PDB-3e8e: Crystal structures of the kinase domain of PKA in complex with AT...

Basic information

• Entry: Database: PDB / ID: 3e8e
• Title: Crystal structures of the kinase domain of PKA in complex with ATP-competitive inhibitors

Components

• PKI inhibitor peptide
• cAMP-dependent protein kinase catalytic subunit alphaCAMP-dependent pathway

• Keywords: TRANSFERASE / PKA / AKT2 / kinase / PKI / bovine / inhibitor / ATP-binding / cAMP / Lipoprotein / Myristate / Nucleotide-binding / Nucleus / Phosphoprotein / Serine/threonine-protein kinase

Function / homology

Function:

CD209 (DC-SIGN) signaling / HDL assembly / Regulation of insulin secretion / Rap1 signalling / Ion homeostasis / PKA activation in glucagon signalling / DARPP-32 events / CREB1 phosphorylation through the activation of Adenylate Cyclase / GPER1 signaling / Factors involved in megakaryocyte development and platelet production / Loss of Nlp from mitotic centrosomes / Recruitment of mitotic centrosome proteins and complexes / Loss of proteins required for interphase microtubule organization from the centrosome / Anchoring of the basal body to the plasma membrane / AURKA Activation by TPX2 / RET signaling / Interleukin-3, Interleukin-5 and GM-CSF signaling / Recruitment of NuMA to mitotic centrosomes / VEGFA-VEGFR2 Pathway / PKA activation / GLI3 is processed to GLI3R by the proteasome / MAPK6/MAPK4 signaling / Regulation of PLK1 Activity at G2/M Transition / Hedgehog 'off' state / cAMP-dependent protein kinase inhibitor activity / cAMP-dependent protein kinase / cAMP-dependent protein kinase activity / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / cAMP-dependent protein kinase complex / AMP-activated protein kinase activity / Vasopressin regulates renal water homeostasis via Aquaporins / protein kinase A regulatory subunit binding / mesoderm formation / sperm flagellum / protein kinase A signaling / negative regulation of TORC1 signaling / acrosomal vesicle / neuromuscular junction / cellular response to heat / peptidyl-serine phosphorylation / protein kinase activity / protein domain specific binding / protein phosphorylation / protein serine kinase activity / protein serine/threonine kinase activity / perinuclear region of cytoplasm / mitochondrion / ATP binding / nucleus / plasma membrane / cytosol / cytoplasm

Domain/homology:

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. / Transferase(Phosphotransferase) domain 1 / Transferase(Phosphotransferase); domain 1 / Phosphorylase Kinase; domain 1 / Phosphorylase Kinase; domain 1 / 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 / 2-Layer Sandwich / Orthogonal Bundle / Mainly Alpha / Alpha Beta

Component:

Chem-G98 / cAMP-dependent protein kinase catalytic subunit alpha / cAMP-dependent protein kinase inhibitor alpha

• Biological species: Bos taurus (cattle)
• Method: X-RAY DIFFRACTION / SYNCHROTRON / Resolution: 2 Å
• Authors: Concha, N.O. / Elkins, P.A. / Smallwood, A. / Ward, P.

Citation

Journal: Bioorg.Med.Chem.Lett. / Year: 2009
Title: Aminofurazans as potent inhibitors of AKT kinase
Authors: Rouse, M.B. / Seefeld, M.A. / Leber, J.D. / McNulty, K.C. / Sun, L. / Miller, W.H. / Zhang, S. / Minthorn, E.A. / Concha, N.O. / Choudhry, A.E. / Schaber, M.D. / Heerding, D.A.

#1: Journal: To be Published
Title: Discovery of 5-pyrrolopyridinyl-2-thiophenecarboxamides as potent AKT kinase inhibitors
Authors: Seefeld, M.A. / Rouse, M.B. / McNulty, K.C. / Sun, L. / Wang, J. / Yamashita, D.S. / Choudhry, A. / Schaber, M.D. / Kumar, R. / Kahana, J. / Zhang, S.Y. / Minthorn, E.A. / Koretke, K.K. / Concha, N.O. / Heerding, D.A.

History

Deposition	Aug 19, 2008	Deposition site: RCSB / Processing site: RCSB
Revision 1.0	Nov 18, 2008	Provider: repository / Type: Initial release
Revision 1.1	Jul 13, 2011	Group: Version format compliance
Revision 1.2	Nov 19, 2014	Group: Non-polymer description
Revision 1.3	Oct 25, 2017	Group: Refinement description / Category: software Item: _software.classification / _software.contact_author / _software.contact_author_email / _software.date / _software.language / _software.location / _software.name / _software.type / _software.version

Assembly

Deposited unit

A: cAMP-dependent protein kinase catalytic subunit alpha

G: PKI inhibitor peptide

B: cAMP-dependent protein kinase catalytic subunit alpha

C: PKI inhibitor peptide

E: cAMP-dependent protein kinase catalytic subunit alpha

F: PKI inhibitor peptide

I: cAMP-dependent protein kinase catalytic subunit alpha

J: PKI inhibitor peptide

L: cAMP-dependent protein kinase catalytic subunit alpha

N: PKI inhibitor peptide

P: cAMP-dependent protein kinase catalytic subunit alpha

Q: PKI inhibitor peptide

hetero molecules

Summary:

• 261 kDa, 12 polymers

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	260,858	18
Polymers	258,077	12
Non-polymers	2,781	6
Water	6,053	336

1

A: cAMP-dependent protein kinase catalytic subunit alpha

G: PKI inhibitor peptide

hetero molecules

Summary:

• defined by author&software
• 43.5 kDa, 2 polymers

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	43,476	3
Polymers	43,013	2
Non-polymers	464	1
Water	36	2

Symmetry operations:

Type	Name	Symmetry operation	Number
identity operation	1_555	x,y,z	1

Calculated values:

Buried area	2120 Å2
ΔGint	-5 kcal/mol
Surface area	16890 Å2
Method	PISA

2

B: cAMP-dependent protein kinase catalytic subunit alpha

C: PKI inhibitor peptide

hetero molecules

Summary:

• defined by author&software
• 43.5 kDa, 2 polymers

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	43,476	3
Polymers	43,013	2
Non-polymers	464	1
Water	36	2

Symmetry operations:

Type	Name	Symmetry operation	Number
identity operation	1_555	x,y,z	1

Calculated values:

Buried area	2110 Å2
ΔGint	-1 kcal/mol
Surface area	16350 Å2
Method	PISA

3

E: cAMP-dependent protein kinase catalytic subunit alpha

F: PKI inhibitor peptide

hetero molecules

Summary:

• defined by author&software
• 43.5 kDa, 2 polymers

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	43,476	3
Polymers	43,013	2
Non-polymers	464	1
Water	36	2

Symmetry operations:

Type	Name	Symmetry operation	Number
identity operation	1_555	x,y,z	1

Calculated values:

Buried area	1980 Å2
ΔGint	-5 kcal/mol
Surface area	17070 Å2
Method	PISA

4

I: cAMP-dependent protein kinase catalytic subunit alpha

J: PKI inhibitor peptide

hetero molecules

Summary:

• defined by author&software
• 43.5 kDa, 2 polymers

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	43,476	3
Polymers	43,013	2
Non-polymers	464	1
Water	36	2

Symmetry operations:

Type	Name	Symmetry operation	Number
identity operation	1_555	x,y,z	1

Calculated values:

Buried area	2080 Å2
ΔGint	-3 kcal/mol
Surface area	17000 Å2
Method	PISA

5

L: cAMP-dependent protein kinase catalytic subunit alpha

N: PKI inhibitor peptide

hetero molecules

Summary:

• defined by author&software
• 43.5 kDa, 2 polymers

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	43,476	3
Polymers	43,013	2
Non-polymers	464	1
Water	36	2

Symmetry operations:

Type	Name	Symmetry operation	Number
identity operation	1_555	x,y,z	1

Calculated values:

Buried area	2070 Å2
ΔGint	-2 kcal/mol
Surface area	16480 Å2
Method	PISA

6

P: cAMP-dependent protein kinase catalytic subunit alpha

Q: PKI inhibitor peptide

hetero molecules

Summary:

• defined by author&software
• 43.5 kDa, 2 polymers

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	43,476	3
Polymers	43,013	2
Non-polymers	464	1
Water	36	2

Symmetry operations:

Type	Name	Symmetry operation	Number
identity operation	1_555	x,y,z	1

Calculated values:

Buried area	2130 Å2
ΔGint	-3 kcal/mol
Surface area	16670 Å2
Method	PISA

Unit cell

Length a, b, c (Å)	82.578, 95.848, 179.097
Angle α, β, γ (deg.)	90.00, 102.53, 90.00
Int Tables number	4
Space group name H-M	P1211

Noncrystallographic symmetry (NCS)

NCS domain:

ID	Ens-ID	Details
1	1	A
2	1	B
3	1	E
4	1	I
5	1	L
6	1	P
1	2	G
2	2	C
3	2	F
4	2	J
5	2	N
6	2	Q

NCS domain segments:

Dom-ID	Component-ID	Ens-ID	Refine code	Auth asym-ID	Auth seq-ID
1	1	1	5	A	17 - 350
2	1	1	5	B	17 - 350
3	1	1	5	E	17 - 350
4	1	1	5	I	17 - 350
5	1	1	5	L	17 - 350
6	1	1	5	P	17 - 350
1	1	2	5	G	7 - 23
2	1	2	5	C	7 - 23
3	1	2	5	F	7 - 23
4	1	2	5	J	7 - 23
5	1	2	5	N	7 - 23
6	1	2	5	Q	7 - 23

NCS ensembles :

ID
1
2

Components

#1: Protein

A B E I L P
cAMP-dependent protein kinase catalytic subunit alpha / CAMP-dependent pathway / PKA C-alpha

Details:

Mass: 40786.395 Da / Num. of mol.: 6 / Fragment: CAMP-DEPENDENT PKA kinase domain
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bos taurus (cattle) / Gene: PKA, PRKACA / Plasmid: pDEST14 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(AI) / References: UniProt: P00517, cAMP-dependent protein kinase

#2: Protein/peptide

G C F J N Q
PKI inhibitor peptide

Details:

Mass: 2226.411 Da / Num. of mol.: 6 / Source method: obtained synthetically
Details: THE PEPTIDE WAS CHEMICALLY SYNTHESIZED. IT IS FOUND NATURALLY IN BOVINE.
References: UniProt: Q3SX13

#3: Chemical

A-351 B-351 E-351 I-351 L-351 P-351
ChemComp-G98 / 4-[2-(4-amino-2,5-dihydro-1,2,5-oxadiazol-3-yl)-6-{[(1S)-3-amino-1-phenylpropyl]oxy}-1-ethyl-1H-imidazo[4,5-c]pyridin-4-yl]-2-methylbut-3-yn-2-ol

Details:

Mass: 463.532 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C₂₄H₂₉N₇O₃

#4: Water

ChemComp-HOH / water / Water

Details:

Mass: 18.015 Da / Num. of mol.: 336 / Source method: isolated from a natural source / Formula: H₂O

Experimental details

Experiment

• Experiment: Method: X-RAY DIFFRACTION / Number of used crystals: 1

Sample preparation

• Crystal: Density Matthews: 2.68 ų/Da / Density % sol: 54.12 %
• Crystal grow: Temperature: 273 K / Method: vapor diffusion; Details: 13-17% PEG 3K-20K, 100 mM MES pH 6.5 or acetate 4.6, 1-15% methanol. Seeded, pH 4.5 - 6.5, vapor diffusion, temperature 273K; PH range: 4.5 - 6.5

Data collection

• Diffraction: Mean temperature: 100 K
• Diffraction source: Source: SYNCHROTRON / Site: APS / Beamline: 17-ID / Wavelength: 1 Å
• Detector: Type: ADSC QUANTUM 210 / Detector: CCD / Date: Oct 1, 2005 / Details: un-focused beam
• Radiation: Monochromator: GRAPHITE / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
• Radiation wavelength: Wavelength: 1 Å / Relative weight: 1
• Reflection: Resolution: 1.72→42.03 Å / Num. obs: 211201 / % possible obs: 73.7 % / Observed criterion σ(I): 2 / Redundancy: 3.5 % / R_merge(I) obs: 0.09
• Reflection shell: Resolution: 1.72→1.78 Å / Redundancy: 2.1 % / R_merge(I) obs: 0.82 / % possible all: 17.3

Processing

Software

Name	Version	Classification	NB
DENZO		data reduction
SCALEPACK		data scaling
REFMAC	5.2.0019	refinement
PDB_EXTRACT	3.006	data extraction
JDirector		data collection
HKL-2000		data reduction
AMoRE		phasing

Refinement

Resolution: 2→42.03 Å / Cor.coef. F_o:F_c: 0.94 / Cor.coef. F_o:F_c free: 0.919 / Occupancy max: 1 / Occupancy min: 0.5 / SU B: 8.838 / SU ML: 0.168 / Cross valid method: THROUGHOUT / ESU R: 0.23 / ESU R Free: 0.196 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS

	Rfactor	Num. reflection	% reflection	Selection details
Rfree	0.2794	8636	5 %	RANDOM
Rwork	0.24311	-	-	-
obs	0.24492	163336	93.48 %	-

• Solvent computation: Ion probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK

Displacement parameters

B_iso mean: 31.635 Ų

	Baniso -1	Baniso -2	Baniso -3
1-	-2.55 Å2	0 Å2	-1.53 Å2
2-	-	3.37 Å2	0 Å2
3-	-	-	-1.48 Å2

Refinement step

Cycle: LAST / Resolution: 2→42.03 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	17910	0	204	336	18450

Refine LS restraints

Refine-ID	Type	Dev ideal	Dev ideal target	Number
X-RAY DIFFRACTION	r_bond_refined_d	0.007	0.022	18613
X-RAY DIFFRACTION	r_bond_other_d	0.001	0.02	12911
X-RAY DIFFRACTION	r_angle_refined_deg	1.234	1.967	25160
X-RAY DIFFRACTION	r_angle_other_deg	0.795	3	31296
X-RAY DIFFRACTION	r_dihedral_angle_1_deg	5.423	5	2179
X-RAY DIFFRACTION	r_dihedral_angle_2_deg	35.979	23.974	926
X-RAY DIFFRACTION	r_dihedral_angle_3_deg	14.354	15.046	3275
X-RAY DIFFRACTION	r_dihedral_angle_4_deg	18.431	15	108
X-RAY DIFFRACTION	r_chiral_restr	0.062	0.2	2612
X-RAY DIFFRACTION	r_gen_planes_refined	0.003	0.02	20434
X-RAY DIFFRACTION	r_gen_planes_other	0.001	0.02	4002
X-RAY DIFFRACTION	r_nbd_refined	0.19	0.2	3796
X-RAY DIFFRACTION	r_nbd_other	0.176	0.2	13029
X-RAY DIFFRACTION	r_nbtor_refined	0.179	0.2	9084
X-RAY DIFFRACTION	r_nbtor_other	0.082	0.2	9320
X-RAY DIFFRACTION	r_xyhbond_nbd_refined	0.141	0.2	668
X-RAY DIFFRACTION	r_xyhbond_nbd_other	0.038	0.2	1
X-RAY DIFFRACTION	r_metal_ion_refined
X-RAY DIFFRACTION	r_metal_ion_other
X-RAY DIFFRACTION	r_symmetry_vdw_refined	0.176	0.2	13
X-RAY DIFFRACTION	r_symmetry_vdw_other	0.288	0.2	56
X-RAY DIFFRACTION	r_symmetry_hbond_refined	0.163	0.2	9
X-RAY DIFFRACTION	r_symmetry_hbond_other
X-RAY DIFFRACTION	r_symmetry_metal_ion_refined
X-RAY DIFFRACTION	r_symmetry_metal_ion_other
X-RAY DIFFRACTION	r_mcbond_it	0.454	1.5	11789
X-RAY DIFFRACTION	r_mcbond_other	0.112	1.5	4401
X-RAY DIFFRACTION	r_mcangle_it	0.69	2	17509
X-RAY DIFFRACTION	r_scbond_it	0.905	3	8743
X-RAY DIFFRACTION	r_scangle_it	1.334	4.5	7644
X-RAY DIFFRACTION	r_rigid_bond_restr
X-RAY DIFFRACTION	r_sphericity_free
X-RAY DIFFRACTION	r_sphericity_bonded

Refine LS restraints NCS

Refine-ID: X-RAY DIFFRACTION

Ens-ID	Dom-ID	Auth asym-ID	Number	Type	Rms dev position (Å)	Weight position
1	1	A	1970	medium positional	0.22	0.5
1	2	B	1970	medium positional	0.24	0.5
1	3	E	1970	medium positional	0.21	0.5
1	4	I	1970	medium positional	0.28	0.5
1	5	L	1970	medium positional	0.19	0.5
1	6	P	1970	medium positional	0.22	0.5
2	1	G	100	medium positional	0.14	0.5
2	2	C	100	medium positional	0.2	0.5
2	3	F	100	medium positional	0.24	0.5
2	4	J	100	medium positional	0.18	0.5
2	5	N	100	medium positional	0.33	0.5
2	6	Q	100	medium positional	0.44	0.5
1	1	A	2766	loose positional	0.4	5
1	2	B	2766	loose positional	0.43	5
1	3	E	2766	loose positional	0.51	5
1	4	I	2766	loose positional	0.66	5
1	5	L	2766	loose positional	0.47	5
1	6	P	2766	loose positional	0.42	5
2	1	G	133	loose positional	0.54	5
2	2	C	133	loose positional	0.66	5
2	3	F	133	loose positional	0.6	5
2	4	J	133	loose positional	0.44	5
2	5	N	133	loose positional	0.66	5
2	6	Q	133	loose positional	0.59	5
1	1	A	1970	medium thermal	0.42	2
1	2	B	1970	medium thermal	0.37	2
1	3	E	1970	medium thermal	0.54	2
1	4	I	1970	medium thermal	0.57	2
1	5	L	1970	medium thermal	0.68	2
1	6	P	1970	medium thermal	0.67	2
2	1	G	100	medium thermal	0.44	2
2	2	C	100	medium thermal	0.47	2
2	3	F	100	medium thermal	0.58	2
2	4	J	100	medium thermal	0.33	2
2	5	N	100	medium thermal	0.27	2
2	6	Q	100	medium thermal	0.69	2
1	1	A	2766	loose thermal	0.9	10
1	2	B	2766	loose thermal	0.85	10
1	3	E	2766	loose thermal	1.06	10
1	4	I	2766	loose thermal	1.08	10
1	5	L	2766	loose thermal	1.19	10
1	6	P	2766	loose thermal	1.21	10
2	1	G	133	loose thermal	1.31	10
2	2	C	133	loose thermal	1.11	10
2	3	F	133	loose thermal	1.24	10
2	4	J	133	loose thermal	1.27	10
2	5	N	133	loose thermal	0.69	10
2	6	Q	133	loose thermal	1.34	10

LS refinement shell

Resolution: 2→2.052 Å / Total num. of bins used: 20

	Rfactor	Num. reflection	% reflection
Rfree	0.379	463	-
Rwork	0.318	9514	-
obs	-	-	73.57 %