Database: EMDB / ID: EMD-25153
|Title||Human Trio residues 1284-1959 in complex with Rac1|
|Map data||TrioN-Rac1 complex|
|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 / regulation of nitric oxide biosynthetic process / Azathioprine ADME / regulation of stress fiber assembly / negative regulation of fibroblast migration / sphingosine-1-phosphate receptor signaling pathway / thioesterase binding / 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 / positive regulation of cell-substrate adhesion / Ephrin signaling / 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-matrix adhesion / substrate adhesion-dependent cell spreading / cell motility / actin filament organization / 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 species||Homo sapiens (human)|
|Method||single particle reconstruction / cryo EM / Resolution: 2.86 Å|
|Authors||Chen C-L / Ravala SK / Bandekar SJ / Cash J / Tesmer JJG|
|Funding support|| United States, 2 items |
|Citation||Journal: 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.
Downloads & links
|File||Download / File: emd_25153.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 1.08 Å|
|Symmetry||Space group: 1|
-Entire : Human Trio residues 1284-1959 in complex with Rac1
|Entire||Name: Human Trio residues 1284-1959 in complex with Rac1|
-Supramolecule #1: Human Trio residues 1284-1959 in complex with Rac1
|Supramolecule||Name: Human Trio residues 1284-1959 in complex with Rac1 / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #1-#2|
|Source (natural)||Organism: Homo sapiens (human)|
|Recombinant expression||Organism: Escherichia coli (E. coli) / Recombinant strain: Rosetta (DE3) pLysS|
-Macromolecule #1: Triple functional domain protein
|Macromolecule||Name: 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 weight||Theoretical: 75.592969 KDa|
|Recombinant expression||Organism: Escherichia coli (E. coli)|
|Sequence||String: 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
-Macromolecule #2: Ras-related C3 botulinum toxin substrate 1
|Macromolecule||Name: 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 weight||Theoretical: 21.811453 KDa|
|Recombinant expression||Organism: Escherichia coli (E. coli)|
|Sequence||String: 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
-Macromolecule #3: water
|Macromolecule||Name: water / type: ligand / ID: 3 / Number of copies: 24 / Formula: HOH|
|Molecular weight||Theoretical: 18.015 Da|
|Chemical component information|
|Processing||single particle reconstruction|
|Buffer||pH: 8 |
|Grid||Model: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE|
|Vitrification||Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: Blot force 10.|
|Microscope||FEI TITAN KRIOS|
|Electron beam||Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN|
|Electron optics||C2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 81000|
|Sample stage||Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN|
|Alignment procedure||Coma free - Residual tilt: 0.01 mrad|
|Image recording||Film 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.
Model: Titan Krios / Image courtesy: FEI Company
-Atomic model buiding 1
|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.|
|Refinement||Space: REAL / Protocol: RIGID BODY FIT / Target criteria: correlation coefficient|
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