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- PDB-6ypp: Crystal structure of the cAMP-dependent protein kinase A cocrysta... -

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Basic information

Entry
Database: PDB / ID: 6ypp
TitleCrystal structure of the cAMP-dependent protein kinase A cocrystallized with PKI (5-24). Soaking of aminofasudil and displacing it with the fragment isoquinoline.
Components
  • cAMP-dependent protein kinase catalytic subunit alphaCAMP-dependent pathway
  • cAMP-dependent protein kinase inhibitor alphaCAMP-dependent pathway
KeywordsTRANSFERASE / phosphotransferase / signalling pathways / glycogen metabolism / serine/threonine kinase
Function / homology
Function and homology information


regulation of protein processing / protein localization to lipid droplet / regulation of bicellular tight junction assembly / cellular response to parathyroid hormone stimulus / cAMP-dependent protein kinase inhibitor activity / cAMP-dependent protein kinase / cellular response to cold / sperm capacitation / regulation of osteoblast differentiation / cAMP-dependent protein kinase activity ...regulation of protein processing / protein localization to lipid droplet / regulation of bicellular tight junction assembly / cellular response to parathyroid hormone stimulus / cAMP-dependent protein kinase inhibitor activity / cAMP-dependent protein kinase / cellular response to cold / sperm capacitation / regulation of osteoblast differentiation / cAMP-dependent protein kinase activity / negative regulation of glycolytic process through fructose-6-phosphate / ciliary base / cAMP-dependent protein kinase complex / AMP-activated protein kinase activity / negative regulation of protein import into nucleus / postsynaptic modulation of chemical synaptic transmission / cellular response to glucagon stimulus / protein kinase A regulatory subunit binding / axoneme / protein kinase A catalytic subunit binding / plasma membrane raft / mesoderm formation / sperm flagellum / regulation of proteasomal protein catabolic process / negative regulation of smoothened signaling pathway / positive regulation of gluconeogenesis / negative regulation of TORC1 signaling / regulation of G2/M transition of mitotic cell cycle / protein export from nucleus / protein serine/threonine/tyrosine kinase activity / acrosomal vesicle / positive regulation of protein export from nucleus / neural tube closure / cellular response to glucose stimulus / neuromuscular junction / adenylate cyclase-activating G protein-coupled receptor signaling pathway / positive regulation of insulin secretion / mRNA processing / cellular response to heat / manganese ion binding / dendritic spine / regulation of cell cycle / nuclear speck / protein domain specific binding / phosphorylation / protein serine kinase activity / centrosome / protein serine/threonine kinase activity / glutamatergic synapse / ubiquitin protein ligase binding / protein kinase binding / perinuclear region of cytoplasm / magnesium ion binding / negative regulation of transcription by RNA polymerase II / mitochondrion / ATP binding / nucleus / cytosol / cytoplasm
Similarity search - Function
cAMP-dependent protein kinase inhibitor / cAMP-dependent protein kinase inhibitor / cAMP-dependent protein kinase catalytic subunit / Extension to Ser/Thr-type protein kinases / AGC-kinase, C-terminal / AGC-kinase C-terminal domain profile. / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / Protein kinase domain / Serine/Threonine protein kinases, catalytic domain ...cAMP-dependent protein kinase inhibitor / cAMP-dependent protein kinase inhibitor / cAMP-dependent protein kinase catalytic subunit / Extension to Ser/Thr-type protein kinases / AGC-kinase, C-terminal / AGC-kinase C-terminal domain profile. / 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
ISOQUINOLINE / cAMP-dependent protein kinase catalytic subunit alpha / cAMP-dependent protein kinase inhibitor alpha
Similarity search - Component
Biological speciesCricetulus griseus (Chinese hamster)
Mus musculus (house mouse)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.75 Å
AuthorsOebbeke, M. / Gerber, H.-D. / Heine, A. / Klebe, G.
CitationJournal: To Be Published
Title: Fluorescence-based displacement experiments
Authors: Oebbeke, M. / Hassaan, E. / Heine, A. / Klebe, G.
History
DepositionApr 16, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 28, 2021Provider: repository / Type: Initial release
Revision 1.1Jan 24, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: cAMP-dependent protein kinase catalytic subunit alpha
B: cAMP-dependent protein kinase inhibitor alpha
hetero molecules


Theoretical massNumber of molelcules
Total (without water)43,4693
Polymers43,3402
Non-polymers1291
Water3,369187
1


  • Idetical with deposited unit
  • defined by software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1910 Å2
ΔGint6 kcal/mol
Surface area15270 Å2
MethodPISA
Unit cell
Length a, b, c (Å)52.499, 71.658, 97.779
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121
Space group name HallP2ac2ab
Symmetry operation#1: x,y,z
#2: x+1/2,-y+1/2,-z
#3: -x,y+1/2,-z+1/2
#4: -x+1/2,-y,z+1/2

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Components

#1: Protein cAMP-dependent protein kinase catalytic subunit alpha / CAMP-dependent pathway / PKA C-alpha


Mass: 41113.766 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Cricetulus griseus (Chinese hamster) / Gene: PRKACA / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P25321, cAMP-dependent protein kinase
#2: Protein/peptide cAMP-dependent protein kinase inhibitor alpha / CAMP-dependent pathway / PKI-alpha / cAMP-dependent protein kinase inhibitor / muscle/brain isoform


Mass: 2226.411 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Mus musculus (house mouse) / References: UniProt: P63248
#3: Chemical ChemComp-ISQ / ISOQUINOLINE / Isoquinoline


Mass: 129.159 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C9H7N / Feature type: SUBJECT OF INVESTIGATION / Comment: alkaloid*YM
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 187 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.24 Å3/Da / Density % sol: 45.14 %
Crystal growTemperature: 277 K / Method: vapor diffusion, sitting drop / pH: 6.9
Details: 0,2 mM PKA in 100 mM MES-BIS-Tris-Buffer, 1 mM dithiothreitol, 0.1 mM sodium EDTA, 75 mM LiCl, 0.2 mM Mega 8 and 23 % methanol (v/v) 0.003 mL drop volume, 0.5 mL reservoir volume Soaking of ...Details: 0,2 mM PKA in 100 mM MES-BIS-Tris-Buffer, 1 mM dithiothreitol, 0.1 mM sodium EDTA, 75 mM LiCl, 0.2 mM Mega 8 and 23 % methanol (v/v) 0.003 mL drop volume, 0.5 mL reservoir volume Soaking of aminofasudil: in buffer with 10% of ligand in DMSO (50 mM stock) and 30% MPD Displacement with isoquinoline: in buffer with 10% of ligand in DMSO and 30% MPD

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: BESSY / Beamline: 14.1 / Wavelength: 0.9184 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Jun 2, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9184 Å / Relative weight: 1
ReflectionResolution: 1.747→48.89 Å / Num. obs: 37708 / % possible obs: 98.9 % / Redundancy: 4.2 % / Biso Wilson estimate: 21.94 Å2 / Rsym value: 0.047 / Net I/σ(I): 20.67
Reflection shellResolution: 1.75→1.85 Å / Mean I/σ(I) obs: 2.56 / Num. unique obs: 5905 / Rsym value: 0.515

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Processing

Software
NameVersionClassification
PHASERphasing
PHENIX1.17.1_3660refinement
XDSdata reduction
XDSdata scaling
Cootmodel building
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 6F14

6f14


Resolution: 1.75→48.89 Å / SU ML: 0.1743 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 19.3249
RfactorNum. reflection% reflection
Rfree0.2029 1886 5 %
Rwork0.1774 --
obs0.1787 37708 98.88 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso mean: 25.62 Å2
Refinement stepCycle: LAST / Resolution: 1.75→48.89 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2813 0 10 187 3010
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00872911
X-RAY DIFFRACTIONf_angle_d0.91013952
X-RAY DIFFRACTIONf_chiral_restr0.0539423
X-RAY DIFFRACTIONf_plane_restr0.0061522
X-RAY DIFFRACTIONf_dihedral_angle_d22.67951045
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.75-1.790.28511380.22722617X-RAY DIFFRACTION96.03
1.79-1.850.25371430.21392720X-RAY DIFFRACTION99.07
1.85-1.910.23251430.19942723X-RAY DIFFRACTION99.41
1.91-1.970.21951450.18512740X-RAY DIFFRACTION99.62
1.97-2.050.21821450.17272758X-RAY DIFFRACTION99.76
2.05-2.150.21191440.16472737X-RAY DIFFRACTION99.76
2.15-2.260.19291440.16252734X-RAY DIFFRACTION98.83
2.26-2.40.18821450.1542766X-RAY DIFFRACTION98.91
2.4-2.590.19161460.15982766X-RAY DIFFRACTION99.66
2.59-2.850.18441450.16682757X-RAY DIFFRACTION99.28
2.85-3.260.18851470.17242798X-RAY DIFFRACTION98.56
3.26-4.110.17421470.17042798X-RAY DIFFRACTION98.86
4.11-48.890.22621540.19642908X-RAY DIFFRACTION97.73
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
10.379930341489-0.467301018677-0.469390916642.767867376811.762961554051.973892341220.116952478618-0.02936522144450.0169171230893-0.104285622538-0.0869816576486-0.140303738755-0.1682015924360.09768719205240.006399216522770.17282933297-0.02695432397320.02354431141610.2302765317610.01509940469370.24187572944219.98672763416.279279717419.778912316
20.927406194911-0.129488000937-0.5676895330550.550107457580.3738071727361.214103067240.001388233001790.01251855285430.00492858535505-0.110489969546-0.0551476529246-0.05666502636950.0005890166033290.08246025671160.05765274871890.1673914200150.01522607050850.003447736531540.1497498415270.0003029515976790.19119196734412.04752741769.7966127097922.6281176848
31.70019299476-0.52702487364-0.01977583976852.1644883883-0.7081369253052.24967537035-0.00684079249214-0.1324641115550.02910715958240.1059030564170.05770380177950.145409888325-0.0368806607468-0.113327489968-0.05359459287070.119734165270.00949527245610.03288218239490.15533812224-0.006143035125150.157583732095-2.7575495891310.798994321637.2927277653
40.592340864096-0.317050783321-0.7309778769530.6436103494490.452693698261.14499263317-0.0619147828719-0.0130980968202-0.0477759479174-0.114318270285-0.03842033143480.02291285290670.01225157019080.1174196610190.1129298075830.175771488531-0.02259882096690.01098580053230.1722388345090.02037506369580.23469442790615.34161968514.8115010822520.4948137524
54.86667795391.35972101810.3762858923046.20577746639-1.656231650167.481330275340.306195221048-0.801276983579-0.3652046185520.573739559869-0.4562377400950.1062336350520.478446599713-0.6682996292810.1198446516730.398566482769-0.173545081059-0.00115032809680.389310901449-0.007478140480290.488172321024-7.48220489627-6.574588983633.2155999129
66.86181972003-1.943450401530.6214478793483.900231275961.509089204520.9282497910660.06699709727740.508507202013-0.510919037192-0.754272380978-0.0812769086682-0.00187060291396-0.0440544365249-0.0194816214610.07082332538880.414166700391-0.07074434331470.02421006067770.291475580748-0.009318732184680.26391309162-1.56503764211-0.46551677838223.5866036428
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection details
1X-RAY DIFFRACTION1chain 'A' and (resid 13 through 54 )
2X-RAY DIFFRACTION2chain 'A' and (resid 55 through 198 )
3X-RAY DIFFRACTION3chain 'A' and (resid 199 through 297 )
4X-RAY DIFFRACTION4chain 'A' and (resid 298 through 350 )
5X-RAY DIFFRACTION5chain 'B' and (resid 6 through 13 )
6X-RAY DIFFRACTION6chain 'B' and (resid 14 through 21 )

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