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- PDB-8fcb: Cryo-EM structure of the human TRPV4 - RhoA in complex with GSK10... -

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Basic information

Entry
Database: PDB / ID: 8fcb
TitleCryo-EM structure of the human TRPV4 - RhoA in complex with GSK1016790A
Components
  • Transforming protein RhoA
  • Transient receptor potential cation channel subfamily V member 4
KeywordsMEMBRANE PROTEIN / TRPV4 / RhoA / GSK1016790A
Function / homology
Function and homology information


stretch-activated, monoatomic cation-selective, calcium channel activity / blood vessel endothelial cell delamination / osmosensor activity / vasopressin secretion / calcium ion import into cytosol / regulation of response to osmotic stress / positive regulation of microtubule depolymerization / positive regulation of macrophage inflammatory protein 1 alpha production / hyperosmotic salinity response / positive regulation of striated muscle contraction ...stretch-activated, monoatomic cation-selective, calcium channel activity / blood vessel endothelial cell delamination / osmosensor activity / vasopressin secretion / calcium ion import into cytosol / regulation of response to osmotic stress / positive regulation of microtubule depolymerization / positive regulation of macrophage inflammatory protein 1 alpha production / hyperosmotic salinity response / positive regulation of striated muscle contraction / cartilage development involved in endochondral bone morphogenesis / positive regulation of chemokine (C-X-C motif) ligand 1 production / positive regulation of chemokine (C-C motif) ligand 5 production / alpha-beta T cell lineage commitment / aortic valve formation / positive regulation of lipase activity / endothelial tube lumen extension / skeletal muscle satellite cell migration / positive regulation of vascular associated smooth muscle contraction / angiotensin-mediated vasoconstriction involved in regulation of systemic arterial blood pressure / SLIT2:ROBO1 increases RHOA activity / bone trabecula morphogenesis / RHO GTPases Activate Rhotekin and Rhophilins / Roundabout signaling pathway / negative regulation of intracellular steroid hormone receptor signaling pathway / cellular hypotonic salinity response / Axonal growth inhibition (RHOA activation) / Axonal growth stimulation / cleavage furrow formation / cellular hypotonic response / regulation of neural precursor cell proliferation / cortical microtubule organization / regulation of osteoblast proliferation / regulation of modification of postsynaptic actin cytoskeleton / multicellular organismal-level water homeostasis / forebrain radial glial cell differentiation / mitotic cleavage furrow formation / apical junction assembly / negative regulation of cell migration involved in sprouting angiogenesis / positive regulation of vascular permeability / beta selection / cell junction assembly / establishment of epithelial cell apical/basal polarity / cellular response to chemokine / negative regulation of motor neuron apoptotic process / regulation of systemic arterial blood pressure by endothelin / calcium ion import / negative regulation of oxidative phosphorylation / regulation of modification of postsynaptic structure / RHO GTPases Activate ROCKs / RHO GTPases activate CIT / negative regulation of cell size / negative regulation of brown fat cell differentiation / Sema4D induced cell migration and growth-cone collapse / osmosensory signaling pathway / PCP/CE pathway / cell-cell junction assembly / positive regulation of monocyte chemotactic protein-1 production / RHO GTPases activate KTN1 / positive regulation of alpha-beta T cell differentiation / positive regulation of podosome assembly / cell volume homeostasis / cellular response to osmotic stress / apolipoprotein A-I-mediated signaling pathway / wound healing, spreading of cells / Sema4D mediated inhibition of cell attachment and migration / motor neuron apoptotic process / positive regulation of leukocyte adhesion to vascular endothelial cell / odontogenesis / Wnt signaling pathway, planar cell polarity pathway / PI3K/AKT activation / ossification involved in bone maturation / regulation of focal adhesion assembly / androgen receptor signaling pathway / negative chemotaxis / EPHA-mediated growth cone collapse / regulation of aerobic respiration / cortical actin cytoskeleton / apical junction complex / stress fiber assembly / myosin binding / diet induced thermogenesis / TRP channels / positive regulation of cytokinesis / RHOC GTPase cycle / regulation of neuron projection development / positive regulation of macrophage chemotaxis / cellular response to cytokine stimulus / cerebral cortex cell migration / positive regulation of protein serine/threonine kinase activity / ERBB2 Regulates Cell Motility / cleavage furrow / semaphorin-plexin signaling pathway / microtubule polymerization / calcium ion import across plasma membrane / negative regulation of cell-substrate adhesion / ficolin-1-rich granule membrane / RHOA GTPase cycle / mitotic spindle assembly / positive regulation of T cell migration
Similarity search - Function
Transient receptor potential cation channel subfamily V member 4 / Small GTPase Rho / Small GTPase Rho domain profile. / Transient receptor potential cation channel subfamily V member 1-4 / Transient receptor potential cation channel subfamily V / Ankyrin repeat / Rho (Ras homology) subfamily of Ras-like small GTPases / Ras subfamily of RAS small GTPases / Small GTPase / Ras family ...Transient receptor potential cation channel subfamily V member 4 / Small GTPase Rho / Small GTPase Rho domain profile. / Transient receptor potential cation channel subfamily V member 1-4 / Transient receptor potential cation channel subfamily V / Ankyrin repeat / Rho (Ras homology) subfamily of Ras-like small GTPases / Ras subfamily of RAS small GTPases / Small GTPase / Ras family / Ankyrin repeat profile. / Ankyrin repeat region circular profile. / ankyrin repeats / Ankyrin repeat / Rab subfamily of small GTPases / Ankyrin repeat-containing domain superfamily / Ion transport domain / Ion transport protein / Small GTP-binding protein domain / P-loop containing nucleotide triphosphate hydrolases / Rossmann fold / P-loop containing nucleoside triphosphate hydrolase / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
5'-GUANOSINE-DIPHOSPHATE-MONOTHIOPHOSPHATE / Chem-XQ3 / CHOLESTEROL HEMISUCCINATE / Transforming protein RhoA / Transient receptor potential cation channel subfamily V member 4
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.52 Å
AuthorsKwon, D.H. / Lee, S.-Y. / Zhang, F.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK) United States
Citation
Journal: Nat Commun / Year: 2023
Title: TRPV4-Rho GTPase complex structures reveal mechanisms of gating and disease.
Authors: Do Hoon Kwon / Feng Zhang / Brett A McCray / Shasha Feng / Meha Kumar / Jeremy M Sullivan / Wonpil Im / Charlotte J Sumner / Seok-Yong Lee /
Abstract: Crosstalk between ion channels and small GTPases is critical during homeostasis and disease, but little is known about the structural underpinnings of these interactions. TRPV4 is a polymodal, ...Crosstalk between ion channels and small GTPases is critical during homeostasis and disease, but little is known about the structural underpinnings of these interactions. TRPV4 is a polymodal, calcium-permeable cation channel that has emerged as a potential therapeutic target in multiple conditions. Gain-of-function mutations also cause hereditary neuromuscular disease. Here, we present cryo-EM structures of human TRPV4 in complex with RhoA in the ligand-free, antagonist-bound closed, and agonist-bound open states. These structures reveal the mechanism of ligand-dependent TRPV4 gating. Channel activation is associated with rigid-body rotation of the intracellular ankyrin repeat domain, but state-dependent interaction with membrane-anchored RhoA constrains this movement. Notably, many residues at the TRPV4-RhoA interface are mutated in disease and perturbing this interface by introducing mutations into either TRPV4 or RhoA increases TRPV4 channel activity. Together, these results suggest that RhoA serves as an auxiliary subunit for TRPV4, regulating TRPV4-mediated calcium homeostasis and disruption of TRPV4-RhoA interactions can lead to TRPV4-related neuromuscular disease. These insights will help facilitate TRPV4 therapeutics development.
#1: Journal: bioRxiv / Year: 2023
Title: Structural insights into TRPV4-Rho GTPase signaling complex function and disease.
Authors: Do Hoon Kwon / Feng Zhang / Brett A McCray / Meha Kumar / Jeremy M Sullivan / Charlotte J Sumner / Seok-Yong Lee
Abstract: Crosstalk between ion channels and small GTPases is critical during homeostasis and disease , but little is known about the structural underpinnings of these interactions. TRPV4 is a polymodal, ...Crosstalk between ion channels and small GTPases is critical during homeostasis and disease , but little is known about the structural underpinnings of these interactions. TRPV4 is a polymodal, calcium-permeable cation channel that has emerged as a potential therapeutic target in multiple conditions . Gain-of-function mutations also cause hereditary neuromuscular disease . Here, we present cryo-EM structures of human TRPV4 in complex with RhoA in the apo, antagonist-bound closed, and agonist-bound open states. These structures reveal the mechanism of ligand-dependent TRPV4 gating. Channel activation is associated with rigid-body rotation of the intracellular ankyrin repeat domain, but state-dependent interaction with membrane-anchored RhoA constrains this movement. Notably, many residues at the TRPV4-RhoA interface are mutated in disease and perturbing this interface by introducing mutations into either TRPV4 or RhoA increases TRPV4 channel activity. Together, these results suggest that the interaction strength between TRPV4 and RhoA tunes TRPV4-mediated calcium homeostasis and actin remodeling, and that disruption of TRPV4-RhoA interactions leads to TRPV4-related neuromuscular disease, findings that will guide TRPV4 therapeutics development.
History
DepositionDec 1, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 12, 2023Provider: repository / Type: Initial release
Revision 1.1Jun 19, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Transient receptor potential cation channel subfamily V member 4
B: Transient receptor potential cation channel subfamily V member 4
C: Transient receptor potential cation channel subfamily V member 4
D: Transient receptor potential cation channel subfamily V member 4
E: Transforming protein RhoA
F: Transforming protein RhoA
G: Transforming protein RhoA
H: Transforming protein RhoA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)487,49920
Polymers480,7728
Non-polymers6,72612
Water00
1


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

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Components

#1: Protein
Transient receptor potential cation channel subfamily V member 4 / TrpV4 / Osm-9-like TRP channel 4 / OTRPC4 / Transient receptor potential protein 12 / TRP12 / ...TrpV4 / Osm-9-like TRP channel 4 / OTRPC4 / Transient receptor potential protein 12 / TRP12 / Vanilloid receptor-like channel 2 / Vanilloid receptor-like protein 2 / VRL-2 / Vanilloid receptor-related osmotically-activated channel / VR-OAC


Mass: 98393.930 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TRPV4, VRL2, VROAC / Production host: Homo sapiens (human) / References: UniProt: Q9HBA0
#2: Protein
Transforming protein RhoA / Rho cDNA clone 12 / h12


Mass: 21799.158 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RHOA, ARH12, ARHA, RHO12 / Production host: Homo sapiens (human) / References: UniProt: P61586, small monomeric GTPase
#3: Chemical
ChemComp-XQ3 / N-[(2S)-1-{4-[N-(2,4-dichlorobenzene-1-sulfonyl)-L-seryl]piperazin-1-yl}-4-methyl-1-oxopentan-2-yl]-1-benzothiophene-2-carboxamide / GSK1016790A


Mass: 655.613 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C28H32Cl2N4O6S2 / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical
ChemComp-Y01 / CHOLESTEROL HEMISUCCINATE


Mass: 486.726 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C31H50O4
#5: Chemical
ChemComp-GSP / 5'-GUANOSINE-DIPHOSPHATE-MONOTHIOPHOSPHATE


Mass: 539.246 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H16N5O13P3S
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: Human TRPV4 - RhoA complex in GSK1016790A / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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: OTHER
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

Image processing
IDImage recording-ID
11
21
CTF correction
IDEM image processing-IDType
11PHASE FLIPPING AND AMPLITUDE CORRECTION
22PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstruction
IDResolution (Å)Resolution methodNum. of particlesImage processing-IDEntry-IDSymmetry type
13.52FSC 0.143 CUT-OFF20764518FCBPOINT
23.52FSC 0.143 CUT-OFF20764518FCBPOINT
33.3FSC 0.143 CUT-OFF20764518FCBPOINT
43.55FSC 0.143 CUT-OFF20764518FCBPOINT
53.3FSC 0.143 CUT-OFF20764528FCBPOINT
63.52FSC 0.143 CUT-OFF20764528FCBPOINT
73.3FSC 0.143 CUT-OFF20764528FCBPOINT
83.55FSC 0.143 CUT-OFF20764528FCBPOINT
RefinementHighest resolution: 3.52 Å

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