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- PDB-8ugb: Cryo-EM structure of bovine phosphodiesterase 6 bound to udenafil -

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Basic information

Entry
Database: PDB / ID: 8ugb
TitleCryo-EM structure of bovine phosphodiesterase 6 bound to udenafil
Components
  • (Rod cGMP-specific 3',5'-cyclic phosphodiesterase subunit ...) x 2
  • Retinal rod rhodopsin-sensitive cGMP 3',5'-cyclic phosphodiesterase subunit gamma
KeywordsSIGNALING PROTEIN/INHIBITOR / phosphodiesterase / GPCR effector enzyme / SIGNALING PROTEIN / inhibitor / SIGNALING PROTEIN-INHIBITOR complex
Function / homology
Function and homology information


3',5'-cyclic-GMP phosphodiesterase / Inactivation, recovery and regulation of the phototransduction cascade / positive regulation of G protein-coupled receptor signaling pathway / Activation of the phototransduction cascade / ion binding / Ca2+ pathway / response to stimulus / positive regulation of epidermal growth factor receptor signaling pathway / photoreceptor outer segment membrane / entrainment of circadian clock by photoperiod ...3',5'-cyclic-GMP phosphodiesterase / Inactivation, recovery and regulation of the phototransduction cascade / positive regulation of G protein-coupled receptor signaling pathway / Activation of the phototransduction cascade / ion binding / Ca2+ pathway / response to stimulus / positive regulation of epidermal growth factor receptor signaling pathway / photoreceptor outer segment membrane / entrainment of circadian clock by photoperiod / 3',5'-cyclic-GMP phosphodiesterase activity / cGMP binding / 3',5'-cyclic-nucleotide phosphodiesterase activity / visual perception / photoreceptor disc membrane / retina development in camera-type eye / positive regulation of MAPK cascade / molecular adaptor activity / signal transduction / zinc ion binding / metal ion binding
Similarity search - Function
Retinal cGMP phosphodiesterase, gamma subunit / Retinal cGMP phosphodiesterase, gamma subunit superfamily / Retinal cGMP phosphodiesterase, gamma subunit / GAF domain / 3'5'-cyclic nucleotide phosphodiesterase / Domain present in phytochromes and cGMP-specific phosphodiesterases. / 3'5'-cyclic nucleotide phosphodiesterase, catalytic domain / 3'5'-cyclic nucleotide phosphodiesterase, conserved site / 3'5'-cyclic nucleotide phosphodiesterase, catalytic domain superfamily / 3'5'-cyclic nucleotide phosphodiesterase ...Retinal cGMP phosphodiesterase, gamma subunit / Retinal cGMP phosphodiesterase, gamma subunit superfamily / Retinal cGMP phosphodiesterase, gamma subunit / GAF domain / 3'5'-cyclic nucleotide phosphodiesterase / Domain present in phytochromes and cGMP-specific phosphodiesterases. / 3'5'-cyclic nucleotide phosphodiesterase, catalytic domain / 3'5'-cyclic nucleotide phosphodiesterase, conserved site / 3'5'-cyclic nucleotide phosphodiesterase, catalytic domain superfamily / 3'5'-cyclic nucleotide phosphodiesterase / 3'5'-cyclic nucleotide phosphodiesterase domain signature. / 3'5'-cyclic nucleotide phosphodiesterase domain profile. / GAF domain / GAF-like domain superfamily / Metal dependent phosphohydrolases with conserved 'HD' motif. / HD/PDEase domain
Similarity search - Domain/homology
CYCLIC GUANOSINE MONOPHOSPHATE / Udenafil / Retinal rod rhodopsin-sensitive cGMP 3',5'-cyclic phosphodiesterase subunit gamma / Rod cGMP-specific 3',5'-cyclic phosphodiesterase subunit alpha / Rod cGMP-specific 3',5'-cyclic phosphodiesterase subunit beta
Similarity search - Component
Biological speciesBos taurus (cattle)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsAplin, C. / Cerione, R.A.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Eye Institute (NIH/NEI)1R01EY034867-01 United States
CitationJournal: J Biol Chem / Year: 2024
Title: Probing the mechanism by which the retinal G protein transducin activates its biological effector PDE6.
Authors: Cody Aplin / Richard A Cerione /
Abstract: Phototransduction in retinal rods occurs when the G protein-coupled photoreceptor rhodopsin triggers the activation of phosphodiesterase 6 (PDE6) by GTP-bound alpha subunits of the G protein ...Phototransduction in retinal rods occurs when the G protein-coupled photoreceptor rhodopsin triggers the activation of phosphodiesterase 6 (PDE6) by GTP-bound alpha subunits of the G protein transducin (Gα). Recently, we presented a cryo-EM structure for a complex between two GTP-bound recombinant Gα subunits and native PDE6, that included a bivalent antibody bound to the C-terminal ends of Gα and the inhibitor vardenafil occupying the active sites on the PDEα and PDEβ subunits. We proposed Gα-activated PDE6 by inducing a striking reorientation of the PDEγ subunits away from the catalytic sites. However, questions remained including whether in the absence of the antibody Gα binds to PDE6 in a similar manner as observed when the antibody is present, does Gα activate PDE6 by enabling the substrate cGMP to access the catalytic sites, and how does the lipid membrane enhance PDE6 activation? Here, we demonstrate that 2:1 Gα-PDE6 complexes form with either recombinant or retinal Gα in the absence of the Gα antibody. We show that Gα binding is not necessary for cGMP nor competitive inhibitors to access the active sites; instead, occupancy of the substrate binding sites enables Gα to bind and reposition the PDE6γ subunits to promote catalytic activity. Moreover, we demonstrate by reconstituting Gα-stimulated PDE6 activity in lipid bilayer nanodiscs that the membrane-induced enhancement results from an increase in the apparent binding affinity of Gα for PDE6. These findings provide new insights into how the retinal G protein stimulates rapid catalytic turnover by PDE6 required for dim light vision.
History
DepositionOct 5, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 17, 2024Provider: repository / Type: Initial release
Revision 1.1Feb 7, 2024Group: Database references / Category: citation / Item: _citation.journal_volume

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Rod cGMP-specific 3',5'-cyclic phosphodiesterase subunit alpha
B: Rod cGMP-specific 3',5'-cyclic phosphodiesterase subunit beta
C: Retinal rod rhodopsin-sensitive cGMP 3',5'-cyclic phosphodiesterase subunit gamma
D: Retinal rod rhodopsin-sensitive cGMP 3',5'-cyclic phosphodiesterase subunit gamma
hetero molecules


Theoretical massNumber of molelcules
Total (without water)219,18312
Polymers217,2804
Non-polymers1,9038
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Rod cGMP-specific 3',5'-cyclic phosphodiesterase subunit ... , 2 types, 2 molecules AB

#1: Protein Rod cGMP-specific 3',5'-cyclic phosphodiesterase subunit alpha / GMP-PDE alpha / PDE V-B1


Mass: 99461.789 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Bos taurus (cattle)
References: UniProt: P11541, 3',5'-cyclic-GMP phosphodiesterase
#2: Protein Rod cGMP-specific 3',5'-cyclic phosphodiesterase subunit beta / GMP-PDE beta


Mass: 98449.648 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Bos taurus (cattle)
References: UniProt: P23439, 3',5'-cyclic-GMP phosphodiesterase

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Protein , 1 types, 2 molecules CD

#3: Protein Retinal rod rhodopsin-sensitive cGMP 3',5'-cyclic phosphodiesterase subunit gamma / GMP-PDE gamma


Mass: 9684.229 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Bos taurus (cattle)
References: UniProt: P04972, 3',5'-cyclic-GMP phosphodiesterase

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Non-polymers , 4 types, 8 molecules

#4: Chemical ChemComp-PCG / CYCLIC GUANOSINE MONOPHOSPHATE / Cyclic guanosine monophosphate


Mass: 345.205 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H12N5O7P / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#7: Chemical ChemComp-ZUD / Udenafil / (3P)-3-(1-methyl-7-oxo-3-propyl-4,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-5-yl)-N-{2-[(2R)-1-methylpyrrolidin-2-yl]ethyl}-4-propoxybenzene-1-sulfonamide


Mass: 516.656 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C25H36N6O4S / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY

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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: Bovine rod phosphodiesterase 6 bound to udenafil / Type: COMPLEX / Entity ID: #3, #1-#2 / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Bos taurus (cattle)
Buffer solutionpH: 8
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: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 63000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 1400 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 2050380 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 95.37 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00314022
ELECTRON MICROSCOPYf_angle_d0.655218923
ELECTRON MICROSCOPYf_chiral_restr0.06042042
ELECTRON MICROSCOPYf_plane_restr0.00582422
ELECTRON MICROSCOPYf_dihedral_angle_d6.67421842

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