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- PDB-8t1e: Closed-state cryo-EM structure of full-length human TRPV4 in the ... -

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Basic information

Entry
Database: PDB / ID: 8t1e
TitleClosed-state cryo-EM structure of full-length human TRPV4 in the presence of 4a-PDD
ComponentsTransient receptor potential cation channel subfamily V member 4,Enhanced green fluorescent protein
KeywordsMEMBRANE PROTEIN / transient receptor potential V family member 4 / TRP / channel / TRPV4 / TRP channels / agonist / 4a-PDD / 4alpha-Phorbol 12 / 13-didecanoate / closed state
Function / homology
Function and homology information


stretch-activated, monoatomic cation-selective, calcium channel activity / blood vessel endothelial cell delamination / regulation of response to osmotic stress / osmosensor activity / vasopressin secretion / positive regulation of striated muscle contraction / calcium ion import into cytosol / negative regulation of brown fat cell differentiation / positive regulation of microtubule depolymerization / positive regulation of macrophage inflammatory protein 1 alpha production ...stretch-activated, monoatomic cation-selective, calcium channel activity / blood vessel endothelial cell delamination / regulation of response to osmotic stress / osmosensor activity / vasopressin secretion / positive regulation of striated muscle contraction / calcium ion import into cytosol / negative regulation of brown fat cell differentiation / positive regulation of microtubule depolymerization / positive regulation of macrophage inflammatory protein 1 alpha production / hyperosmotic salinity response / positive regulation of chemokine (C-X-C motif) ligand 1 production / positive regulation of chemokine (C-C motif) ligand 5 production / cartilage development involved in endochondral bone morphogenesis / cellular hypotonic salinity response / cortical microtubule organization / cellular hypotonic response / multicellular organismal-level water homeostasis / osmosensory signaling pathway / positive regulation of vascular permeability / positive regulation of monocyte chemotactic protein-1 production / cell-cell junction assembly / cell volume homeostasis / cellular response to osmotic stress / calcium ion import / regulation of aerobic respiration / TRP channels / cortical actin cytoskeleton / diet induced thermogenesis / positive regulation of macrophage chemotaxis / beta-tubulin binding / microtubule polymerization / calcium ion import across plasma membrane / alpha-tubulin binding / monoatomic cation channel activity / response to mechanical stimulus / cytoplasmic microtubule / SH2 domain binding / bioluminescence / protein kinase C binding / actin filament organization / generation of precursor metabolites and energy / filopodium / adherens junction / response to insulin / positive regulation of JNK cascade / calcium ion transmembrane transport / calcium channel activity / positive regulation of interleukin-6 production / ruffle membrane / intracellular calcium ion homeostasis / positive regulation of inflammatory response / calcium ion transport / actin filament binding / glucose homeostasis / lamellipodium / negative regulation of neuron projection development / actin binding / cellular response to heat / actin cytoskeleton organization / growth cone / positive regulation of cytosolic calcium ion concentration / microtubule binding / High laminar flow shear stress activates signaling by PIEZO1 and PECAM1:CDH5:KDR in endothelial cells / response to hypoxia / positive regulation of ERK1 and ERK2 cascade / calmodulin binding / cilium / apical plasma membrane / focal adhesion / lipid binding / protein kinase binding / negative regulation of transcription by RNA polymerase II / cell surface / endoplasmic reticulum / ATP binding / metal ion binding / identical protein binding / membrane / plasma membrane
Similarity search - Function
Transient receptor potential cation channel subfamily V member 4 / Transient receptor potential cation channel subfamily V member 1-4 / Transient receptor potential cation channel subfamily V / Green fluorescent protein, GFP / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein / Ankyrin repeat / Ankyrin repeat profile. / Ankyrin repeat region circular profile. ...Transient receptor potential cation channel subfamily V member 4 / Transient receptor potential cation channel subfamily V member 1-4 / Transient receptor potential cation channel subfamily V / Green fluorescent protein, GFP / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein / Ankyrin repeat / Ankyrin repeat profile. / Ankyrin repeat region circular profile. / ankyrin repeats / Ankyrin repeat / Ankyrin repeat-containing domain superfamily / Ion transport domain / Ion transport protein
Similarity search - Domain/homology
Chem-9ZR / Chem-YJ0 / Enhanced green fluorescent protein / Transient receptor potential cation channel subfamily V member 4
Similarity search - Component
Biological speciesHomo sapiens (human)
Human betaherpesvirus 5
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.77 Å
AuthorsTalyzina, I.A. / Nadezhdin, K.D. / 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 1.1Oct 16, 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
B: Transient receptor potential cation channel subfamily V member 4,Enhanced green fluorescent protein
C: Transient receptor potential cation channel subfamily V member 4,Enhanced green fluorescent protein
D: Transient receptor potential cation channel subfamily V member 4,Enhanced green fluorescent protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)546,34947
Polymers510,8724
Non-polymers35,47743
Water64936
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, 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 / 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.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human), (gene. exp.) Human betaherpesvirus 5
Gene: TRPV4, VRL2, VROAC, egfp / Cell line (production host): HEK293S / Production host: Homo sapiens (human) / References: UniProt: Q9HBA0, UniProt: C5MKY7
#2: Chemical...
ChemComp-9ZR / [(2~{R})-2-[(~{Z})-hexadec-9-enoyl]oxy-3-[oxidanyl-[2-(trimethyl-$l^{4}-azanyl)ethoxy]phosphoryl]oxy-propyl] (~{Z})-docos-13-enoate


Mass: 815.175 Da / Num. of mol.: 32 / Source method: obtained synthetically / Formula: C46H89NO8P
#3: Chemical
ChemComp-YJ0 / (2R)-2-{[(4-O-hexopyranosyl-beta-D-glucopyranosyl)oxy]methyl}-4-{[(25R)-5beta,14beta,17beta-spirostan-3beta-yl]oxy}butyl 4-O-alpha-D-glucopyranosyl-beta-D-glucopyranoside


Mass: 1165.315 Da / Num. of mol.: 8 / Source method: isolated from a natural source / Formula: C56H92O25
#4: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Na
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 36 / Source method: isolated from a natural source / Formula: H2O
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: full-length human TRPV4 in the presence of 4a-PDD / Type: COMPLEX / Entity ID: #1 / 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
510 mMGTPgammaS1
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: 750 nm / Cs: 2.7 mm
Image recordingAverage exposure time: 2.4 sec. / Electron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 15624
Image scansWidth: 11520 / Height: 8184

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Processing

EM softwareName: PHENIX / Version: 1.11.1_2575: / Category: model refinement
CTF correctionType: NONE
Particle selectionNum. of particles selected: 3935964
3D reconstructionResolution: 2.77 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 75146 / Symmetry type: POINT
Atomic model buildingSpace: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00745916
ELECTRON MICROSCOPYf_angle_d1.43383612
ELECTRON MICROSCOPYf_dihedral_angle_d12.83718152
ELECTRON MICROSCOPYf_chiral_restr0.0953320
ELECTRON MICROSCOPYf_plane_restr0.0066352

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