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- PDB-9iqx: Cryo-EM structure of the human TRPV4-RhoA in complex with AH001 -

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Basic information

Entry
Database: PDB / ID: 9iqx
TitleCryo-EM structure of the human TRPV4-RhoA in complex with AH001
Components
  • Transforming protein RhoA
  • Transient receptor potential cation channel subfamily V member 4
KeywordsMEMBRANE PROTEIN / Antagonist / Complex / Hydrolase
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 / alpha-beta T cell lineage commitment / aortic valve formation / mitotic cleavage furrow formation / positive regulation of lipase activity / bone trabecula morphogenesis / 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 / RHO GTPases Activate Rhotekin and Rhophilins / Roundabout signaling pathway / negative regulation of intracellular steroid hormone receptor signaling pathway / Axonal growth inhibition (RHOA activation) / Axonal growth stimulation / cellular hypotonic salinity response / cleavage furrow formation / regulation of neural precursor cell proliferation / cellular hypotonic response / cortical microtubule organization / regulation of modification of postsynaptic actin cytoskeleton / regulation of osteoblast proliferation / forebrain radial glial cell differentiation / multicellular organismal-level water homeostasis / apical junction assembly / regulation of modification of postsynaptic structure / cell junction assembly / negative regulation of cell migration involved in sprouting angiogenesis / cellular response to chemokine / establishment of epithelial cell apical/basal polarity / positive regulation of vascular permeability / beta selection / regulation of systemic arterial blood pressure by endothelin / osmosensory signaling pathway / negative regulation of oxidative phosphorylation / negative regulation of motor neuron apoptotic process / RHO GTPases Activate ROCKs / negative regulation of cell size / RHO GTPases activate CIT / cell-cell junction assembly / Sema4D induced cell migration and growth-cone collapse / positive regulation of monocyte chemotactic protein-1 production / PCP/CE pathway / cellular response to osmotic stress / RHO GTPases activate KTN1 / calcium ion import / cell volume homeostasis / positive regulation of podosome assembly / apolipoprotein A-I-mediated signaling pathway / positive regulation of alpha-beta T cell differentiation / Sema4D mediated inhibition of cell attachment and migration / wound healing, spreading of cells / positive regulation of leukocyte adhesion to vascular endothelial cell / PI3K/AKT activation / Wnt signaling pathway, planar cell polarity pathway / odontogenesis / motor neuron apoptotic process / ossification involved in bone maturation / regulation of aerobic respiration / regulation of focal adhesion assembly / TRP channels / negative chemotaxis / apical junction complex / cortical actin cytoskeleton / EPHA-mediated growth cone collapse / stress fiber assembly / positive regulation of cytokinesis / diet induced thermogenesis / positive regulation of macrophage chemotaxis / myosin binding / RHOC GTPase cycle / regulation of neuron projection development / cellular response to cytokine stimulus / cerebral cortex cell migration / microtubule polymerization / ERBB2 Regulates Cell Motility / cleavage furrow / semaphorin-plexin signaling pathway / calcium ion import across plasma membrane / androgen receptor signaling pathway / ficolin-1-rich granule membrane / mitotic spindle assembly / RHOA GTPase cycle / negative regulation of cell-substrate adhesion / alpha-tubulin binding / Rho protein signal transduction
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 / Rab subfamily of small GTPases / Ankyrin repeat / 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
GUANOSINE-5'-DIPHOSPHATE / Chem-P5S / : / 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: 3.37 Å
AuthorsYuan, Z. / Ruan, S.S. / Li, S.L.
Funding support China, 2items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)82425104 China
National Natural Science Foundation of China (NSFC)82150208 China
CitationJournal: Circulation / Year: 2025
Title: Inactivation of RhoA for Hypertension Treatment Through the TRPV4-RhoA-RhoGDI1 Axis.
Authors: Jiawen Wang / Zhen Yuan / Na Yu / Qian Jiao / Honglei Zhou / Wenjie Liao / Jiwei Shan / Shanshan Ruan / Yi Zhao / Ya Mo / Luyao Qi / Tiejun Li / Jianjun Fu / Bowen Ke / Yufang Xu / Xuhong ...Authors: Jiawen Wang / Zhen Yuan / Na Yu / Qian Jiao / Honglei Zhou / Wenjie Liao / Jiwei Shan / Shanshan Ruan / Yi Zhao / Ya Mo / Luyao Qi / Tiejun Li / Jianjun Fu / Bowen Ke / Yufang Xu / Xuhong Qian / Jian Zhang / Zhenjiang Zhao / Shiliang Li / Rui Wang / Honglin Li
Abstract: BACKGROUND: The RhoA (Ras homolog family member A) signaling pathway is pivotal in regulating vascular smooth muscle cells (VSMCs) function and blood pressure homeostasis. Current inhibitors of the ...BACKGROUND: The RhoA (Ras homolog family member A) signaling pathway is pivotal in regulating vascular smooth muscle cells (VSMCs) function and blood pressure homeostasis. Current inhibitors of the RhoA signaling pathway are limited in hypertension treatment, suffering from poor efficacy, insufficient specificity, and developmental challenges.
METHODS: Cryo-electron microscopy (EM), proximity ligation assay (PLA), and site-directed mutagenesis were used to explore the mechanism of RhoA activity regulation. VSMC, hypertensive animal models, ...METHODS: Cryo-electron microscopy (EM), proximity ligation assay (PLA), and site-directed mutagenesis were used to explore the mechanism of RhoA activity regulation. VSMC, hypertensive animal models, and Myh11-CRE (smooth muscle-specific RhoGDI1 knockout) mice were used to investigate the role of the TRPV4 (transient receptor potential cation channel subfamily V member 4)-RhoA-RhoGDI1 (Rho GDP dissociation inhibitor 1) axis in hypertension.
RESULTS: AH001 ((R)-1-(3-ethylphenyl) ethane-1,2-diol) was identified as a novel inhibitor of the RhoA signaling pathway. It targets the TRPV4-RhoA-RhoGDI1 axis to effectively sequester inactive RhoA- ...RESULTS: AH001 ((R)-1-(3-ethylphenyl) ethane-1,2-diol) was identified as a novel inhibitor of the RhoA signaling pathway. It targets the TRPV4-RhoA-RhoGDI1 axis to effectively sequester inactive RhoA-GDP in the plasma membrane and cytoplasm, which is distinct from typical RhoA inhibition modes. The cryo-EM structure of the TRPV4-RhoA complex showed that AH001-bound TRPV4 adopts a closed state with RhoA in an inactive GDP-bound state. Functional studies further revealed that AH001 reduced the pool of active RhoA by enhancing TRPV4-RhoA binding and facilitating RhoGDI1-RhoA interaction in VSMC. This inhibition notably decreased both acute and long-term blood pressure and prevented vascular remodeling in Ang II-induced hypertensive mice and spontaneously hypertensive rats. However, these antihypertensive effects were weakened in and Myh11-CRE mice. Additionally, AH001 effectively inhibited VSMC contraction via the RhoA/ROCK (Rho-associated protein kinase)/MYPT1 (myosin phosphatase target subunit 1)/MLC (myosin light chain 2) signaling pathway and suppressed VSMC phenotype switching to myofibroblasts through the RhoA/ROCK/LIMK1 (LIM domain kinase)/cofilin/MRTF-A (myocardin-related transcription factor A)/SRF (serum response factor) signaling cascade. TRPV4 and RhoGDI1 knockdown attenuated AH001's inhibition of VSMC contraction and phenotypic switching to myofibroblasts.
CONCLUSIONS: This study revealed a novel mode of RhoA signaling inhibition targeting the TRPV4-RhoA-RhoGDI1 axis, offering new insights for future antihypertensive drug development and proposing ...CONCLUSIONS: This study revealed a novel mode of RhoA signaling inhibition targeting the TRPV4-RhoA-RhoGDI1 axis, offering new insights for future antihypertensive drug development and proposing innovative strategies for targeting challenging Rho GTPases.
History
DepositionJul 13, 2024Deposition site: PDBJ / Processing site: PDBC
Revision 1.0May 28, 2025Provider: repository / Type: Initial release
Revision 1.0May 28, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0May 28, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0May 28, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0May 28, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0May 28, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Jul 2, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)338,60514
Polymers335,7536
Non-polymers2,8528
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, C2 symmetry
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 2 types, 6 molecules CDABEF

#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: 73038.719 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.: 2
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

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Non-polymers , 4 types, 8 molecules

#3: Chemical ChemComp-P5S / O-[(R)-{[(2R)-2,3-bis(octadecanoyloxy)propyl]oxy}(hydroxy)phosphoryl]-L-serine / phosphatidyl serine


Mass: 792.075 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C42H82NO10P
#4: Chemical ChemComp-U6L / (1~{R})-1-(3-ethylphenyl)ethane-1,2-diol / SCHEMBL24015313


Mass: 166.217 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H14O2
#5: Chemical ChemComp-GDP / GUANOSINE-5'-DIPHOSPHATE


Type: RNA linking / Mass: 443.201 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H15N5O11P2 / Comment: GDP, energy-carrying molecule*YM
#6: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg

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Details

Has ligand of interestY
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: Cryo-EM structure of the human TRPV4-RhoA in complex with AH001
Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.5
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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Calibrated magnification: 64000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOCONTINUUM (6k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.19.2_4158: / Category: model refinement
CTF correctionType: NONE
3D reconstructionResolution: 3.37 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 71000 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00322694
ELECTRON MICROSCOPYf_angle_d0.61530776
ELECTRON MICROSCOPYf_dihedral_angle_d5.0283092
ELECTRON MICROSCOPYf_chiral_restr0.0413530
ELECTRON MICROSCOPYf_plane_restr0.0063842

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