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- PDB-8t1c: Cryo-EM structure of human TRPV4 ankyrin repeat domain in complex... -

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Basic information

Entry
Database: PDB / ID: 8t1c
TitleCryo-EM structure of human TRPV4 ankyrin repeat domain in complex with GTPase RhoA
Components
  • Transforming protein RhoA
  • Transient receptor potential cation channel subfamily V member 4-Enhanced green fluorescent protein chimera
KeywordsMEMBRANE PROTEIN / transient receptor potential V family member 4 / TRP / channel / TRPV4 / TRP channels / GTPase / transforming protein RhoA / RhoA / ankyrin repeat domain / GDP / guanosine diphosphate
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 / diet induced thermogenesis / regulation of osteoblast proliferation / regulation of modification of postsynaptic actin cytoskeleton / mitotic cleavage furrow formation / apical junction assembly / negative regulation of cell migration involved in sprouting angiogenesis / calcium ion import / establishment of epithelial cell apical/basal polarity / positive regulation of alpha-beta T cell differentiation / cell junction assembly / cellular response to chemokine / positive regulation of vascular permeability / negative regulation of oxidative phosphorylation / positive regulation of monocyte chemotactic protein-1 production / regulation of modification of postsynaptic structure / RHO GTPases Activate ROCKs / 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 / Wnt signaling pathway, planar cell polarity pathway / positive regulation of leukocyte adhesion to vascular endothelial cell / 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 / positive regulation of macrophage chemotaxis / cellular response to cytokine stimulus / TRP channels / positive regulation of protein serine/threonine kinase activity / ERBB2 Regulates Cell Motility / cleavage furrow / microtubule polymerization / semaphorin-plexin signaling pathway / negative regulation of cell-substrate adhesion / calcium ion import across plasma membrane / mitotic spindle assembly / ficolin-1-rich granule membrane / RHOA GTPase cycle / response to mechanical stimulus / endothelial cell migration / positive regulation of T cell migration / alpha-tubulin binding / Rho protein signal transduction / positive regulation of stress fiber assembly / RHO GTPases activate PKNs / GPVI-mediated activation cascade / beta-tubulin binding / substrate adhesion-dependent cell spreading / cytoplasmic microtubule / PTK6 Regulates RHO GTPases, RAS GTPase and MAP kinases / monoatomic cation channel activity / negative regulation of reactive oxygen species biosynthetic process / 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 / Green fluorescent protein, GFP / Ankyrin repeat / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein ...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 / Green fluorescent protein, GFP / Ankyrin repeat / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein / 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
GUANOSINE-5'-DIPHOSPHATE / Enhanced green fluorescent protein / Transforming protein RhoA / Transient receptor potential cation channel subfamily V member 4
Similarity search - Component
Biological speciesHomo sapiens (human)
Human cytomegalovirus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.49 Å
AuthorsNadezhdin, K.D. / Talyzina, I.A. / Neuberger, A. / Sobolevsky, A.I.
Funding support United States, Germany, 6items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIH/NIAMS)R01 AR078814 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)R01 CA206573 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)R01 NS083660 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)R01 NS107253 United States
German Research Foundation (DFG)464295817 Germany
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)R01 HL096647 United States
CitationJournal: Nat Commun / Year: 2023
Title: Structure of human TRPV4 in complex with GTPase RhoA.
Authors: Kirill D Nadezhdin / Irina A Talyzina / Aravind Parthasarathy / Arthur Neuberger / David X Zhang / Alexander I Sobolevsky /
Abstract: Transient receptor potential (TRP) channel TRPV4 is a polymodal cellular sensor that responds to moderate heat, cell swelling, shear stress, and small-molecule ligands. It is involved in ...Transient receptor potential (TRP) channel TRPV4 is a polymodal cellular sensor that responds to moderate heat, cell swelling, shear stress, and small-molecule ligands. It is involved in thermogenesis, regulation of vascular tone, bone homeostasis, renal and pulmonary functions. TRPV4 is implicated in neuromuscular and skeletal disorders, pulmonary edema, and cancers, and represents an important drug target. The cytoskeletal remodeling GTPase RhoA has been shown to suppress TRPV4 activity. Here, we present a structure of the human TRPV4-RhoA complex that shows RhoA interaction with the membrane-facing surface of the TRPV4 ankyrin repeat domains. The contact interface reveals residues that are mutated in neuropathies, providing an insight into the disease pathogenesis. We also identify the binding sites of the TRPV4 agonist 4α-PDD and the inhibitor HC-067047 at the base of the S1-S4 bundle, and show that agonist binding leads to pore opening, while channel inhibition involves a π-to-α transition in the pore-forming helix S6. Our structures elucidate the interaction interface between hTRPV4 and RhoA, as well as residues at this interface that are involved in TRPV4 disease-causing mutations. They shed light on TRPV4 activation and inhibition and provide a template for the design of future therapeutics for treatment of TRPV4-related diseases.
History
DepositionJun 2, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 5, 2023Provider: repository / Type: Initial release
Revision 1.1Nov 6, 2024Group: Data collection / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Transient receptor potential cation channel subfamily V member 4-Enhanced green fluorescent protein chimera
E: Transforming protein RhoA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)149,9603
Polymers149,5172
Non-polymers4431
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Transient receptor potential cation channel subfamily V member 4-Enhanced green fluorescent protein chimera / 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: 127717.938 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human), (gene. exp.) Human cytomegalovirus
Gene: TRPV4, VRL2, VROAC, egfp / Cell line (production host): HEK293S / Production host: Homo sapiens (human) / References: UniProt: Q9HBA0, UniProt: C5MKY7
#2: Protein Transforming protein RhoA / Rho cDNA clone 12 / h12


Mass: 21799.158 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RHOA, ARH12, ARHA, RHO12 / Cell line (production host): HEK293S / Production host: Homo sapiens (human) / References: UniProt: P61586, small monomeric GTPase
#3: Chemical ChemComp-GDP / GUANOSINE-5'-DIPHOSPHATE


Type: RNA linking / Mass: 443.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O11P2 / Comment: GDP, energy-carrying molecule*YM
Has ligand of interestN
Has protein modificationY

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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 ankyrin repeat domain in complex with GTPase RhoA
Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.51 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: Human embryonic kidney 293 / Plasmid: pEG BacMam
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMsodium chlorideNaCl1
220 mMtris(hydroxymethyl)aminomethane1
31 mMbeta-Mercaptoethanol1
40.01 %glyco-diosgenin1
SpecimenConc.: 3.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: human TRPV4
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: 295 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1500 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm
Image recordingAverage exposure time: 2.5 sec. / Electron dose: 58 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5456
Image scansWidth: 5760 / Height: 4092

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Processing

EM softwareName: PHENIX / Version: 1.11.1_2575: / Category: model refinement
CTF correctionType: NONE
Particle selectionNum. of particles selected: 1285028
3D reconstructionResolution: 3.49 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 86562 / Symmetry type: POINT
Atomic model buildingSpace: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0066097
ELECTRON MICROSCOPYf_angle_d1.20311031
ELECTRON MICROSCOPYf_dihedral_angle_d12.832427
ELECTRON MICROSCOPYf_chiral_restr0.156463
ELECTRON MICROSCOPYf_plane_restr0.005926

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