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- EMDB-40959: Cryo-EM structure of human TRPV4 ankyrin repeat domain in complex... -

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Entry
Database: EMDB / ID: EMD-40959
TitleCryo-EM structure of human TRPV4 ankyrin repeat domain in complex with GTPase RhoA
Map data
Sample
  • Complex: human TRPV4 ankyrin repeat domain in complex with GTPase RhoA
    • Protein or peptide: Transient receptor potential cation channel subfamily V member 4-Enhanced green fluorescent protein chimera
    • Protein or peptide: Transforming protein RhoA
  • Ligand: GUANOSINE-5'-DIPHOSPHATE
Keywordstransient receptor potential V family member 4 / TRP / channel / TRPV4 / TRP channels / membrane protein / 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 nucleoside triphosphate hydrolase
Similarity search - Domain/homology
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
Methodsingle particle reconstruction / cryo EM / Resolution: 3.49 Å
AuthorsNadezhdin KD / Talyzina IA / Neuberger A / Sobolevsky AI
Funding support United States, Germany, 6 items
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, 2023-
Header (metadata) releaseJul 5, 2023-
Map releaseJul 5, 2023-
UpdateNov 6, 2024-
Current statusNov 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_40959.png

Downloads & links

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Map

FileDownload / File: emd_40959.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 320 pix.
= 265.6 Å		0.83 Å/pix.
x 320 pix.
= 265.6 Å		0.83 Å/pix.
x 320 pix.
= 265.6 Å

Surface

Projections

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.83 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.45
Minimum - Maximum-2.2633567 - 2.8912475
Average (Standard dev.)-0.000033028107 (±0.04397476)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 265.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_40959_half_map_1.map
Projections & Slices
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Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_40959_half_map_2.map
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : human TRPV4 ankyrin repeat domain in complex with GTPase RhoA

EntireName: human TRPV4 ankyrin repeat domain in complex with GTPase RhoA
Components
  • Complex: human TRPV4 ankyrin repeat domain in complex with GTPase RhoA
    • Protein or peptide: Transient receptor potential cation channel subfamily V member 4-Enhanced green fluorescent protein chimera
    • Protein or peptide: Transforming protein RhoA
  • Ligand: GUANOSINE-5'-DIPHOSPHATE

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Supramolecule #1: human TRPV4 ankyrin repeat domain in complex with GTPase RhoA

SupramoleculeName: human TRPV4 ankyrin repeat domain in complex with GTPase RhoA
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 510 KDa

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Macromolecule #1: Transient receptor potential cation channel subfamily V member 4-...

MacromoleculeName: Transient receptor potential cation channel subfamily V member 4-Enhanced green fluorescent protein chimera
type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Human cytomegalovirus
Molecular weightTheoretical: 127.717938 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MADSSEGPRA GPGEVAELPG DESGTPGGEA FPLSSLANLF EGEDGSLSPS PADASRPAGP GDGRPNLRMK FQGAFRKGVP NPIDLLEST LYESSVVPGP KKAPMDSLFD YGTYRHHSSD NKRWRKKIIE KQPQSPKAPA PQPPPILKVF NRPILFDIVS R GSTADLDG ...String:
MADSSEGPRA GPGEVAELPG DESGTPGGEA FPLSSLANLF EGEDGSLSPS PADASRPAGP GDGRPNLRMK FQGAFRKGVP NPIDLLEST LYESSVVPGP KKAPMDSLFD YGTYRHHSSD NKRWRKKIIE KQPQSPKAPA PQPPPILKVF NRPILFDIVS R GSTADLDG LLPFLLTHKK RLTDEEFREP STGKTCLPKA LLNLSNGRND TIPVLLDIAE RTGNMREFIN SPFRDIYYRG QT ALHIAIE RRCKHYVELL VAQGADVHAQ ARGRFFQPKD EGGYFYFGEL PLSLAACTNQ PHIVNYLTEN PHKKADMRRQ DSR GNTVLH ALVAIADNTR ENTKFVTKMY DLLLLKCARL FPDSNLEAVL NNDGLSPLMM AAKTGKIGIF QHIIRREVTD EDTR HLSRK FKDWAYGPVY SSLYDLSSLD TCGEEASVLE ILVYNSKIEN RHEMLAVEPI NELLRDKWRK FGAVSFYINV VSYLC AMVI FTLTAYYQPL EGTPPYPYRT TVDYLRLAGE VITLFTGVLF FFTNIKDLFM KKCPGVNSLF IDGSFQLLYF IYSVLV IVS AALYLAGIEA YLAVMVFALV LGWMNALYFT RGLKLTGTYS IMIQKILFKD LFRFLLVYLL FMIGYASALV SLLNPCA NM KVCNEDQTNC TVPTYPSCRD SETFSTFLLD LFKLTIGMGD LEMLSSTKYP VVFIILLVTY IILTFVLLLN MLIALMGE T VGQVSKESKH IWKLQWATTI LDIERSFPVF LRKAFRSGEM VTVGKSSDGT PDRRWCFRVD EVNWSHWNQN LGIINEDPG KNETYQYYGF SHTVGRLRRD RWSSVVPRVV ELNKNSNPDE VVVPLDSMGN PRCDGHQQGY PRKWRTDDAP LLVPRGSAAA AVSKGEELF TGVVPILVEL DGDVNGHKFS VSGEGEGDAT YGKLTLKFIC TTGKLPVPWP TLVTTLTYGV QCFSRYPDHM K QHDFFKSA MPEGYVQERT IFFKDDGNYK TRAEVKFEGD TLVNRIELKG IDFKEDGNIL GHKLEYNYNS HNVYIMADKQ KN GIKVNFK IRHNIEDGSV QLADHYQQNT PIGDGPVLLP DNHYLSTQSK LSKDPNEKRD HMVLLEFVTA AGITLGMDEL YKS GLRSWS HPQFEK

UniProtKB: Transient receptor potential cation channel subfamily V member 4, Enhanced green fluorescent protein

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Macromolecule #2: Transforming protein RhoA

MacromoleculeName: Transforming protein RhoA / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: small monomeric GTPase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 21.799158 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MAAIRKKLVI VGDGACGKTC LLIVFSKDQF PEVYVPTVFE NYVADIEVDG KQVELALWDT AGQEDYDRLR PLSYPDTDVI LMCFSIDSP DSLENIPEKW TPEVKHFCPN VPIILVGNKK DLRNDEHTRR ELAKMKQEPV KPEEGRDMAN RIGAFGYMEC S AKTKDGVR ...String:
MAAIRKKLVI VGDGACGKTC LLIVFSKDQF PEVYVPTVFE NYVADIEVDG KQVELALWDT AGQEDYDRLR PLSYPDTDVI LMCFSIDSP DSLENIPEKW TPEVKHFCPN VPIILVGNKK DLRNDEHTRR ELAKMKQEPV KPEEGRDMAN RIGAFGYMEC S AKTKDGVR EVFEMATRAA LQARRGKKKS GCLVL

UniProtKB: Transforming protein RhoA

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Macromolecule #3: GUANOSINE-5'-DIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-DIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 1 / Formula: GDP
Molecular weightTheoretical: 443.201 Da
Chemical component information

ChemComp-GDP:
GUANOSINE-5'-DIPHOSPHATE / GDP, energy-carrying molecule*YM

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration3.2 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
150.0 mMNaClsodium chloride
20.0 mMtris(hydroxymethyl)aminomethane
1.0 mMbeta-Mercaptoethanol
0.01 %glyco-diosgenin
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV
Detailshuman TRPV4

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 5456 / Average exposure time: 2.5 sec. / Average electron dose: 58.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1285028
Startup modelType of model: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.49 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 86562
Initial angle assignmentType: OTHER
Final angle assignmentType: OTHER
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL
Output model

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

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