain dopaminergic neuron differentiation / ruffle organization / epithelial cell morphogenesis / cell projection assembly / positive regulation of bicellular tight junction assembly / regulation of lamellipodium assembly / thioesterase binding / regulation of stress fiber assembly / regulation of neuron migration / negative regulation of fibroblast migration / RHO GTPases activate CIT / cell-cell junction organization / sphingosine-1-phosphate receptor signaling pathway / Nef and signal transduction / motor neuron axon guidance / PCP/CE pathway / RHO GTPases activate KTN1 / Activation of RAC1 / MET activates RAP1 and RAC1 / positive regulation of neutrophil chemotaxis / regulation of nitric oxide biosynthetic process / DCC mediated attractive signaling / Azathioprine ADME / regulation of small GTPase mediated signal transduction / Sema4D mediated inhibition of cell attachment and migration / hyperosmotic response / positive regulation of cell-substrate adhesion / Ephrin signaling / CD28 dependent Vav1 pathway / presynaptic active zone / positive regulation of ruffle assembly / superoxide anion generation / Wnt signaling pathway, planar cell polarity pathway / lamellipodium assembly / extrinsic component of membrane / regulation of receptor signaling pathway via JAK-STAT / Activation of RAC1 downstream of NMDARs / small GTPase-mediated signal transduction / NRAGE signals death through JNK / dendrite morphogenesis / negative regulation of fat cell differentiation / regulation of cell size / Rho GDP-dissociation inhibitor binding / positive regulation of Rho protein signal transduction / establishment or maintenance of cell polarity / positive regulation of dendritic spine development / RHOJ GTPase cycle / synaptic transmission, GABAergic / positive regulation of actin filament polymerization / Rac protein signal transduction / pericentriolar material / RHO GTPases activate PAKs / semaphorin-plexin signaling pathway / neuron projection morphogenesis / CDC42 GTPase cycle / ficolin-1-rich granule membrane / regulation of postsynapse assembly / Sema3A PAK dependent Axon repulsion / RHOG GTPase cycle / RHO GTPases Activate NADPH Oxidases / EPH-ephrin mediated repulsion of cells / regulation of neuronal synaptic plasticity / anatomical structure morphogenesis / RHOA GTPase cycle / positive regulation of focal adhesion assembly / regulation of synaptic vesicle endocytosis
Similarity search - Function
Rho GDP/GTP exchange factor Kalirin/TRIO / : / : / SH3-RhoGEF linking unstructured region / Kalirin-like, spectrin repeats / Kalirin, SH3 / : / Divergent CRAL/TRIO domain / CRAL-TRIO lipid binding domain profile. / Domain in homologues of a S. cerevisiae phosphatidylinositol transfer protein (Sec14p) ...Rho GDP/GTP exchange factor Kalirin/TRIO / : / : / SH3-RhoGEF linking unstructured region / Kalirin-like, spectrin repeats / Kalirin, SH3 / : / Divergent 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 / : / SOS1/NGEF-like PH domain / Spectrin repeat / Spectrin repeat / Spectrin/alpha-actinin / Spectrin repeats / Small GTPase Rho / Small GTPase Rho domain profile. / 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 profile. / Pleckstrin homology domain. / Pleckstrin homology domain / SH3 domain / Rho (Ras homology) subfamily of Ras-like small GTPases / Immunoglobulin subtype 2 / Immunoglobulin C-2 Type / 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 / PH-like domain superfamily / Immunoglobulin subtype / Immunoglobulin / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / Ig-like domain profile. / Immunoglobulin-like domain / 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.86 Å
AuthorsChen C-L / Ravala SK / Bandekar SJ / Cash J / Tesmer JJG
Funding support United States, 2 items
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, 2021-
Header (metadata) releaseJul 6, 2022-
Map releaseJul 6, 2022-
UpdateJun 5, 2024-
Current statusJun 5, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_25153.png

Downloads & links

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Map

FileDownload / File: emd_25153.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationTrioN-Rac1 complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 200 pix.
= 216. Å		1.08 Å/pix.
x 200 pix.
= 216. Å		1.08 Å/pix.
x 200 pix.
= 216. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.08 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.6
Minimum - Maximum-3.8982356 - 6.2453713
Average (Standard dev.)-0.00026100618 (±0.10754663)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 216.00002 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Human Trio residues 1284-1959 in complex with Rac1

EntireName: Human Trio residues 1284-1959 in complex with Rac1
Components
  • Complex: Human Trio residues 1284-1959 in complex with Rac1
    • Protein or peptide: Triple functional domain protein
    • Protein or peptide: Ras-related C3 botulinum toxin substrate 1
  • Ligand: water

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Supramolecule #1: Human Trio residues 1284-1959 in complex with Rac1

SupramoleculeName: Human Trio residues 1284-1959 in complex with Rac1 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Triple functional domain protein

MacromoleculeName: Triple functional domain protein / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: non-specific serine/threonine protein kinase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 75.592969 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: SNAEEKRKSA RRKEFIMAEL IQTEKAYVRD LRECMDTYLW EMTSGVEEIP PGIVNKELII FGNMQEIYEF HNNIFLKELE KYEQLPEDV GHCFVTWADK FQMYVTYCKN KPDSTQLILE HAGSYFDEIQ QRHGLANSIS SYLIKPVQRI TKYQLLLKEL L TCCEEGKG ...String:
SNAEEKRKSA RRKEFIMAEL IQTEKAYVRD LRECMDTYLW EMTSGVEEIP PGIVNKELII FGNMQEIYEF HNNIFLKELE KYEQLPEDV GHCFVTWADK FQMYVTYCKN KPDSTQLILE HAGSYFDEIQ QRHGLANSIS SYLIKPVQRI TKYQLLLKEL L TCCEEGKG EIKDGLEVML SVPKRANDAM HLSMLEGFDE NIESQGELIL QESFQVWDPK TLIRKGRERH LFLFEMSLVF SK EVKDSSG RSKYLYKSKL FTSELGVTEH VEGDPCKFAL WVGRTPTSDN KIVLKASSIE NKQDWIKHIR EVIQERTIHL KGA LKEPIH IPKTAPATRQ KGRRDGEDLD SQGDGSSQPD TISIASRTSQ NTLDSDKLSG GCELTVVIHD FTACNSNELT IRRG QTVEV LERPHDKPDW CLVRTTDRSP AAEGLVPCGS LCIAHSRSSM EMEGIFNHKD SLSVSSNDAS PPASVASLQP HMIGA QSSP GPKRPGNTLR KWLTSPVRRL SSGKADGHVK KLAHKHKKSR EVRKSADAGS QKDSDDSAAT PQDETVEERG RNEGLS SGT LSKSSSSGMQ SCGEEEGEEG ADAVPLPPPM AIQQHSLLQP DSQDDKASSR LLVRPTSSET PSAAELVSAI EELVKSK MA LEDRPSSLLV DQGDSSSPSF NPSDNSLLSS SSPIDEM

UniProtKB: Triple functional domain protein

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Macromolecule #2: Ras-related C3 botulinum toxin substrate 1

MacromoleculeName: Ras-related C3 botulinum toxin substrate 1 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 21.811453 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: GEFMQAIKCV VVGDGAVGKT CLLISYTTNA FPGEYIPTVF DNYSANVMVD GKPVNLGLWD TAGQEDYDRL RPLSYPQTDV FLICFSLVS PASFENVRAK WYPEVRHHCP NTPIILVGTK LDLRDDKDTI EKLKEKKLTP ITYPQGLAMA KEIGAVKYLE C SALTQRGL ...String:
GEFMQAIKCV VVGDGAVGKT CLLISYTTNA FPGEYIPTVF DNYSANVMVD GKPVNLGLWD TAGQEDYDRL RPLSYPQTDV FLICFSLVS PASFENVRAK WYPEVRHHCP NTPIILVGTK LDLRDDKDTI EKLKEKKLTP ITYPQGLAMA KEIGAVKYLE C SALTQRGL KTVFDEAIRA VLCPPPVKKR KRKCLLL

UniProtKB: Ras-related C3 botulinum toxin substrate 1

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Macromolecule #3: water

MacromoleculeName: water / type: ligand / ID: 3 / Number of copies: 24 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.25 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4S(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)
200.0 mMNaClSodium Chloride
2.0 mMC4H10O2S2dithiothreitol
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: Blot force 10.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Alignment procedureComa free - Residual tilt: 0.01 mrad
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 11520 pixel / Digitization - Dimensions - Height: 8184 pixel / Number real images: 2817 / Average exposure time: 3.12 sec. / Average electron dose: 55.0 e/Å2
Details: 3514 movies were initially collected. After motion correction and CTF estimation, 697 micrographs were rejected due to poor CTF fitting.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 81000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE / Details: Ab-initio model generated from cryoSPARC
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 2.86 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.1) / Number images used: 922202
Initial angle assignmentType: COMMON LINE / Software - Name: cryoSPARC (ver. 3.3.1)
Final angle assignmentType: COMMON LINE / Software - Name: cryoSPARC (ver. 3.3.1)
Final 3D classificationNumber classes: 2 / Software - Name: cryoSPARC (ver. 3.3.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

2nz8

chain_id: A, source_name: PDB, initial_model_type: experimental model

2nz8

chain_id: B, source_name: PDB, initial_model_type: experimental model
DetailsThe 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.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: correlation coefficient
Output model

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

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