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- PDB-9edd: Reset Type-I Protein Kinase A Holoenzyme -

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Basic information

Entry
Database: PDB / ID: 9edd
TitleReset Type-I Protein Kinase A Holoenzyme
Components
  • cAMP-dependent protein kinase catalytic subunit alpha
  • cAMP-dependent protein kinase type I-alpha regulatory subunit
KeywordsSIGNALING PROTEIN / Kinase / Regulator
Function / homology
Function and homology information


PKA-mediated phosphorylation of CREB / PKA-mediated phosphorylation of key metabolic factors / sperm head-tail coupling apparatus / ROBO receptors bind AKAP5 / HDL assembly / PKA activation in glucagon signalling / DARPP-32 events / CREB1 phosphorylation through the activation of Adenylate Cyclase / GPER1 signaling / channel activator activity ...PKA-mediated phosphorylation of CREB / PKA-mediated phosphorylation of key metabolic factors / sperm head-tail coupling apparatus / ROBO receptors bind AKAP5 / HDL assembly / PKA activation in glucagon signalling / DARPP-32 events / CREB1 phosphorylation through the activation of Adenylate Cyclase / GPER1 signaling / channel activator activity / Factors involved in megakaryocyte development and platelet production / Regulation of glycolysis by fructose 2,6-bisphosphate metabolism / mitochondrial protein catabolic process / PKA activation / nucleotide-activated protein kinase complex / cell communication by electrical coupling involved in cardiac conduction / Hedgehog 'off' state / high-density lipoprotein particle assembly / Rap1 signalling / negative regulation of cAMP/PKA signal transduction / cAMP-dependent protein kinase inhibitor activity / cAMP-dependent protein kinase / regulation of protein processing / cAMP-dependent protein kinase activity / sarcomere organization / protein localization to lipid droplet / cAMP-dependent protein kinase complex / regulation of bicellular tight junction assembly / cellular response to parathyroid hormone stimulus / Loss of phosphorylation of MECP2 at T308 / CREB1 phosphorylation through the activation of Adenylate Cyclase / negative regulation of interleukin-2 production / PKA activation / regulation of osteoblast differentiation / cellular response to cold / sperm capacitation / negative regulation of glycolytic process through fructose-6-phosphate / Triglyceride catabolism / ciliary base / High laminar flow shear stress activates signaling by PIEZO1 and PECAM1:CDH5:KDR in endothelial cells / Vasopressin regulates renal water homeostasis via Aquaporins / protein kinase A regulatory subunit binding / negative regulation of activated T cell proliferation / protein kinase A catalytic subunit binding / intracellular potassium ion homeostasis / mesoderm formation / RET signaling / cAMP/PKA signal transduction / Interleukin-3, Interleukin-5 and GM-CSF signaling / immunological synapse / plasma membrane raft / axoneme / PKA activation in glucagon signalling / Regulation of MECP2 expression and activity / DARPP-32 events / regulation of cardiac conduction / regulation of macroautophagy / cardiac muscle cell proliferation / regulation of cardiac muscle contraction / sperm flagellum / postsynaptic modulation of chemical synaptic transmission / regulation of cardiac muscle contraction by regulation of the release of sequestered calcium ion / vascular endothelial cell response to laminar fluid shear stress / renal water homeostasis / cAMP binding / Hedgehog 'off' state / Ion homeostasis / negative regulation of TORC1 signaling / regulation of proteasomal protein catabolic process / sperm midpiece / multivesicular body / cellular response to epinephrine stimulus / calcium channel complex / positive regulation of gluconeogenesis / protein serine/threonine/tyrosine kinase activity / Mitochondrial protein degradation / cellular response to glucagon stimulus / Loss of Nlp from mitotic centrosomes / Loss of proteins required for interphase microtubule organization from the centrosome / Recruitment of mitotic centrosome proteins and complexes / acrosomal vesicle / CD209 (DC-SIGN) signaling / positive regulation of calcium-mediated signaling / Recruitment of NuMA to mitotic centrosomes / Anchoring of the basal body to the plasma membrane / regulation of heart rate / FCGR3A-mediated IL10 synthesis / protein export from nucleus / positive regulation of phagocytosis / AURKA Activation by TPX2 / positive regulation of protein export from nucleus / negative regulation of smoothened signaling pathway / neural tube closure / Regulation of insulin secretion / neuromuscular junction / cellular response to glucose stimulus / Degradation of GLI1 by the proteasome / positive regulation of cholesterol biosynthetic process / Degradation of GLI2 by the proteasome / GLI3 is processed to GLI3R by the proteasome
Similarity search - Function
cAMP-dependent protein kinase regulatory subunit / cAMP-dependent protein kinase regulatory subunit, dimerization-anchoring domain / Regulatory subunit of type II PKA R-subunit / RIIalpha, Regulatory subunit portion of type II PKA R-subunit / : / Cyclic nucleotide-binding domain signature 2. / cAMP-dependent protein kinase catalytic subunit / Cyclic nucleotide-binding domain signature 1. / Cyclic nucleotide-binding, conserved site / Cyclic nucleotide-monophosphate binding domain ...cAMP-dependent protein kinase regulatory subunit / cAMP-dependent protein kinase regulatory subunit, dimerization-anchoring domain / Regulatory subunit of type II PKA R-subunit / RIIalpha, Regulatory subunit portion of type II PKA R-subunit / : / Cyclic nucleotide-binding domain signature 2. / cAMP-dependent protein kinase catalytic subunit / Cyclic nucleotide-binding domain signature 1. / Cyclic nucleotide-binding, conserved site / Cyclic nucleotide-monophosphate binding domain / Cyclic nucleotide-binding domain / cAMP/cGMP binding motif profile. / Cyclic nucleotide-binding domain / Cyclic nucleotide-binding domain superfamily / Extension to Ser/Thr-type protein kinases / AGC-kinase, C-terminal / AGC-kinase C-terminal domain profile. / RmlC-like jelly roll fold / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / Protein kinase domain / Serine/Threonine protein kinases, catalytic domain / Protein kinase, ATP binding site / Protein kinases ATP-binding region signature. / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily
Similarity search - Domain/homology
ADENOSINE-5'-TRIPHOSPHATE / cAMP-dependent protein kinase type I-alpha regulatory subunit / cAMP-dependent protein kinase catalytic subunit alpha
Similarity search - Component
Biological speciesHomo sapiens (human)
Bos taurus (domestic cattle)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 6.3 Å
AuthorsVenkatakrishnan, V. / Buckley, T. / Laremore, T.N. / Armache, J.P. / Anand, G.S.
Funding support United States, 1items
OrganizationGrant numberCountry
Not funded United States
CitationJournal: J Am Chem Soc / Year: 2025
Title: Multiplicity of Regulatory Subunit Conformations Defines Structural Ensemble of Reset Protein Kinase A Holoenzyme.
Authors: Varun Venkatakrishnan / Tatiana N Laremore / Theresa S C Buckley / Jean-Paul Armache / Ganesh S Anand /
Abstract: How protein kinase A (PKA) is reset to a basal state following 3'5'-cyclic adenosine monophosphate (cAMP)-mediated activation is unknown. Here we describe the mechanism of cAMP-PKA type I signal ...How protein kinase A (PKA) is reset to a basal state following 3'5'-cyclic adenosine monophosphate (cAMP)-mediated activation is unknown. Here we describe the mechanism of cAMP-PKA type I signal termination leading to a reset of PKA by holoenzyme formation through the obligatory action of phosphodiesterases (PDEs). We report a catalytic subunit (Cα)-assisted mechanism for the reset of type I PKA and describe for the first time multiple structures of the reset PKA holoenzyme (RIα:Cα) that capture an ensemble of multiple conformational end-states through integrative electron microscopy and structural mass spectrometry approaches. Together these complementary methods highlight the large conformational dynamics of the regulatory subunit (RIα) within the tetrameric reset PKA holoenzyme. The cAMP-free reset PKA holoenzyme adopts multiple distinct conformations of RIα with contributions from the N-terminal linker and CNB-B dynamics. Our findings highlight the interplay between RIα, Cα, and PDEs (PDE8) in cAMP-PKA signalosomes to offer a new paradigm for PDE-mediated regulation of cAMP-PKA signaling.
History
DepositionNov 16, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 23, 2025Provider: repository / Type: Initial release
Revision 1.1May 14, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: cAMP-dependent protein kinase catalytic subunit alpha
B: cAMP-dependent protein kinase type I-alpha regulatory subunit
hetero molecules


Theoretical massNumber of molelcules
Total (without water)88,6233
Polymers88,1162
Non-polymers5071
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 cAMP-dependent protein kinase catalytic subunit alpha / PKA C-alpha


Mass: 40657.316 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PRKACA, PKACA / Production host: Escherichia coli (E. coli) / References: UniProt: P17612, cAMP-dependent protein kinase
#2: Protein cAMP-dependent protein kinase type I-alpha regulatory subunit


Mass: 47458.473 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bos taurus (domestic cattle) / Gene: PRKAR1A / Production host: Escherichia coli (E. coli) / References: UniProt: P00514
#3: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
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: Reset type-I PKA holoenzyme complex of regulatory and catalytic subunits
Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7
Buffer component
IDConc.NameFormulaBuffer-ID
150 mM3-(N-morpholino)propanesulfonic acidMOPS1
250 mMSodium ChlorideNaCl1
35 mMMagnesium ChlorideMgCl21
40.1 mMAdenosine 5-TriphosphateATP1
51 mM2-MercaptoethanolB-ME1
SpecimenConc.: 0.6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: SPOT SCAN
Electron lensMode: OTHER / Nominal defocus max: 2200 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 49.66 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of real images: 4559

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.2particle selection
4cryoSPARC4.2CTF correction
7UCSF ChimeraX1.8model fitting
8Coot0.9model fitting
11cryoSPARC4.2final Euler assignment
12cryoSPARC4.2classification
13cryoSPARC4.23D reconstruction
14Coot0.9model refinement
15PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2683140
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 6.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 12483 / Symmetry type: POINT
Atomic model buildingB value: 467.63 / Protocol: RIGID BODY FIT / Space: REAL
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
13FHI

3fhi

13FHI1PDBexperimental model
22QCS

2qcs

12QCS2PDBexperimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0064715
ELECTRON MICROSCOPYf_angle_d1.1176396
ELECTRON MICROSCOPYf_dihedral_angle_d8.862639
ELECTRON MICROSCOPYf_chiral_restr0.064697
ELECTRON MICROSCOPYf_plane_restr0.008822

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