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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 / calcium ion import into cytosol / cartilage development involved in endochondral bone morphogenesis / positive regulation of microtubule depolymerization / positive regulation of macrophage inflammatory protein 1 alpha production / positive regulation of striated muscle contraction / positive regulation of chemokine (C-C motif) ligand 5 production / positive regulation of chemokine (C-X-C motif) ligand 1 production ...stretch-activated, monoatomic cation-selective, calcium channel activity / blood vessel endothelial cell delamination / osmosensor activity / calcium ion import into cytosol / cartilage development involved in endochondral bone morphogenesis / positive regulation of microtubule depolymerization / positive regulation of macrophage inflammatory protein 1 alpha production / positive regulation of striated muscle contraction / positive regulation of chemokine (C-C motif) ligand 5 production / positive regulation of chemokine (C-X-C motif) ligand 1 production / alpha-beta T cell lineage commitment / aortic valve formation / beta selection / positive regulation of lipase activity / endothelial tube lumen extension / skeletal muscle satellite cell migration / bone trabecula morphogenesis / SLIT2:ROBO1 increases RHOA activity / RHO GTPases Activate Rhotekin and Rhophilins / Roundabout signaling pathway / cellular hypotonic salinity response / Axonal growth inhibition (RHOA activation) / Axonal growth stimulation / cleavage furrow formation / cellular hypotonic response / negative regulation of cell size / cortical microtubule organization / multicellular organismal-level water homeostasis / regulation of osteoblast proliferation / regulation of modification of postsynaptic actin cytoskeleton / diet induced thermogenesis / mitotic cleavage furrow formation / apical junction assembly / negative regulation of cell migration involved in sprouting angiogenesis / positive regulation of alpha-beta T cell differentiation / cell junction assembly / calcium ion import / establishment of epithelial cell apical/basal polarity / cellular response to chemokine / positive regulation of vascular permeability / negative regulation of oxidative phosphorylation / regulation of modification of postsynaptic structure / RHO GTPases Activate ROCKs / positive regulation of monocyte chemotactic protein-1 production / negative regulation of brown fat cell differentiation / RHO GTPases activate CIT / cell volume homeostasis / odontogenesis / PCP/CE pathway / cell-cell junction assembly / Sema4D induced cell migration and growth-cone collapse / RHO GTPases activate KTN1 / osmosensory signaling pathway / apolipoprotein A-I-mediated signaling pathway / cellular response to osmotic stress / wound healing, spreading of cells / Sema4D mediated inhibition of cell attachment and migration / positive regulation of leukocyte adhesion to vascular endothelial cell / Wnt signaling pathway, planar cell polarity pathway / PI3K/AKT activation / ossification involved in bone maturation / regulation of focal adhesion assembly / negative chemotaxis / regulation of aerobic respiration / EPHA-mediated growth cone collapse / cortical actin cytoskeleton / apical junction complex / stress fiber assembly / myosin binding / positive regulation of cytokinesis / RHOC GTPase cycle / cellular response to cytokine stimulus / positive regulation of macrophage chemotaxis / TRP channels / ERBB2 Regulates Cell Motility / positive regulation of protein serine/threonine kinase activity / cleavage furrow / microtubule polymerization / semaphorin-plexin signaling pathway / negative regulation of cell-substrate adhesion / calcium ion import across plasma membrane / ficolin-1-rich granule membrane / mitotic spindle assembly / RHOA GTPase cycle / endothelial cell migration / response to mechanical stimulus / positive regulation of T cell migration / alpha-tubulin binding / Rho protein signal transduction / beta-tubulin binding / positive regulation of stress fiber assembly / RHO GTPases activate PKNs / GPVI-mediated activation cascade / cytoplasmic microtubule / PTK6 Regulates RHO GTPases, RAS GTPase and MAP kinases / monoatomic cation channel activity / negative regulation of reactive oxygen species biosynthetic process / substrate adhesion-dependent cell spreading / cytoplasmic microtubule organization / positive regulation of neuron differentiation
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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