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- PDB-2k31: Solution Structure of cGMP-binding GAF domain of Phosphodiesterase 5 -

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Entry
Database: PDB / ID: 2k31
TitleSolution Structure of cGMP-binding GAF domain of Phosphodiesterase 5
ComponentsPhosphodiesterase 5A, cGMP-specific
KeywordsHYDROLASE / cyclic nucleotide phosphodiesterase / PDE5 / GAF domain / cGMP
Function / homology
Function and homology information


cGMP effects / RHOBTB1 GTPase cycle / cyclic-nucleotide phosphodiesterase activity / cGMP metabolic process / positive regulation of oocyte development / 3',5'-cyclic-GMP phosphodiesterase / calmodulin-activated dual specificity 3',5'-cyclic-GMP, 3',5'-cyclic-AMP phosphodiesterase activity / calmodulin-activated 3',5'-cyclic-GMP phosphodiesterase activity / regulation of nitric oxide mediated signal transduction / Smooth Muscle Contraction ...cGMP effects / RHOBTB1 GTPase cycle / cyclic-nucleotide phosphodiesterase activity / cGMP metabolic process / positive regulation of oocyte development / 3',5'-cyclic-GMP phosphodiesterase / calmodulin-activated dual specificity 3',5'-cyclic-GMP, 3',5'-cyclic-AMP phosphodiesterase activity / calmodulin-activated 3',5'-cyclic-GMP phosphodiesterase activity / regulation of nitric oxide mediated signal transduction / Smooth Muscle Contraction / negative regulation of cardiac muscle contraction / positive regulation of G protein-coupled receptor signaling pathway / oocyte development / relaxation of cardiac muscle / cGMP catabolic process / positive regulation of cardiac muscle hypertrophy / 3',5'-cyclic-nucleotide phosphodiesterase activity / cGMP binding / T cell proliferation / negative regulation of T cell proliferation / positive regulation of MAP kinase activity / signal transduction / metal ion binding
Similarity search - Function
GAF domain / 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 / 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 / Metal dependent phosphohydrolases with conserved 'HD' motif. / HD/PDEase domain / GAF-like domain superfamily / Beta-Lactamase / 2-Layer Sandwich / Alpha Beta
Similarity search - Domain/homology
GUANOSINE-3',5'-MONOPHOSPHATE / cGMP-specific 3',5'-cyclic phosphodiesterase / cGMP-specific 3',5'-cyclic phosphodiesterase
Similarity search - Component
Biological speciesMus musculus (house mouse)
MethodSOLUTION NMR / simulated annealing
AuthorsHeikaus, C.C. / Stout, J.R. / Sekharan, M.R. / Eakin, C.M. / Rajagopal, P. / Brzovic, P.S. / Beavo, J.A. / Klevit, R.E.
CitationJournal: J.Biol.Chem. / Year: 2008
Title: Solution Structure of the cGMP Binding GAF Domain from Phosphodiesterase 5: Insights into Nucleotide Selectivity, Dimerization, and cGMP-Dependent Conformational Change.
Authors: Heikaus, C.C. / Stout, J.R. / Sekharan, M.R. / Eakin, C.M. / Rajagopal, P. / Brzovic, P.S. / Beavo, J.A. / Klevit, R.E.
History
DepositionApr 16, 2008Deposition site: BMRB / Processing site: RCSB
Revision 1.0Jun 3, 2008Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Version format compliance
Revision 1.2Feb 19, 2020Group: Database references / Derived calculations / Other
Category: database_2 / pdbx_database_status ...database_2 / pdbx_database_status / pdbx_struct_assembly / pdbx_struct_oper_list / struct_ref_seq_dif
Item: _pdbx_database_status.status_code_cs / _struct_ref_seq_dif.details
Revision 1.3Oct 20, 2021Group: Database references / Derived calculations / Category: database_2 / struct_ref_seq_dif / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id
Revision 1.4May 8, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2 / Item: _database_2.pdbx_DOI
Remark 650HELIX DETERMINATION METHOD: AUTHOR
Remark 700SHEET DETERMINATION METHOD: AUTHOR

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Phosphodiesterase 5A, cGMP-specific
hetero molecules


Theoretical massNumber of molelcules
Total (without water)20,3142
Polymers19,9681
Non-polymers3451
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)20 / 100structures with the lowest energy
RepresentativeModel #1lowest energy

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Components

#1: Protein Phosphodiesterase 5A, cGMP-specific


Mass: 19968.471 Da / Num. of mol.: 1 / Fragment: CGMP-BINDING GAF DOMAIN (UNP residues 154-320) / Mutation: A295E, I302E
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Pde5a / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3)star
References: UniProt: Q0VBW0, UniProt: Q8CG03*PLUS, 3',5'-cyclic-GMP phosphodiesterase
#2: Chemical ChemComp-35G / GUANOSINE-3',5'-MONOPHOSPHATE


Mass: 345.205 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H12N5O7P

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Experimental details

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Experiment

ExperimentMethod: SOLUTION NMR
NMR experiment
Conditions-IDExperiment-IDSolution-IDType
1122D 1H-15N HSQC
1242D 1H-13C HSQC
1312D 1H-1H NOESY
1433D HNCO
1533D HNCA
1633D HN(CA)CB
1733D CBCA(CO)NH
1843D (H)CCH-COSY
1933D HN(COCA)CB
11043D (H)CCH-TOCSY
11143D 1H-15N NOESY
11263D 1H-13C NOESY
11333D HN(CO)CA
11452D 1H-15N HSQC

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Sample preparation

Details
Solution-IDContentsSolvent system
10.5 - 1.2 mM GUANOSINE-3',5'-MONOPHOSPHATE, 0.5 - 1.2 mM mPDE5_GAFA, 150 mM sodium chloride, 1 mM PMSF, 1-5 mM DTT, 0.1 mM EDTA, 25 mM sodium phosphate, 90% H2O/10% D2O90% H2O/10% D2O
20.5 - 1.2 mM GUANOSINE-3',5'-MONOPHOSPHATE, 0.5 - 1.2 mM [U-100% 15N] mPDE5_GAFA, 150 mM sodium chloride, 1 mM PMSF, 1-5 mM DTT, 0.1 mM EDTA, 25 mM sodium phosphate, 90% H2O/10% D2O90% H2O/10% D2O
30.5 - 1.2 mM GUANOSINE-3',5'-MONOPHOSPHATE, 0.5 - 1.2 mM [U-100% 13C; U-100% 15N; 80% 2H] mPDE5_GAFA, 150 mM sodium chloride, 1 mM PMSF, 1-5 mM DTT, 0.1 mM EDTA, 25 mM sodium phosphate, 90% H2O/10% D2O90% H2O/10% D2O
40.5 - 1.2 mM GUANOSINE-3',5'-MONOPHOSPHATE, 0.5 - 1.2 mM [U-100% 13C; U-100% 15N] mPDE5_GAFA, 150 mM sodium chloride, 1 mM PMSF, 1-5 mM DTT, 0.1 mM EDTA, 25 mM sodium phosphate, 90% H2O/10% D2O90% H2O/10% D2O
50.5 - 1.2 mM GUANOSINE-3',5'-MONOPHOSPHATE, 0.5 - 1.2 mM [U-100% 15N] mPDE5_GAFA, 150 mM sodium chloride, 1 mM PMSF, 1-5 mM DTT, 0.1 mM EDTA, 25 mM sodium phosphate, 100% D2O100% D2O
60.5 - 1.2 mM GUANOSINE-3',5'-MONOPHOSPHATE, 0.5 - 1.2 mM [U-100% 13C; U-100% 15N] mPDE5_GAFA, 150 mM sodium chloride, 1 mM PMSF, 1-5 mM DTT, 0.1 mM EDTA, 25 mM sodium phosphate, 100% D2O100% D2O
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
0.5 mMGUANOSINE-3',5'-MONOPHOSPHATE1
0.5 mMmPDE5_GAFA1
150 mMsodium chloride1
1 mMPMSF1
1 mMDTT1
0.1 mMEDTA1
25 mMsodium phosphate1
0.5 mMGUANOSINE-3',5'-MONOPHOSPHATE2
0.5 mMmPDE5_GAFA[U-100% 15N]2
150 mMsodium chloride2
1 mMPMSF2
1 mMDTT2
0.1 mMEDTA2
25 mMsodium phosphate2
0.5 mMGUANOSINE-3',5'-MONOPHOSPHATE3
0.5 mMmPDE5_GAFA[U-100% 13C; U-100% 15N; 80% 2H]3
150 mMsodium chloride3
1 mMPMSF3
1 mMDTT3
0.1 mMEDTA3
25 mMsodium phosphate3
0.5 mMGUANOSINE-3',5'-MONOPHOSPHATE4
0.5 mMmPDE5_GAFA[U-100% 13C; U-100% 15N]4
150 mMsodium chloride4
1 mMPMSF4
1 mMDTT4
0.1 mMEDTA4
25 mMsodium phosphate4
0.5 mMGUANOSINE-3',5'-MONOPHOSPHATE5
0.5 mMmPDE5_GAFA[U-100% 15N]5
150 mMsodium chloride5
1 mMPMSF5
1 mMDTT5
0.1 mMEDTA5
25 mMsodium phosphate5
0.5 mMGUANOSINE-3',5'-MONOPHOSPHATE6
0.5 mMmPDE5_GAFA[U-100% 13C; U-100% 15N]6
150 mMsodium chloride6
1 mMPMSF6
1 mMDTT6
0.1 mMEDTA6
25 mMsodium phosphate6
Sample conditionsIonic strength: 150 / pH: 7 / Pressure: ambient atm / Temperature: 310 K

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NMR measurement

NMR spectrometer
TypeManufacturerModelField strength (MHz)Spectrometer-ID
Bruker DMXBrukerDMX5001
Bruker DMXBrukerDMX6002
Varian INOVAVarianINOVA6003
Varian INOVAVarianINOVA8004
Varian INOVAVarianINOVA9005

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Processing

NMR software
NameVersionDeveloperClassification
CNS1.1Brunger, Adams, Clore, Gros, Nilges and Readstructure solution
CYANA2Guntert, Mumenthaler and Wuthrichstructure solution
NMRDrawDelaglio, Grzesiek, Vuister, Zhu, Pfeifer and Baxprocessing
NMRViewJohnson, One Moon Scientificchemical shift assignment
NMRViewJohnson, One Moon Scientificpeak picking
NMRViewJohnson, One Moon Scientificdata analysis
ModelFreePalmerdata analysis
TALOSCornilescu, Delaglio and Baxdata analysis
CYANA2Guntert, Mumenthaler and Wuthrichrefinement
RefinementMethod: simulated annealing / Software ordinal: 1
NMR representativeSelection criteria: lowest energy
NMR ensembleConformer selection criteria: structures with the lowest energy
Conformers calculated total number: 100 / Conformers submitted total number: 20

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