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- EMDB-42220: Cryo-EM structure of bovine phosphodiesterase 6 bound to udenafil -

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Basic information

Entry
Database: EMDB / ID: EMD-42220
TitleCryo-EM structure of bovine phosphodiesterase 6 bound to udenafil
Map data
Sample
  • Complex: Bovine rod phosphodiesterase 6 bound to udenafil
    • Protein or peptide: Retinal rod rhodopsin-sensitive cGMP 3',5'-cyclic phosphodiesterase subunit gamma
    • Protein or peptide: Rod cGMP-specific 3',5'-cyclic phosphodiesterase subunit alpha
    • Protein or peptide: Rod cGMP-specific 3',5'-cyclic phosphodiesterase subunit beta
  • Ligand: CYCLIC GUANOSINE MONOPHOSPHATE
  • Ligand: ZINC ION
  • Ligand: MAGNESIUM ION
  • Ligand: Udenafil
Keywordsphosphodiesterase / 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 / Ca2+ pathway / positive regulation of epidermal growth factor receptor signaling pathway / photoreceptor outer segment membrane / entrainment of circadian clock by photoperiod / cGMP binding / 3',5'-cyclic-GMP phosphodiesterase activity ...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 / Ca2+ pathway / positive regulation of epidermal growth factor receptor signaling pathway / photoreceptor outer segment membrane / entrainment of circadian clock by photoperiod / cGMP binding / 3',5'-cyclic-GMP phosphodiesterase activity / 3',5'-cyclic-AMP phosphodiesterase activity / cAMP-mediated signaling / visual perception / photoreceptor disc membrane / retina development in camera-type eye / molecular adaptor activity / 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 / 3'5'-cyclic nucleotide phosphodiesterase / 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. ...Retinal cGMP phosphodiesterase, gamma subunit / Retinal cGMP phosphodiesterase, gamma subunit superfamily / Retinal cGMP phosphodiesterase, gamma subunit / 3'5'-cyclic nucleotide phosphodiesterase / 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 / Domain present in phytochromes and cGMP-specific phosphodiesterases. / GAF domain / GAF-like domain superfamily / Metal dependent phosphohydrolases with conserved 'HD' motif. / HD/PDEase domain
Similarity search - Domain/homology
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsAplin C / Cerione RA
Funding support United States, 1 items
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, 2023-
Header (metadata) releaseJan 17, 2024-
Map releaseJan 17, 2024-
UpdateFeb 7, 2024-
Current statusFeb 7, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_42220.png

Downloads & links

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Map

FileDownload / File: emd_42220.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.31 Å/pix.
x 256 pix.
= 335.36 Å		1.31 Å/pix.
x 256 pix.
= 335.36 Å		1.31 Å/pix.
x 256 pix.
= 335.36 Å

Surface

Projections

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.31 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.276
Minimum - Maximum-0.59358925 - 1.3507295
Average (Standard dev.)-0.00036745085 (±0.030057142)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 335.36 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_42220_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_42220_half_map_2.map
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : Bovine rod phosphodiesterase 6 bound to udenafil

EntireName: Bovine rod phosphodiesterase 6 bound to udenafil
Components
  • Complex: Bovine rod phosphodiesterase 6 bound to udenafil
    • Protein or peptide: Retinal rod rhodopsin-sensitive cGMP 3',5'-cyclic phosphodiesterase subunit gamma
    • Protein or peptide: Rod cGMP-specific 3',5'-cyclic phosphodiesterase subunit alpha
    • Protein or peptide: Rod cGMP-specific 3',5'-cyclic phosphodiesterase subunit beta
  • Ligand: CYCLIC GUANOSINE MONOPHOSPHATE
  • Ligand: ZINC ION
  • Ligand: MAGNESIUM ION
  • Ligand: Udenafil

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Supramolecule #1: Bovine rod phosphodiesterase 6 bound to udenafil

SupramoleculeName: Bovine rod phosphodiesterase 6 bound to udenafil / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #3, #1-#2
Source (natural)Organism: Bos taurus (cattle)

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Macromolecule #1: Rod cGMP-specific 3',5'-cyclic phosphodiesterase subunit alpha

MacromoleculeName: Rod cGMP-specific 3',5'-cyclic phosphodiesterase subunit alpha
type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: 3',5'-cyclic-GMP phosphodiesterase
Source (natural)Organism: Bos taurus (cattle)
Molecular weightTheoretical: 99.461789 KDa
SequenceString: MGEVTAEEVE KFLDSNVSFA KQYYNLRYRA KVISDLLGPR EAAVDFSNYH ALNSVEESEI IFDLLRDFQD NLQAEKCVFN VMKKLCFLL QADRMSLFMY RARNGIAELA TRLFNVHKDA VLEECLVAPD SEIVFPLDMG VVGHVALSKK IVNVPNTEED E HFCDFVDT ...String:
MGEVTAEEVE KFLDSNVSFA KQYYNLRYRA KVISDLLGPR EAAVDFSNYH ALNSVEESEI IFDLLRDFQD NLQAEKCVFN VMKKLCFLL QADRMSLFMY RARNGIAELA TRLFNVHKDA VLEECLVAPD SEIVFPLDMG VVGHVALSKK IVNVPNTEED E HFCDFVDT LTEYQTKNIL ASPIMNGKDV VAIIMVVNKV DGPHFTENDE EILLKYLNFA NLIMKVFHLS YLHNCETRRG QI LLWSGSK VFEELTDIER QFHKALYTVR AFLNCDRYSV GLLDMTKQKE FFDVWPVLMG EAPPYAGPRT PDGREINFYK VID YILHGK EDIKVIPNPP PDHWALVSGL PTYVAQNGLI CNIMNAPSED FFAFQKEPLD ESGWMIKNVL SMPIVNKKEE IVGV ATFYN RKDGKPFDEM DETLMESLTQ FLGWSVLNPD TYELMNKLEN RKDIFQDMVK YHVKCDNEEI QTILKTREVY GKEPW ECEE EELAEILQGE LPDADKYEIN KFHFSDLPLT ELELVKCGIQ MYYELKVVDK FHIPQEALVR FMYSLSKGYR RITYHN WRH GFNVGQTMFS LLVTGKLKRY FTDLEALAMV TAAFCHDIDH RGTNNLYQMK SQNPLAKLHG SSILERHHLE FGKTLLR DE SLNIFQNLNR RQHEHAIHMM DIAIIATDLA LYFKKRTMFQ KIVDQSKTYE TQQEWTQYMM LDQTRKEIVM AMMMTACD L SAITKPWEVQ SKVALLVAAE FWEQGDLERT VLQQNPIPMM DRNKADELPK LQVGFIDFVC TFVYKEFSRF HEEITPMLD GITNNRKEWK ALADEYETKM KGLEEEKQKQ QAANQAAAGS QHGGKQPGGG PASKSCCVQ

UniProtKB: Rod cGMP-specific 3',5'-cyclic phosphodiesterase subunit alpha

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Macromolecule #2: Rod cGMP-specific 3',5'-cyclic phosphodiesterase subunit beta

MacromoleculeName: Rod cGMP-specific 3',5'-cyclic phosphodiesterase subunit beta
type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: 3',5'-cyclic-GMP phosphodiesterase
Source (natural)Organism: Bos taurus (cattle)
Molecular weightTheoretical: 98.449648 KDa
SequenceString: MSLSEGQVHR FLDQNPGFAD QYFGRKLSPE DVANACEDGC PEGCTSFREL CQVEESAALF ELVQDMQENV NMERVVFKIL RRLCSILHA DRCSLFMYRQ RNGVAELATR LFSVQPDSVL EDCLVPPDSE IVFPLDIGVV GHVAQTKKMV NVQDVMECPH F SSFADELT ...String:
MSLSEGQVHR FLDQNPGFAD QYFGRKLSPE DVANACEDGC PEGCTSFREL CQVEESAALF ELVQDMQENV NMERVVFKIL RRLCSILHA DRCSLFMYRQ RNGVAELATR LFSVQPDSVL EDCLVPPDSE IVFPLDIGVV GHVAQTKKMV NVQDVMECPH F SSFADELT DYVTRNILAT PIMNGKDVVA VIMAVNKLDG PCFTSEDEDV FLKYLNFGTL NLKIYHLSYL HNCETRRGQV LL WSANKVF EELTDIERQF HKAFYTVRAY LNCDRYSVGL LDMTKEKEFF DVWPVLMGEA QAYSGPRTPD GREILFYKVI DYI LHGKED IKVIPSPPAD HWALASGLPT YVAESGFICN IMNAPADEMF NFQEGPLDDS GWIVKNVLSM PIVNKKEEIV GVAT FYNRK DGKPFDEQDE VLMESLTQFL GWSVLNTDTY DKMNKLENRK DIAQDMVLYH VRCDREEIQL ILPTRERLGK EPADC EEDE LGKILKEVLP GPAKFDIYEF HFSDLECTEL ELVKCGIQMY YELGVVRKFQ IPQEVLVRFL FSVSKGYRRI TYHNWR HGF NVAQTMFTLL MTGKLKSYYT DLEAFAMVTA GLCHDIDHRG TNNLYQMKSQ NPLAKLHGSS ILERHHLEFG KFLLSEE TL NIYQNLNRRQ HEHVIHLMDI AIIATDLALY FKKRTMFQKI VDESKNYEDR KSWVEYLSLE TTRKEIVMAM MMTACDLS A ITKPWEVQSK VALLVAAEFW EQGDLERTVL DQQPIPMMDR NKAAELPKLQ VGFIDFVCTF VYKEFSRFHE EILPMFDRL QNNRKEWKAL ADEYEAKVKA LEEDQKKETT AKKVGTEICN GGPAPRSSTC RIL

UniProtKB: Rod cGMP-specific 3',5'-cyclic phosphodiesterase subunit beta

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Macromolecule #3: Retinal rod rhodopsin-sensitive cGMP 3',5'-cyclic phosphodiestera...

MacromoleculeName: Retinal rod rhodopsin-sensitive cGMP 3',5'-cyclic phosphodiesterase subunit gamma
type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO / EC number: 3',5'-cyclic-GMP phosphodiesterase
Source (natural)Organism: Bos taurus (cattle)
Molecular weightTheoretical: 9.684229 KDa
SequenceString:
MNLEPPKAEI RSATRVMGGP VTPRKGPPKF KQRQTRQFKS KPPKKGVQGF GDDIPGMEGL GTDITVICPW EAFNHLELHE LAQYGII

UniProtKB: Retinal rod rhodopsin-sensitive cGMP 3',5'-cyclic phosphodiesterase subunit gamma

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Macromolecule #4: CYCLIC GUANOSINE MONOPHOSPHATE

MacromoleculeName: CYCLIC GUANOSINE MONOPHOSPHATE / type: ligand / ID: 4 / Number of copies: 2 / Formula: PCG
Molecular weightTheoretical: 345.205 Da
Chemical component information

ChemComp-PCG:
CYCLIC GUANOSINE MONOPHOSPHATE

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Macromolecule #5: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 5 / Number of copies: 2 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #6: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 6 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #7: Udenafil

MacromoleculeName: Udenafil / type: ligand / ID: 7 / Number of copies: 2 / Formula: ZUD
Molecular weightTheoretical: 516.656 Da
Chemical component information

ChemComp-ZUD:
Udenafil

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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

BufferpH: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.4000000000000001 µm / Nominal magnification: 63000
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 2050380
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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