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PDBsum entry 4fbx
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Transferase/transferase inhibitor
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PDB id
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4fbx
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References listed in PDB file
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Key reference
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Title
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A subnanomolar fluorescent probe for protein kinase ck2 interaction studies.
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Authors
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E.Enkvist,
K.Viht,
N.Bischoff,
J.Vahter,
S.Saaver,
G.Raidaru,
O.G.Issinger,
K.Niefind,
A.Uri.
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Ref.
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Org Biomol Chem, 2012,
10,
8645-8653.
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PubMed id
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Abstract
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Up-regulation of an acidophilic protein kinase, CK2, has been established in
several types of cancer. This cognition has made CK2 an important target for
drug development for cancer chemotherapy. The characterization of potential drug
candidates, determination of the structure and clarification of the functions of
CK2 could be facilitated by the application of small-molecule fluorescent probes
that bind to the active site of the enzyme with high affinity and selectivity.
We have used a bisubstrate approach for the development of a highly potent
inhibitor of CK2. 4,5,6,7-Tetrabromo-1H-benzimidazole was conjugated with
peptides containing multiple aspartate residues via different linkers. The
design of the inhibitors was by crystallographic analysis of the complex of an
inhibitor with the catalytic subunit of the enzyme (CK2α). The inhibitory
potency of the synthesized compounds was established in a kinetic assay that
used thin layer chromatography for the measurement of the rate of
phosphorylation of fluorescently labelled peptide 5-TAMRA-RADDSDDDDD. The most
potent inhibitor, ARC-1502 (K(i) = 0.5 nM), revealed high selectivity for CK2α
in a panel of 140 protein kinases. Labelling of ARC-1502 with PromoFluor-647
gave the fluorescent probe ARC-1504 that possessed subnanomolar affinity towards
both CK2α and the holoenzyme. The probe was used in a fluorescence
anisotropy-based binding assay to measure the concentration of CK2α and
characterize non-labelled ligands binding to the active site of CK2α.
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Secondary reference #1
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Title
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Crystal structure of a c-Terminal deletion mutant of human protein kinase ck2 catalytic subunit.
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Authors
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I.Ermakova,
B.Boldyreff,
O.G.Issinger,
K.Niefind.
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Ref.
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J Mol Biol, 2003,
330,
925-934.
[DOI no: ]
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PubMed id
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Figure 2.
Figure 2. Aspects of the rhCK2a^DC structure. (a) Folding
of the monomer drawn in rainbow colors. A view was chosen that
shows the attachment of the N-terminal segment to the activation
segment and to helix aC. The equivalents of these regions and
the b4/b5 loop as found in maize CK2a (PDB code 1LP4) are drawn
in black. (b) Stereo picture of the cosubstrate-binding site.
The AMPPNP molecule is covered with s[A]-weighted (F[o] -F[c])
OMIT density[30] colored in blue and drawn above a cutoff level
of 2.7s above the average. The OMIT map was calculated with CNS
[31] after a 2000 K simulated annealing run in which the AMPPNP
molecule had been omitted. The surrounding protein is embedded
in the final s[A]-weighted (2F[o] -F[c]) electron density (green
color; cutoff level 1.4s above the average). ATP as bound to
CAPK is drawn in black after superimposition of the protein
matrices. (c) Stereo picture of the b4/b5 loop covered by
s[A]-weighted (2F[o] -F[c]) electron density with a cutoff level
of 1.0s above the average. For comparison, the equivalent region
of rhCK2a^DC within the CK2 holoenzyme is drawn in black.
Furthermore a part of the CK2b-dimer is shown in black to
illustrate that the b4/b5-loop conformation in isolated
rhCK2a^DC is not compatible with CK2b binding. All parts of the
Figure were prepared with BOBSCRIPT[41] and RASTER3D. [42]
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Figure 3.
Figure 3. Structures for comparison. (a) The CK2 holoenzyme
(PDB file 1JWH). The complex is formed by a central CK2b dimer
(blue and red) with a Zn2+-containing interface and by two
rhCK2a^DC subunits composed of an N-terminal (green) and a
C-terminal (yellow) domain, respectively. The activation segment
and the helix aC are indicated by black color. (b) Active
CDK2[35] (PDB code 1FIN) in complex with a cyclin A fragment
(gray). The activation segment in active CDK2 is drawn in
magenta. To illustrate the structural changes upon cyclin
A-mediated activation the activation segment, the helix aC and
the ATP-binding loop of inactive, cyclin-free CDK2 [34] (PDB
code 1HCL) are added in blue color.
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The above figures are
reproduced from the cited reference
with permission from Elsevier
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Secondary reference #2
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Title
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The catalytic subunit of human protein kinase ck2 structurally deviates from its maize homologue in complex with the nucleotide competitive inhibitor emodin.
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Authors
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J.Raaf,
K.Klopffleisch,
O.G.Issinger,
K.Niefind.
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Ref.
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J Mol Biol, 2008,
377,
1-8.
[DOI no: ]
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PubMed id
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Figure 1.
Fig. 1. Structural plasticity of CK2α in the ATP binding
region. (a) Main-chain RMSDs after global 3D fit. Black curve:
hsCK2α^1–335/emodin complex fitted on
hsCK2α^1–335/AMPPNP/sulfate (PDB file: 2PVR);^23 red curve:
hsCK2α^1–335/emodin complex fitted on zmCK2α/emodin (PDB
file: 1F0Q^11). The conformational deviations at the ATP-binding
loop and at the hinge region are discussed in the text while
those at the β4–β5 loop (part of the interface to CK2β) and
the so-called CMGC insert (protein docking module typical for
all CMGC kinases) are not important in the context of this
study. The RMSDs were minimized with a least-squares algorithm
implemented in BRAGI.^24 (b) Pairwise structural comparisons of
the hinge regions of various maize and human CK2α
structures specified by their PDB codes. The given values are
minimized RMSDs (in angstroms) for all main-chain atoms from
Phe113 to Phe121 calculated with the program LSQKAB from the
CCP4 suite.^19 Two conformational clusters are indicated by a
colored background. (c) Stereo picture to illustrate the
structural adaptations of hsCK2α^1–335 upon ligand binding.
The picture shows the hsCK2α^1–335/emodin structure of this
study (yellow carbon atoms) and—after structural
superimposition—the hsCK2α^1–335/AMPPNP/sulfate complex of
PDB file 2PVR^23 (black carbon atoms). The drawn parts of the
hsCK2α^1–335/emodin structure are covered with electron
density (contour level of 1 σ) in different colors (green for
the enzyme, blue for emodin, and red for water). The electron
density around Glu114 is weaker because this side chain occurs
in two alternate conformations (only one conformation is shown).
The figure was drawn with BobScript^25 and Raster3D.^26 (d)
Stereo picture to demonstrate the major structural differences
between the emodin complexes of hsCK2α^1–335 (yellow carbon
atoms and backbone traces) and zmCK2α (PDB file 1F0Q;^11 gray
carbon atoms and backbone traces). The division of the hinge
region into two conformational clusters, which is apparent from
(b), is illustrated by the backbone traces of the seven
structures included in the computational analysis. Two pieces of
the final electron density are drawn with a contour level of 1
σ. The purple dotted line indicates a hydrogen bond between
His160 and Arg47 across the ATP-binding cleft of zmCK2α.
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Figure 2.
Fig. 2. Emodin binding to CK2α. (a) Stereo picture to
illustrate the differences in emodin orientation and binding
between hsCK2α^1–335 (carbon atoms and glycine-rich loop in
yellow) and zmCK2α (PDB file 1F0Q;^11 carbon atoms and
glycine-rich loop in gray). Some parts of the
hsCK2α^1–335/emodin complex are covered by the final electron
density (contour level 1 σ). The purple dotted lines indicate
hydrogen bonds. Distances are given in angstroms. The figure was
drawn with BobScript^25 and Raster3D.^26 (b and c) Main
interactions of emodin with human (a) and maize (b) CK2α. The
electron density of the emodin molecule as bound to
hsCK2α^1–335 was drawn with a contour level of 1 σ. The
purple dotted lines indicate hydrogen bonds; the black dotted
lines indicate van der Waals interactions. Distances are given
in angstroms. Hydrogen bonds play a major role in inhibitor
binding in the hsCK2α^1–335/emodin structure (b) but not in
the maize CK2α/emodin structure (c).
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The above figures are
reproduced from the cited reference
with permission from Elsevier
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Secondary reference #3
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Title
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Protein kinase ck2 in health and disease: protein kinase ck2: from structures to insights.
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Authors
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K.Niefind,
J.Raaf,
O.G.Issinger.
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Ref.
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Cell Mol Life Sci, 2009,
66,
1800-1816.
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PubMed id
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