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- PDB-9ax5: Cryo-EM structure of Phospholipase C epsilon PH-C terminus in com... -

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Basic information

Entry
Database: PDB / ID: 9ax5
TitleCryo-EM structure of Phospholipase C epsilon PH-C terminus in complex with RhoA-GTP
Components
  • 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase epsilon-1
  • Transforming protein RhoA
KeywordsMEMBRANE PROTEIN / GPCR signaling / complex / phospholipase / PIP2 hydrolysis / G protein
Function / homology
Function and homology information


diacylglycerol biosynthetic process / Synthesis of IP3 and IP4 in the cytosol / phosphoinositide phospholipase C / alpha-beta T cell lineage commitment / aortic valve formation / positive regulation of lipase activity / 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 ...diacylglycerol biosynthetic process / Synthesis of IP3 and IP4 in the cytosol / phosphoinositide phospholipase C / alpha-beta T cell lineage commitment / aortic valve formation / positive regulation of lipase activity / 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 / bone trabecula morphogenesis / RHO GTPases Activate Rhotekin and Rhophilins / Roundabout signaling pathway / negative regulation of intracellular steroid hormone receptor signaling pathway / phosphatidylinositol metabolic process / Axonal growth inhibition (RHOA activation) / Axonal growth stimulation / cleavage furrow formation / phosphatidylinositol-4,5-bisphosphate phospholipase C activity / regulation of neural precursor cell proliferation / regulation of osteoblast proliferation / regulation of modification of postsynaptic actin cytoskeleton / forebrain radial glial cell differentiation / mitotic cleavage furrow formation / C-type glycerophospholipase activity / apical junction assembly / negative regulation of cell migration involved in sprouting angiogenesis / glomerulus development / cell junction assembly / beta selection / establishment of epithelial cell apical/basal polarity / cellular response to chemokine / negative regulation of motor neuron apoptotic process / regulation of systemic arterial blood pressure by endothelin / negative regulation of oxidative phosphorylation / negative regulation of cell size / regulation of modification of postsynaptic structure / RHO GTPases Activate ROCKs / RHO GTPases activate CIT / PCP/CE pathway / Sema4D induced cell migration and growth-cone collapse / RHO GTPases activate KTN1 / positive regulation of podosome assembly / positive regulation of alpha-beta T cell differentiation / apolipoprotein A-I-mediated signaling pathway / Sema4D mediated inhibition of cell attachment and migration / wound healing, spreading of cells / PI3K/AKT activation / motor neuron apoptotic process / positive regulation of leukocyte adhesion to vascular endothelial cell / Wnt signaling pathway, planar cell polarity pathway / odontogenesis / ossification involved in bone maturation / regulation of focal adhesion assembly / androgen receptor signaling pathway / negative chemotaxis / EPHA-mediated growth cone collapse / apical junction complex / stress fiber assembly / myosin binding / positive regulation of cytokinesis / RHOC GTPase cycle / regulation of neuron projection development / cellular response to cytokine stimulus / phosphatidylinositol-mediated signaling / cerebral cortex cell migration / positive regulation of protein serine/threonine kinase activity / ERBB2 Regulates Cell Motility / cleavage furrow / semaphorin-plexin signaling pathway / ficolin-1-rich granule membrane / negative regulation of cell-substrate adhesion / RHOA GTPase cycle / mitotic spindle assembly / positive regulation of T cell migration / endothelial cell migration / skeletal muscle tissue development / PTK6 Regulates RHO GTPases, RAS GTPase and MAP kinases / lipid catabolic process / positive regulation of lamellipodium assembly / GPVI-mediated activation cascade / Rho protein signal transduction / RHO GTPases activate PKNs / negative regulation of reactive oxygen species biosynthetic process / positive regulation of stress fiber assembly / cytoplasmic microtubule organization / release of sequestered calcium ion into cytosol / EPHB-mediated forward signaling / positive regulation of neuron differentiation / substrate adhesion-dependent cell spreading / substantia nigra development / guanyl-nucleotide exchange factor activity / regulation of cell migration / secretory granule membrane / regulation of microtubule cytoskeleton organization / cell-matrix adhesion / small monomeric GTPase / TGF-beta receptor signaling in EMT (epithelial to mesenchymal transition) / cell periphery
Similarity search - Function
1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase epsilon-1 / : / : / Ras association (RalGDS/AF-6) domain / Phosphoinositide phospholipase C family / Phospholipase C, phosphatidylinositol-specific, Y domain / Phosphatidylinositol-specific phospholipase C, Y domain / Phosphatidylinositol-specific phospholipase Y-box domain profile. / Phospholipase C, catalytic domain (part); domain Y / Ras association (RalGDS/AF-6) domain ...1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase epsilon-1 / : / : / Ras association (RalGDS/AF-6) domain / Phosphoinositide phospholipase C family / Phospholipase C, phosphatidylinositol-specific, Y domain / Phosphatidylinositol-specific phospholipase C, Y domain / Phosphatidylinositol-specific phospholipase Y-box domain profile. / Phospholipase C, catalytic domain (part); domain Y / Ras association (RalGDS/AF-6) domain / Phosphatidylinositol-specific phospholipase C, X domain / Phosphatidylinositol-specific phospholipase C, X domain / Phospholipase C, catalytic domain (part); domain X / Ras-associating (RA) domain profile. / Phosphatidylinositol-specific phospholipase X-box domain profile. / Ras-associating (RA) domain / Ras guanine nucleotide exchange factor domain superfamily / Ras guanine-nucleotide exchange factor, catalytic domain superfamily / RasGEF domain / Ras guanine-nucleotide exchange factors catalytic domain profile. / Guanine nucleotide exchange factor for Ras-like small GTPases / Ras guanine-nucleotide exchange factors catalytic domain / PLC-like phosphodiesterase, TIM beta/alpha-barrel domain superfamily / Small GTPase Rho / Small GTPase Rho domain profile. / Protein kinase C conserved region 2 (CalB) / C2 domain / C2 domain / C2 domain profile. / C2 domain superfamily / Rho (Ras homology) subfamily of Ras-like small GTPases / Ras subfamily of RAS small GTPases / Small GTPase / Ras family / Rab subfamily of small GTPases / EF-hand domain pair / Small GTP-binding protein domain / Ubiquitin-like domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
GUANOSINE-5'-TRIPHOSPHATE / Transforming protein RhoA / 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase epsilon-1
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsOhri, V. / Lyon, A.M.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)1R01HL141076-01 United States
CitationJournal: Commun Biol / Year: 2025
Title: RhoA allosterically activates phospholipase Cε via its EF hands.
Authors: Vaani Ohri / Kadidia Samassekou / Kaushik Muralidharan / Elisabeth E Garland-Kuntz / Isaac J Fisher / William C Hogan / Bailey M Davis / Angeline M Lyon /
Abstract: Phospholipase Cε (PLCε) cleaves phosphatidylinositol lipids to increase intracellular Ca and activate protein kinase C (PKC) in response to stimulation of cell surface receptors. PLCε is activated ...Phospholipase Cε (PLCε) cleaves phosphatidylinositol lipids to increase intracellular Ca and activate protein kinase C (PKC) in response to stimulation of cell surface receptors. PLCε is activated via direct binding of small GTPases at the cytoplasmic leaflets of cellular membranes. In the cardiovascular system, the RhoA GTPase regulates PLCε to initiate a pathway that protects against ischemia/reperfusion injuries, but the underlying molecular mechanism is not known. We present here the cryo-electron microscopy (cryo-EM) reconstruction of RhoA bound to PLCε, showing that the G protein binds a unique insertion within the PLCε EF hands. Deletion of or mutations to this PLCε insertion decrease RhoA-dependent activation without impacting its regulation by other G proteins. Together, our data support a model wherein RhoA binding to PLCε allosterically activates the lipase and increases its interactions with the membrane, resulting in maximum activity and cardiomyocyte survival.
History
DepositionMar 5, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 12, 2025Provider: repository / Type: Initial release
Revision 1.0Mar 12, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Mar 12, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase epsilon-1
C: Transforming protein RhoA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)190,3405
Polymers189,7522
Non-polymers5883
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase epsilon-1 / Phosphoinositide phospholipase C-epsilon-1 / Phospholipase C-epsilon-1 / PLC-epsilon-1


Mass: 165182.938 Da / Num. of mol.: 1 / Fragment: PH-C terminal residues 837-2281
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Plce1, Plce / Plasmid: pFastBacHTA / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: Q99P84, phosphoinositide phospholipase C
#2: Protein Transforming protein RhoA / Rho cDNA clone 12 / h12


Mass: 24569.055 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RHOA, ARH12, ARHA, RHO12 / Plasmid: pFastBacHTA / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P61586, small monomeric GTPase
#3: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE


Mass: 523.180 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: C10H16N5O14P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: GTP, energy-carrying molecule*YM
#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationN

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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

Component
IDNameTypeEntity IDParent-IDSource
1PLCe PH-C in complex with RhoA-GTPCOMPLEX#1-#20MULTIPLE SOURCES
2PH-C fragment (837-2281) of PLC epsilonCOMPLEX#11RECOMBINANT
3RhoA-GTPCOMPLEX#21RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.189 MDaNO
210.165 MDaNO
310.024 MDaNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Rattus norvegicus (Norway rat)10116
23Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDPlasmid
12Spodoptera frugiperda (fall armyworm)7108pFastBacHTA
23Trichoplusia ni (cabbage looper)7111pFastBacHTA
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
120 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acidHEPES1
2150 mMsodium chlorideNaCl1
32 mMdithiothreitolDTT1
40.1 mMethylenediaminetetraacetic acidEDTA1
50.1 mMegtazic acidEGTA1
61 mMmagnesium dichlorideMgCl21
70.04 mMguanosine triphosphateGTP1
80.5 mMcalcium chlorideCaCl21
92.5 mM3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonateCHAPS1
SpecimenConc.: 0.6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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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 / Nominal magnification: 81000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 3.2 sec. / Electron dose: 57.8 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 6345

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1329298
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 209463 / Symmetry type: POINT

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