Download Structural Characterization of the GSK

doi:10.1016/j.jmb.2003.08.031
J. Mol. Biol. (2003) 333, 393–407
Structural Characterization of the GSK-3b Active Site
Using Selective and Non-selective
ATP-mimetic Inhibitors
J. A. Bertrand1*, S. Thieffine2, A. Vulpetti1, C. Cristiani2, B. Valsasina2
S. Knapp1, H. M. Kalisz2 and M. Flocco1
1
Department of Chemistry
and
2
Department of Biology
Pharmacia Italia S.p.A.
Discovery Research Oncology
Viale Pasteur, 10, 20014
Nerviano, Italy
GSK-3b is a regulatory serine/threonine kinase with a plethora of cellular
targets. Consequently, selective small molecule inhibitors of GSK-3b may
have a variety of therapeutic uses including the treatment of neurodegenerative diseases, type II diabetes and cancer. In order to characterize
the active site of GSK-3b, we determined crystal structures of
unphosphorylated GSK-3b in complex with selective and non-selective
ATP-mimetic inhibitors. Analysis of the inhibitors’ interactions with
GSK-3b in the structures reveals how the enzyme can accommodate a
number of diverse molecular scaffolds. In addition, a conserved water
molecule near Thr138 is identified that can serve a functional role in
inhibitor binding. Finally, a comparison of the interactions made by selective and non-selective inhibitors highlights residues on the edge of the
ATP binding-site that can be used to obtain inhibitor selectivity. Information gained from these structures provides a promising route for the
design of second-generation GSK-3b inhibitors.
q 2003 Elsevier Ltd. All rights reserved.
*Corresponding author
Keywords: glycogen synthase kinase-3; selective inhibitors; signal
transduction; structure-based design; X-ray crystallography
Introduction
Over 20 years ago, glycogen synthase kinase-3
(GSK-3) was discovered as one of many protein
kinases that phosphorylate and inactivate glycogen
synthase.1 Subsequently, molecular cloning of
GSK-3 in mammalian tissues revealed two closely
related isoforms, GSK-3a and GSK-3b,2,3 that are
related by a high degree of similarity in the kinase
catalytic domain (97%) but diverge at the N and C
termini. Nonetheless, both GSK-3a and GSK-3b
Abbreviations used: AMP-PNP, adenyl
imidodiphosphate; CDK2, cyclin-dependent kinase 2;
CDKs, cyclin-dependent kinases; ERK2, extracellular
signal-regulated protein kinase 2; FGFR, fibroblast
growth factor receptor; GSK-3b, glycogen synthase
kinase 3b; I-5, 2-chloro-5-{[4-(3-chlorophenyl)-2,5-dioxo2,5-dihydro-1H-pyrrol-3-yl]amino}benzoic acid; PKA,
protein kinase A; RMSD, root-mean-square deviation;
SU5402, 3-[(3-(2-carboxyethyl)-4-methylpyrrol-2yl)methylene]-2-indolinone; VEGFR, vascular
endothelial growth factor receptor.
E-mail address of the corresponding author:
[email protected]
contain a conserved N-terminal serine residue
(Ser21 for GSK-3a and Ser9 for GSK-3b) whose
phosphorylation is important for the regulation of
the enzymatic activity.4
Despite the specificity suggested by its name,
GSK-3 is involved in a diverse number of regulatory pathways by the phosphorylation of several
different cellular targets. Medical interest in GSK-3
stems from its involvement in animal development, metabolic control, neurodegeneration and
oncogenesis.
In
the
phosphatidylinositide
3-kinase-dependent pathway, insulin stimulation
inhibits GSK-3 by activating protein kinase B
(PKB; also called Akt) which, in turn, phosphorylates GSK-3 at the conserved N-terminal serine.4
Thus, insulin stimulation inhibits GSK-3 and, in so
doing, allows the dephosphorylation and
activation of glycogen synthase. GSK-3 is also
recognized as a key component of the Wnt signaling pathway, which is essential for pattern
development during embryonic development and
regulation of cell proliferation (reviewed in Refs.
5,6). In addition, GSK-3 has been implicated in
Alzheimer’s disease by its potential role in the
0022-2836/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.
394
Structures of GSK-3b Complexes
Figure 1. Diagram showing the chemical structures of AMP-PNP, staurosporine, indirubin-3-monoxime, alsterpaullone and I-5.
abnormal hyperphosphorylation of microtubuleassociated protein tau.7 Recent reviews give a
more detailed description of the roles played by
GSK-3 in the different cellular pathways and
pathologies.8,9 Small molecule inhibitors of GSK-3
may have a therapeutic potential in a number of
different human diseases such as cancer, type II
diabetes, chronic inflammation processes, stroke
and neurological diseases such as bipolar disorders
or Alzheimer’s disease.10
To date, three groups have published structures
of GSK-3b. These include two structures of the
unphosphorylated apoenzyme,11,12 one structure of
the Tyr216 monophosphorylated enzyme complexed with a peptide that inhibits b-catenin
phosphorylation13 and one structure of the Tyr216
monophosphorylated enzyme complexed with a
peptide from axin.14 Analysis of the GSK-3b structure provided insight into both its regulation of
the kinase activity and its preference for pre-phosphorylated substrates. Many protein kinases are
activated by the phosphorylation of residues
within the activation loop. For example, ERK2
activation consists of the phosphorylation of two
residues in the activation loop, Thr183 and
Tyr185.15 In GSK-3b, the activation loop residue
395
Structures of GSK-3b Complexes
equivalent to Tyr185 in ERK2 is Tyr216 and its
phosphorylation has been reported in vivo.16 It is
thought that this residue plays a role in the opening and closing of the substrate-binding site.13 In
contrast to ERK2, GSK-3b does not contain a phosphorylated threonine in the activation loop. However, the site expected for the “missing”
phosphothreonine is present in GSK-3b and is
used for the recognition of the primed phosphorylation on substrates with the sequence motif
SxxxS(P) (where S(P) represents a phosphoserine).
In general, GSK-3 has a preference for substrates
that contain a “priming” phosphate at position
n þ 4 (n is the site of GSK-3 phosphorylation) and
these substrates are phosphorylated more readily
than those that lack the “priming” phosphate. In
comparison with ERK2, the GSK-3 primed substrate’s n þ 4 phosphate would occupy the site
generally occupied by the phosphothreonine of
the activation loop. In addition, the “missing”
phosphothreonine
site
also
provides
an
explanation for the inhibition that results from
Ser9 phosphorylation.11,12 Presumably, after Ser9
phosphorylation the N terminus of the protein is
bound in the active site as a pseudo-substrate,
blocking the active site. Although there are several
structures of GSK-3b, no information is available
concerning how ATP-mimetic inhibitors interact
with the enzyme. We present here five structures
of GSK-3b in complex with selective and nonselective ATP-mimetic inhibitors (Figure 1). The
structural information obtained from these complexes provides a useful tool for the design of
second-generation GSK-3b specific inhibitors.
Results and Discussion
General structure
The five structures of GSK-3b presented here
were solved by the difference Fourier method
using the coordinates of the unphosphorylated
GSK-3b.11 As expected, the overall structures of
the enzyme (Figure 2) are similar to that of the
apoenzyme. In fact, the RMSD calculated for 333
structurally equivalent atoms in the apoenzyme
(PDB code 1H8F) and the inhibitor complexes
range from 0.3 Å to 0.6 Å. Several sections of the
structure lacked interpretable electron density,
indicating a high degree of disorder. These include
the N-terminal segment 1– 34, the loop 120 –124
and the C-terminal segment 386 –420. In addition,
the electron density was weak for the region 285 –
295. The crystals contain two molecules of GSK-3b
in the asymmetric unit and, in general, the two
molecules are treated as identical unless specified
otherwise. The chemical structures of the ATPmimetic inhibitors co-crystallized with GSK-3b are
shown in Figure 1 and the corresponding X-ray
data collection and refinement statistics are in
Table 1.
AMP-PNP complex
The 2.4 Å crystal structure of GSK-3b with the
non-hydrolyzable ATP analog adenyl imidodiphosphate (AMP-PNP) provides a logical starting point for understanding how ATP-mimetic
inhibitors interact with GSK-3b. Difference electron
density shows the AMP-PNP molecule and the two
magnesium ions bound in the GSK-3b active site
(Figure 3(a)). Interestingly, all three nucleotide
phosphate groups and the two magnesium ions
are all well defined in the binary complex. As
observed in the previous structures of protein
kinases with ATP-analogs, AMP-PNP binds in the
cleft formed between the N- and C-terminal lobes
of GSK-3b, with the adenine group making hydrogen bonds with the hinge residues Asp133 and
Val135 (Figures 2 and 4(a)). In addition to the
polar interactions, the adenine group also makes
hydrophobic interactions with GSK-3b residues
Ile62, Val70, Ala83, Val110, Leu132, Tyr134 and
Leu188. The ribose group of AMP-PNP interacts
with GSK-3b through a single hydrogen bond
between O3 and the carbonyl oxygen of Glu185.
No direct hydrogen bonds are observed between
O2 and GSK-3b residues. This is in contrast to
what was observed for several other protein kinase
0
0
Figure 2. Stereo view showing the a-carbon trace of the GSK-3b complex with AMP-PNP and magnesium. Every
15th a-carbon and the magnesium ions are shown as spheres and AMP-PNP is shown as ball-and-stick.
396
Structures of GSK-3b Complexes
Table 1. Crystal structure data and refinement statistics
Data collection
GSK-3b complex
Space group
Cell parameters (Å)
a
b
c
X-ray source
Resolution (Å)
No. observations
Total
Unique
Completeness (%)
Rsym
I=sI
Refinement
Resolution range (Å)
No. reflections
Working set (%)
Test set (%)
Rcryst =Rfree
RMSD
Bond lengths (Å)
Bond angles (8)
AMP-PNP
P21 21 21
Staurosporine
P21 21 21
Indirubin-3-monoxime
P21 21 21
Alsterpaullone
P21 21 21
I-5
P21 21 21
82.69
85.21
178.14
ID14eh2 ESRF
2.40
83.73
87.05
177.80
ID14eh4 ESRF
2.20
83.43
86.44
178.82
ID14eh2 ESRF
2.10
82.67
85.99
178.19
ID14eh2 ESRF
2.30
84.17
86.66
178.65
ID14eh4 ESRF
2.77
206,766
50,028
98.9 (100.0)
0.076 (0.518)
15.9 (3.1)
185,970
63,705
95.4 (97.9)
0.060 (0.419)
12.6 (1.8)
334,335
76,152
99.8 (100.0)
0.060 (0.423)
21.3 (3.6)
194,732
57,037
99.6 (99.9)
0.060 (0.512)
18.1 (2.4)
106,100
33,530
98.5 (99.6)
0.093 (0.569)
10.8 (1.9)
30 –2.40
30–2.20
30–2.10
30–2.30
30 –2.77
47,034
2,468
0.206/0.233
60,456
3,206
0.230/0.252
72,232
3,844
0.229/0.245
54,132
2,846
0.225/0.248
31,837
1,660
0.212/0.251
0.010
1.5
0.010
1.4
0.012
1.5
0.010
1.4
0.009
1.4
Numbers in parenthesis correspond to the shell of data at highest resolution.
complexes with nucleotides, in which a hydrogen
bond is formed with a conserved residue shortly
after the hinge. In GSK-3b, this residue is Thr138
and although it seems possible for a threonine to
make this type of hydrogen bond, it is not the case
in this structure. Instead, the Og of Thr138 is
directed away from the ribose group, making a
hydrogen bond with the backbone nitrogen of
Arg141.
The position and interactions made by the AMPPNP phosphate groups are indicative of a
productive binding-mode. Indeed, a superposition
of the active phosphorylated structure of CDK2
with AMP-PNP and peptide substrate17 (PDB
accession code 1QMZ) onto the GSK-3b structure
reveals that all the critical residues for phosphoryl-transfer occupy similar positions in the
two structures. Lys85 hydrogen bonds to the aand b-phosphates of AMP-PNP and also forms a
salt-bridge with a kinase-conserved glutamate
(Glu97) from the C-helix. Mg1 coordinates four
oxygen atoms; one from each of the a- and g-phosphates and one from each of the kinase conserved
residues Asn186 and Asp200. Mg2 shows octahedral coordination of six oxygen atoms; one from
each of the b- and g-phosphates, both carboxylate
oxygen atoms from Asp200 and two water
molecules. In addition, the kinase conserved
residue Lys183 (catalytic loop) extends up towards
the g-phosphate making hydrogen bonds with a
g-phosphate oxygen and a carboxylate oxygen of
another kinase-conserved residue Asp181 (catalytic
loop). No direct interactions are observed between
the AMP-PNP phosphate groups and the nucleotide-binding loop of GSK-3b. Instead the loop
interacts with the phosphate groups through
bridging water molecules and shields the phosphate groups from the bulk solvent. It is also noteworthy to mention that Og of Ser66, one of the
residues from the nucleotide-binding loop, hydrogen bonds to a carboxylate oxygen of Asp264
from the other GSK-3b molecule in the asymmetric
unit. Consequently, the position of the loop may be
partially influenced by the intermolecular contact.
However, the variation in its position in the
different complexes presented here suggests
that the loop adapts to the inhibitors. This adaptation appears to occur independently from the
intermolecular contact, depending only on the
inhibitor.
A superposition of the other reported GSK-3b
structures11 – 13 onto the GSK-3b/AMP-PNP complex reveals several significant differences. Firstly,
the complex with AMP-PNP is the most “closed”
structure with respect to the N- and C-terminal
lobes. Indeed, a rotation that closes the interdomain angle by 4.48 for the two other unphosphorylated structures (PDB accession numbers
1H8F and 1IO9) and 7.28 for the active phosphorylated structure (PDB accession number 1GNG) is
required to fully superimpose these structures
onto the GSK-3b/AMP-PNP complex. Secondly,
all the other structures contain a sulfate or phosphate group in the vicinity of Val214. However, no
tetrahedral shaped molecules are observed in the
vicinity of Val214 in any of the structures with
inhibitors bound in the ATP-site (this work).
Instead, two water molecules are located in the
same region; one water bridges between the backbone nitrogen and Arg180 and the other extends
Structures of GSK-3b Complexes
397
Figure 3. Electron density maps of the inhibitors bound to GSK-3b. The Fo 2 Fc omit map is contoured at 3.3s. The
hinge region that interconnects the N- and C-terminal domains is shown to the left of the bound inhibitors and
Gln185 in the lower right in green. Magnesium ions are shown in dark blue and water molecules in red. Inhibitors
are shown in pink. (a) AMP-PNP, (b) staurosporine, (c) indirubin-3-monoxime, (d) alsterpaullone and (e) I-5.
398
Structures of GSK-3b Complexes
Figure 4. Diagram showing the binding of (a) AMP-PNP, (b) staurosporine, (c) indirubin-3-monoxime and
(d) alsterpaullone to GSK-3b. Residues playing a direct or indirect role in inhibitor binding are shown in a ball-andstick representation. Inhibitor molecules are shown in tan; water molecules and Mg2þ are shown in red and yellow,
respectively. The Ca trace of GSK-3b is shown in blue and green for the N- and C-terminal domains, respectively. To
avoid “cluttering” in the AMP-PNP diagram, the two water molecules coordinating Mg1 are omitted and Asp200,
the residue coordinating Mg1 and Mg2, is unlabelled.
out into the solvent region, making hydrogen
bonds only with the first water molecule. A more
detailed comparison of the AMP-PNP complex
and the activated, Tyr-phosphorylated structure
(1GNG) reveals other differences that are
presumably related to the activation. In the AMPPNP complex, the aromatic ring of Tyr216 is
rotated inwards so that it makes hydrophobic
interactions with Val214. However, in the activated
structure Tyr216 is rotated outward and the
phosphate group interacts with Arg220 and
Arg223. Nevertheless, the main-chain paths for
residues 213– 223 in both the unactivated and the
activated structures are similar, with the largest
deviation being observed for Tyr216.
Staurosporine complex
Staurosporine is a potent inhibitor of GSK-3b,
with a reported IC50 value of 15 nM.18 The 2.2 Å
co-crystal structure of staurosporine with GSK-3b
reveals how the inhibitor binds in the ATP-binding
site (Figures 3(b) and 4(b)). The hinge interactions
include hydrogen bonds from N1 of staurosporine
to the Asp133 carbonyl oxygen and from O5 of
staurosporine to the backbone nitrogen of Val135.
These hinge interactions mimic those observed for
the adenine ring of AMP-PNP. Surprisingly, these
are the only direct hydrogen bonds observed
between GSK-3b and staurosporine. The other
polar interaction is a water-mediated interaction
399
Structures of GSK-3b Complexes
from the methylamino nitrogen (N4) of the glycosidic ring to the carbonyl oxygen of Gln185. This
water-mediated interaction is in contrast to the
direct interaction observed in the other structures
of
protein
kinases
in
complex
with
staurosporine.19 – 23 In addition, the structures of
CDK2, Chk1, Lck and PKA in complex with
staurosporine also contain a hydrogen bond to a
conserved residue shortly after the hinge (Thr138
in GSK-3b). No interaction of this type is observed
in the GSK-3b complex with staurosporine. As
observed in the AMP-PNP complex, Thr138
makes hydrogen bonds to the backbone nitrogen
of Arg141 and to a conserved water molecule. In
the staurosporine complex, this water molecule is
part of a hydrogen-bonding network that starts
with Og of Thr138, passes through four water
molecules and ends with the carbonyl oxygen of
Val135 (Figures 3(b) and 4(b)). Interestingly, this
water network fills the cavity that exists under the
fuzed carbazole moiety of staurosporine. Staurosporine also makes a significant number of
hydrophobic interactions with GSK-3b, especially
through the fuzed carbazole moiety. Indeed,
formation of the GSK-3b/staurosporine complex
buries 891 Å2 of surface area. Residues that contribute to this surface include Ile62, Gly63, Gly65,
Val70, Ala83, Asp133, Tyr134, Gln185, Asn186,
Leu188, Cys199 and Asp200.
A superposition of the AMP-PNP and the
staurosporine complexes with GSK-3b using the
C-terminal domain reveals several differences
with respect to inhibitor binding. One difference is
the angle of binding in the active site. The adenine
plane of AMP-PNP comes in to interact with the
hinge-region of GSK-3b in the “classical” way for
a protein kinase. However, the plane of the fuzed
carbazole moiety of staurosporine interacts with a
significantly different angle. A comparison of the
two planes reveals a difference in angle of approximately 158. The difference in angle also explains
why there are no direct interactions between GSK3b and the glycosidic-ring of staurosporine. This
is because the glycosidic-ring is located slightly
above the ribose-binding pocket. This lack of
occupancy of the ribose pocket for a protein kinase
in complex with staurosporine appears to be
unique for GSK-3b. In the other complexes with
staurosporine, the fuzed carbazole moieties are
coplanar with the adenine ring plane and the
glycosidic rings are bound in the ribose pocket.19 – 23
Another difference that can be observed from the
superposition of the GSK-3b complexes with AMPPNP and staurosporine is the position of the
N-terminal domain. In the complex with staurosporine, the N-terminal domain has rotated back
roughly 108 from its position in the AMP-PNP
complex, increasing the interdomain angle to
allow greater access to the ATP binding-site. Presumably, staurosporine binding has induced the
N-terminal domain movement in order to occupy
a preferred binding mode. However, it is unclear
why this binding mode is preferred over the more
classical mode of staurosporine binding observed
in the other protein kinase/staurosporine
complexes.19 – 23
Indirubin-30 -monoxime
Indirubins have been reported as potent
inhibitors (IC50 values in the 5– 50 nM range) of
GSK-3b.18 Within the series, the two most potent
GSK-3b inhibitors are indirubin-30 -monoxime
(IC50 ¼ 22 nM) and 5-iodoindirubin-30 -monoxime
(IC50 ¼ 9 nM). Indirubins are also described as
potent inhibitors (IC50 values in the 50– 100 nM
range) of cell cycle regulating cyclin-dependent
kinases (CDKs).24 This finding led to crystal structures of both active and inactive CDK2 in the
presence of substituted indirubins; CDK2 (inactive)
with indirubin-30 -monoxime, CDK2 (inactive) with
indirubin-5-sulphonic acid and CDK2/CyclinA
(active) with indirubin-5-sulphonic acid.24,25 Interestingly, these two compounds highlight the
differences in specificity within the class for GSK3b and the CDKs.18 While the 30 -monoxime
substituent is extremely potent in GSK-3b
(IC50 ¼ 22 nM) it is significantly less potent against
CDK2/CyclinA (IC50 ¼ 440 nM). However, the
opposite pattern is reported for the 5-sulphonic
acid substituent, with the compound being more
potent for CDK2/CyclinA (IC50 ¼ 35 nM) than
GSK-3b (IC50 ¼ 280 nM). To determine the basis of
this selectivity pattern, indirubin-30 -monoxime
was co-crystallized with GSK-3b. The 2.1 Å structure reveals a donor – acceptor –donor series of
hydrogen bonds between indirubin-30 -monoxime
and the hinge residues of GSK-3b (Figures 3(c)
and 4(c)). The N1 and O2 atoms form hydrogen
bonds with the carbonyl oxygen of Asp133 and
the backbone nitrogen of Val135, respectively.
These interactions are similar to those observed in
the complexes with AMP-PNP and staurosporine.
However, indirubin-30 -monoxime also makes a
third hydrogen bond with N1 and the carbonyl
oxygen of Val135. This pattern of hinge interactions
was also observed in the structures of CDK2
in complex with indirubin-30 -monoxime and
indirubin-5-sulphonic acid.24,25
In addition to the hinge interactions with
indirubin-30 -monoxime, GSK-3b also interacts
with the inhibitor through a bridging water
molecule; the oxime oxygen hydrogen bonds with
a water molecule that, in turn, hydrogen bonds
with the backbone oxygen of Gln185 (Figure 4(c)).
Another water molecule, that is also hydrogen
bonded to the Og of Thr138, helps to position the
bridging water molecule. Interestingly, both of
these water molecules were also observed in the
staurosporine complex. A final feature that
explains the potency of indirubin-30 -monoxime is
its complementary shape that allows it to bury its
apolar ring system in GSK-3b. Formation of the
GSK-3b/indirubin-30 -monoxime complex buries
683 Å2 of surface area in a tight-fitting pocket. The
pocket sandwiches the inhibitor between Ile62,
0
400
Val70, and Ala83 on the top and Leu188 on the
bottom. The complementary shape of the GSK-3b
hinge segment Leu132-Asp133-Tyr134-Val135Pro136 forms another portion of the pocket. Finally,
Arg141 forms the final piece of the pocket by
orienting its side-chain to shield the edge of the
inhibitor’s aromatic ring from the bulk solvent.
In the case of GSK-3b, modification of the
indirubin scaffold to obtain indirubin-30 -monoxime
gives a significant (27-fold) improvement in
inhibitor potency.18 Based on the structure, this
improvement can be attributed to the bridging
water molecule that links the inhibitor’s hydroxyl
group to the carbonyl oxygen of Gln185. In
addition, the combination of the monoxime group
and the bridging water provide a partial
occupancy of the ribose pocket. Unfortunately,
coordinates for the CDK2 (inactive) indirubin-30 monoxime complex were not available for comparison with GSK-3b.24 However, the minor change
in CDK2/CyclinA potency (fivefold) with respect
to modifying indirubin to obtain indirubin-30 monoxime suggests that CDK2 does not benefit
from the same type of water interaction.
Superposition of CDK2/CyclinA with indirubin5-sulphonic acid25 (PDB accession number 1E9H)
onto GSK-3b with indirubin-30 -monoxime provides
an understanding of the potencies with respect to
indirubin modifications in the 5-position.18,24 In the
case of CDK2/CyclinA, the inhibitor obtains a
large increase in potency by the addition of the
sulphonic acid group in position 5, going from an
IC50 value of 2200 nM for indirubin to 35 nM for
indirubin-5-sulphonic acid (63-fold improvement).
In contrast, for GSK-3b, the inhibitor obtains a significantly smaller increase in potency, going from
an IC50 value of 600 nM for indirubin to 280 nM
for indirubin-5-sulphonic acid (twofold improvement). Moreover, in the case of indirubin-30 monoxime the addition of a sulphonic acid
group in the 5-position actually causes a fourfold
decrease in affinity (IC50 ¼ 22 nM for indirubin-30 monoxime and IC50 ¼ 80 nM for indirubin-30 monoxime-5-sulphonic acid). In the structure of
CDK2/CyclinA with indirubin-5-sulphonic acid,
the sulphonic acid group is directed away from
the hinge and towards the DFG segment (CDK2
residues 145– 147 and GSK-3b residues 200– 202).
CDK2 residue Lys33 (Lys85 in GSK-3b) bridges
between one of the inhibitor’s sulphoryl oxygen
atoms and the side-chain of Glu51 (Glu97 in GSK3b). Another sulphoryl oxygen makes a hydrogen
bond with the backbone nitrogen of Asp145
(Asp200 in GSK-3b). The combination of the open
shape of the pocket and the complementary charge
in the region of the inhibitor’s sulphonic acid
help to explain CDK2’s affinity for indirubin-5sulphonic acid. Analysis of the corresponding
region in GSK-3b reveals that a similar pocket
exists with the correct charge distribution for
accommodating a sulphonic acid group. However,
a minor modification of the shape of the pocket in
GSK-3b and the resulting steric conflict may
Structures of GSK-3b Complexes
explain the difference in affinity for indirubin-5sulphonic acid. This is due to a single residue
change in the amino acid composition of the
pocket. In CDK2, Ala144 helps to form the bottom
of the pocket and makes a van der Waals contact
with an oxygen of the sulphonic group (3.4 Å).
This is the same oxygen that hydrogen bonds with
the backbone nitrogen of Asp145 (see above). However, in GSK-3b the corresponding residue is
Cys199 and the side-chain Sg is directed up into
the pocket. As a result, the pocket contains an
indentation that could potentially clash with a
bulky substituent, such as a sulphonic acid group,
in the 5-position of indirubin. In addition, having
a cysteine side-chain in this position could potentially block the formation of the hydrogen bond
between the sulphonic acid’s oxygen atom and the
adjacent residue’s backbone nitrogen (Asp145 in
CDK2 and Asp200 in GSK-3b). Presumably, the
combination of the indentation in the pocket and
the loss of the hydrogen bond would result in a
shift of the inhibitor to favorably reposition a
bulky 5-position substituent. However, the shift in
position would provide a new environment with
modified characteristics for the substituent. Further
analysis of the GSK-3b IC50 values with respect to
substitution of the indirubin 5-position reinforces
this interpretation. Although a sulfonic acid group
is poorly supported in this position a slight
modification in charge and hydrogen bonding
capabilities to sulfonamide makes a favorable
substituent for GSK-3b (IC50 ¼ 40 nM for
indirubin-5-sulfonamide).
Alsterpaullone
Paullones have been reported as potent ATPcompetitive inhibitors of CDKs with promising
antitumoral properties.26 In addition, a subsequent
modeling study of the paullones bound in the
CDK1 active site helped to explain the relation
between inhibitor modifications and experimental
IC50 values.27 More recently, paullones were also
shown to be potent inhibitors of GSK-3b and
CDK5/p25.28 Alsterpaullone (9-nitropaullone), the
most potent GSK-3b inhibitor of the series (GSK3b IC50 value of 4 nM), was shown to compete
with ATP for binding in the GSK-3b active site. In
addition, an investigation of the selectivity of
alsterpaullone using 25 purified kinases has
shown the compound to be extremely selective for
GSK-3a/b and CDK1/2/5.28 The combination of
high potency and selectivity for GSK-3b led us to
determine the structure of GSK-3b in complex
with alsterpaullone. The 2.3 Å co-crystal structure
shows alsterpaullone bound in the GSK-3b active
site (Figures 3(d) and 4(d)). Alsterpaullone’s hinge
interactions include two direct hydrogen bonds
with Val135 and one water mediated interaction
with Asp133; the N5 and carbonyl oxygen atoms
of alsterpaullone make a pair of hydrogen bonds
with the backbone nitrogen and carbonyl oxygen
of Val135, respectively, and the water molecule
401
Structures of GSK-3b Complexes
bridges between the carbonyl oxygen atoms of
alsterpaullone and Asp133. In addition to the
hinge interactions, alsterpaullone also makes polar
interactions between the nitro group in position 9
and the side-chain amino group of Lys85. The
final polar interaction between alsterpaullone and
GSK-3b occurs through a bridging water molecule;
the water hydrogen bonds with N12 of alsterpaullone and the carbonyl oxygen of Gln185. A water
molecule in this position was already observed in
the GSK-3b structures with staurosporine and
indirubin-30 -monoxime and, in all three cases, the
water is oriented by a hydrogen bond network
that starts with Thr138 Og.
One feature that makes alsterpaullone unique is
the positioning of its coupled ring systems. In the
complex with GSK-3b, the inhibitor’s sevenmembered ring is situated slightly below the
adenine-binding pocket and the pucker of ring B
directs the attached ring systems (Figures 1 and
4(d)). Ring A extends downward, following the
path of the hinge towards the guanidine group of
Arg141. Rings C and D extend away from the
hinge, passing through the ribose pocket and up
towards the side-chain amino group of Lys85. The
shape of alsterpaullone also allows it to bury itself
in the GSK-3b active site. Indeed, formation of the
alsterpaullone/GSK-3b complex buries 676 Å2 of
surface area. The coupled ring systems blanket
over residues Thr138, Leu188, and Cys199. The
complementary shape of rings A and B follows
the contour of the hinge residues Asp133-Tyr134Val135-Pro136 and the side-chain of Arg141 caps
the end of the pocket. Finally, Asp200 and the
side-chains of Lys85 and Leu132 form the surface
around alsterpaullone’s NO2 group. One significant difference from the other complexes with
GSK-3b is the absence of van der Waals interactions from the N-terminal residues Ile62 and
Val70. In the complex with alsterpaullone, the
nucleotide-binding loop is shifted up slightly so
that no direct interactions are made with the
inhibitor. Not surprisingly, several residues from
the nucleotide-binding loop are poorly defined in
the electron density as a result of disorder.
values of less than 50%. Also, although a number
of the other GSK-3b inhibitors have been reported
as selective, the majority also inhibit the CDKs at
a similar level. However, the most potent 3-anilino4-arylmaleimides are not inhibitors of CDK2/
cyclin A.31 This finding led us to investigate the
3-anilino-4-arylmaleimides as inhibitors of GSK3b. For this purpose, 2-chloro-5-{[4-(3-chlorophenyl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-3-yl]amino}benzoic acid (Figure 1), hereinafter referred
to as I-5, was synthesized using the protocols of
Smith and co-workers.31 In order to take advantage
of commercially available starting materials, the
3-chloro substituent of ring B (GSK-3a
IC50 ¼ 76 nM) was chosen over the more potent
2-NO2 (GSK-3a IC50 ¼ 28 nM) or 3-NO2 (GSK-3a
IC50 ¼ 26 nM) ring B substituents. Although Smith
and co-workers reported a GSK-3a IC50 value for
I-5, no value has been reported for GSK-3b. Thus,
in addition to solving the 2.8 Å co-crystal structure
of I-5 and GSK-3b, IC50 (160 nM) and isothermal
titration calorimetry (Figure 5; KD ¼ 40 nM)
measurements were made on GSK-3b using I-5.
Finally efforts were made to further investigate
the selectivity of I-5.
The crystal structure of I-5/GSK-3b shows the
inhibitor bound in the active site, making an
elaborate number of interactions with residues
from both the N- and C-terminal lobes
(Figures 3(e) and 6). Not surprisingly, the hinge
The 3-anilino-4-arylmaleimide inhibitor I-5
Inhibitors containing a maleimide core structure
have been shown to be potent inhibitors of GSK3b.29 – 31 Initial reports on GSK-3b inhibition by
maleimides focused on the bisindoylmaleimide
derivatives of staurosporine that were being
widely used as inhibitors of protein kinase C.30
However, the lack of selectivity of these compounds made them less appealing for GSK-3b
inhibitor development. More recently, 3-anilino-4arylmaleimides have been reported as potent,
selective inhibitors of GSK-3a/b.31 In the same
report, the selectivities of several compounds in
the class were evaluated using a panel of 25
kinases. At an inhibitor concentration of 10 mM,
the majority of the kinases showed inhibition
Figure 5. Titration experiment showing the binding
of I-5 to unphosphorylated GSK-3b. The isothermal
titration calorimetry data were measured in PBS
buffer with 1 mM DTT. The thermodynamic
parameters determined by least-square fitting were, n ¼
1:02; KB ¼ 2:5 ^ 0:4 £ 107 Mol21 ðKD ¼ 40 nMÞ; DHobs ¼
12:2 ^ 0:2 kcal=mol; TDS ¼ 22:5 kcal=mol; DG ¼ 29:7
kcal=mol:
402
Structures of GSK-3b Complexes
Figure 6. Stereo view showing I-5 bound in the GSK-3b active site. Residues playing a direct or indirect role in
inhibitor binding are shown in a ball-and-stick representation. I-5 is shown in light brown; chlorine atoms are in
green and water molecules are in red. The Ca trace of GSK-3b is shown in blue and green for the N- and C-terminal
domains, respectively. For the sake of clarity, Leu132 and Tyr134 are unlabelled and Val110 has been omitted from
the Figure.
interactions between I-5 and GSK-3b are similar to
those observed for staurosporine; N1A of I-5 to
the carbonyl oxygen of Asp133 and O2A of I-5 to
the backbone nitrogen of Val135. Also, ring A of
I-5 is sandwiched between Ala83, on top, and
Leu188, on the bottom. However, besides the ring
A interactions, I-5 makes no other interactions that
resemble those of staurosporine. Instead, the
inhibitor takes advantage of the lack of interconnectivity between the rings to rotate rings B
and C out of plane from ring A. Ring B is rotated
out of the plane from ring A to allow maximum
interactions with Val70, Lys85 and Cys99. Ring C
is also rotated out of plane from the ring A plane,
allowing it to make extensive interactions with the
nucleotide-binding loop. Substituents on rings B
and C contribute to I-5’s binding affinity for GSK3b with several specific interactions. The chlorine
atom of ring B extends up towards the amino
group of Lys85, making van der Waals interactions.
As for ring C, the chlorine atom is directed towards
the nucleotide-binding loop, making interactions
with backbone atoms from Gly63 and Asn64. Ring
C’s carboxylate group makes the most intricate
series of interactions; one carboxylate oxygen
hydrogen bonds with Nh1 and Nh2 of Arg141 and
the other with the Ne2 of Gln185 and a water
molecule (Wat1). Interestingly, Wat1 is held in
place by hydrogen bonds to Og of Thr138, as
observed in several other complexes, and to Oe2 of
Gln185.
The three-dimensional form of I-5 allows it to
bury itself in the GSK-3b active site. In all, formation of the GSK-3b/I-5 complex buries 779 Å2
of surface area. Residues contributing to this
surface include Ile62, Gly63, Asn64, Gly65, Val70,
Ala83, Lys85, Val110, Leu132, Val135, Thr138,
Arg141, Gln185, Asn186, Leu188, Cys199, and
Asp200. One significant difference from the other
complexes is the detailed number of interactions
made with the nucleotide-binding loop. In the
GSK-3b/I-5 complex, the loop folds down towards
the inhibitor and, in so doing, forms pockets
around rings B and C. A superposition of the
AMP-PNP and I-5 complexes using the C-terminal
domain shows how GSK-3b adapts to the inhibitor.
The reorganization of the nucleotide-binding loop
in the I-5 complex begins with the segment Ile62Gly63-Asn64 shifting up slightly to accommodate
the chlorine substituent of ring C. After Asn64, the
loop shifts downwards towards Asp200, with
Gly65-Ser66 occupying a similar position to that of
the b-phosphate group of AMP-PNP. Subsequently,
the loop shifts gradually back up through the segment Phe67-Gly68. In addition to the rearrangement of the nucleotide-binding loop, two residues
in the active site, Arg141 and Gln185, also shift
positions to accommodate I-5. In the AMP-PNP
complex, Arg141 makes charge– charge interactions with both side-chain oxygen atoms of
Glu137 and the carbonyl oxygen of Gln185 interacts with the 30 -OH group of AMP-PNP. However,
in the I-5 complex the side-chains of Arg141 and
Gln185 both rotate around to interact with the
inhibitor’s carboxylate oxygen atoms.
In order to evaluate I-5’s level of selectivity, the
inhibitor was run against an internal kinase
selectivity screen (KSS) that includes 30 different
kinases. Two motivating factors for running I-5
through the KSS were (1) the fact that no information was available concerning the selectivity of
I-5 and (2) the fact that the KSS includes several
kinases that were not present in the panel used by
Smith and co-workers to evaluate four different
Interestingly,
in
3-anilino-4-arylmaleimides.31
addition to GSK-3b (IC50 ¼ 160 nM), I-5 also
inhibits two other protein kinases, VEGFR3 (also
known as Flt-4; IC50 ¼ 560 nM) and FGFR1
403
Structures of GSK-3b Complexes
(IC50 ¼ 1,200 nM). These results were somewhat
surprising given the level of selectivity reported
for the other 3-anilino-4-arylmaleimides.31 One
potential explanation could be due to the fact that
different compounds were tested in different
screens. However, this explanation seems unlikely
considering the slight differences between the compounds tested in the screens; I-5 contains a chlorine
atom in position 3 of ring B while the most similar
compound tested by Smith and co-workers contains a nitro group in the same position. A more
likely explanation for the difference in selectivity
would be due to the differences in the number
and types of protein kinases tested in the screens.
In effect, no members of the VEGFR or FGFR subfamily of kinases were included in the selectivity
screen run by Smith and co-workers. Therefore, it
seems logical that the 3-anilino-4-arylmaleimides,
in general, inhibit VEGFR3 and FGFR1.
The selective inhibition of GSK-3b, VEGFR3 and
FGFR1 by I-5 can be explained on a structural
basis using sequence and structural comparisons
between the three enzymes. Table 2 shows the residues in GSK-3b that interact with I-5, along with
the corresponding residues in the other kinases. In
addition, a superposition of the kinase domain
structures of GSK-3b (I-5 complex) and FGF1R32
(PDB accession number 1FGI) allows us to dock
I-5 into the FGF1R active site (Figure 7). Not surprisingly, based on the IC50 value of 1.2 mM, I-5
fits well into the ATP-site of FGFR1. Only a
minimum amount of rearrangement of the nucleotide-binding loop appears necessary to accommodate the inhibitor. A comparison of the
positions of the interacting atoms in 3-[(3-(2-carboxyethyl)-4-methylpyrrol-2-yl)methylene]-2-indolinone (SU5402), the oxindolinone inhibitor present
in the FGFR1 structure,32 and those of I-5 reinforce
Table 2. Amino acid compositions in the ATP-binding
pocket
GSK-3ba
CDK2
FGFR1
VEGFR3
Ile62b
Gly63b
Asn64b
Val70
Ala83
Lys85
Val110
Leu132
Asp133b
Tyr134
Val135b
Thr138
Arg141
Gln185
Leu188
Cys199
Asp200
†
†
Glu
†
†
†
†
Phe
Glu
Phe
Leu
Asp
Lys
†
†
Ala
†
Leu
†
Glu
†
†
†
Ile
Val
Glu
†
Ala
Asn
Glu
Arg
†
Ala
†
Leu
†
Tyr
†
†
†
†
Val
Glu
Phe
Cys
Asn
Asn
Arg
†
†
†
a
Listed are GSK-3b residues that interact (interatomic distance ,3.9 Å) with I-5.
b
Residues that interact with I-5 using only main-chain
atoms. †, indicates the same residue as in GSK-3b.
the docking results. Clearly, the hinge interactions
made by I-5 would be similar to those made by
the oxindolinone ring of SU5402. However, the surprising result is the similarity in the positions of
the carboxylate groups in the two inhibitors.
Based on the interactions observed between
SU5402’s carboxylate group and the side-chain
nitrogen of FGFR1’s Asn568, we would expect the
carboxylate of I-5 to make a similar interaction
with Asn568. Actually, closer analysis of the superimposed structures reveals that the position
occupied by FGFR1 atom Nd2 of Asn568 corresponds to Wat1 in the GSK-3b structure. Hence,
Asn568 in FGFR1 replaces the combination of
Thr138 and Wat1 in GSK-3b that interact with the
carboxylate of I-5. In addition to Wat1, Arg141 and
Gln185 also interact with I-5’s carboxylate group
in the GSK-3b/I-5 complex. The corresponding
residues in FGFR1 are Glu571 and Arg627,
respectively. Based on the position of these
residues in the FGFR1/SU5402 structure, we
would not expect these residues to behave
differently in a complex with I-5; Glu571 hydrogen
bonds with Nd2 of Asn568 and makes charge–
charge interactions with the guanidino group of
Arg570 while Arg627 extends out away from
Asn568.
The lack of an available structure for VEGFR3
makes it impossible to perform a similar docking
with I-5. However, using the sequence similarities
between the kinase domains of VEGFR3 and
FGFR1 (Table 2) and recognizing the reported similarities in structure between FGFR1 and VEGFR233
we can draw conclusions about the VEGFR3/I-5
complex. Firstly, we would expect I-5 to fit well
into the active site and make similar hinge interactions. Secondly, VEGFR3 would take advantage
of the Asn in the position equivalent to Thr138 in
GSK-3b to interact with I-5’s carboxylate group.
Finally, we would expect I-5 to fit slightly better in
the VEGFR3 active site than in the FGFR1. This
conclusion is based on the fact that VEGFR3 also
has a cysteine in the position that corresponds to
Cys199 in GSK-3b. Therefore, VEGFR3 would be
able to make van der Waals interactions with I-5’s
ring B that would not be possible with the alanine
in FGFR1.
I-5 inhibits GSK-3b, VEGFR3 and FGFR1 but, at
an inhibitor concentration of 10 mM, does not
inhibit CDK2/cyclinA. This finding is likely due
to the fact that CDK2 contains an acidic residue
(Asp86) in the position that corresponds to Thr138
in GSK-3b (Table 2). As mentioned above for
FGFR1 and VEGFR3, an asparagine or, in this
case, an aspartic acid could potentially replace the
interaction made by Thr138 and Wat1. However,
at neutral pH an aspartic acid would not be able
to form a similar hydrogen bond with the I-5’s
carboxyl group. By having an acidic group in this
position, I-5 obtains selectivity against those
kinases that contain an acidic residue in the
equivalent position, a population that corresponds
to roughly 50% of the known kinases. Of course,
404
Structures of GSK-3b Complexes
Figure 7. Stereo view showing I-5 docked into the active site of FGFR1 by the superposition of Ca atoms in the ATPbinding sites (Table 2) of GSK-3b and FGFR1. I-5 is shown in light brown and SU5402 in blue. For the sake of clarity,
Gly485, Glu486 and Val561 are unlabelled and Ile545 has been omitted from the Figure.
for those kinases having non-acidic residues in this
position there are additional factors to consider in
order to obtain a reasonable level of inhibition.
Obviously, a logical way to determine the importance of I-5’s carboxyl group for the selectivity
against CDK2 would be to analyze a similar
3-anilino-4-arylmaleimide that does not contain a
carboxyl group in the 3-position. In the report by
Smith and co-workers,31 two compounds satisfying
these criteria were analyzed for percent inhibition
using the 25 kinases, but unfortunately, no values
were reported for CDK2/cyclinA with these
compounds. Interestingly, Mohammadi and
co-workers used a similar explanation for why
SU5402 is specific for FGFR1.32 Apparently,
SU5402 also takes advantage of its carboxyl group
to obtain selectivity for FGFR1 and against both
the insulin and PDGF receptors. This observation
and other similarities between the two inhibitors
raise the question of whether SU5402 would also
inhibit GSK-3b.
In conclusion, the structures of five GSK-3b complexes with inhibitors provide, for the first time,
information concerning how the enzyme interacts
with selective and non-selective inhibitor scaffolds.
Staurosporine, a non-selective inhibitor, interacts
with GSK-3b in a unique manner that utilizes a
water network to adapt to the enzyme. Indirubin30 -monoxime benefits from both the complementarity between the inhibitor and the enzyme
and the conserved water molecules (Wat1 and
Wat2). Alsterpaullone uses the form of the coupled
ring systems to direct the interacting groups of the
inhibitor towards the hinge, Lys85 and the conserved water molecules (Wat1 and Wat2). I-5 takes
advantage of its ability to independently rotate the
attached ring systems, allowing it to position substituents that interact with the nucleotide-binding
loop, Lys85, Arg141, Gln185 and Wat1. On the
basis of selectivity, indirubin-30 -monoxime and
alsterpaullone can be grouped together because
both inhibitors have similar problems for
selectivity with respect to the CDKs. In contrast,
I-5 does not have selectivity problems with the
CDKs but does have issues with respect to the
tyrosine kinases VEGFR3 and FGFR1. I-5 obtains
selectivity versus the CDKs by taking advantage of
sequence differences just outside of the ATP-binding cleft. Unfortunately, in this case, the strategy
of going outside the ATP-binding cleft to obtain
specificity led to new selectivity issues. However,
future efforts could potentially take into account
other differences between GSK-3b, VEGFR3 and
FGFR1 to obtain selective GSK-3b inhibitors.
Materials and Methods
Construct design, expression and purification
of GSK-3b
DNA encoding for full length GST was amplified by
PCR, incorporating an N-terminal Xba I restriction site
and C-terminal stop codon, a sequence corresponding
to the Prescission site and Bam HI restriction site. The
product was digested with Xba I and Bam HI ligated into
appropriately digested pVL 1392 (Pharmingen, San
Diego, CA), creating the pVL-GST vector.
DNA encoding for full length GSK-3b was amplified
by PCR, incorporating an N-terminal Bgl II restriction
site and C-terminal stop codon and Bgl II restriction site.
The product was digested with Bgl II and ligated into
pVL-GST digested by Bam HI.
Spodoptera frugiperda insect cells, SF21, were transfected with the pVL-GST-GSK-3b plasmid using the
baculogold transfection system (Pharmingen) as
described by the manufacturer’s protocol. One liter of
High Fivee cells (Invitrogen, San Diego, CA) in suspension at 2 £ 106 cells/ml was inoculated with 10 ml of
amplified GSK-3b virus and incubated at 27 8C for 48
hours. Cells were harvested by centrifugation at 1500g
for 15 minutes. Cells were frozen by submersion in dry
ice/alcohol and then thawed when needed for
purification.
405
Structures of GSK-3b Complexes
For the purification of GST-GSK-3b, cells were re-suspended in 100 ml of buffer A (50 mM Hepes (pH 7.2),
10% (v/v) glycerol, 20 mM DTT, 150 mM NaCl, protease
inhibitor cocktail (1 tablet/25 ml of buffer; Roche Biochemicals)) and mechanically homogenized. The lysate
was centrifuged at 25,000g for 45 minutes to remove
insoluble material. The supernatant was passed over a
0.8 mm vacuum filter and the filtrate was loaded onto a
Glutathione Sepharose column (Amersham Biosciences
Biosciences, Piscataway, NJ). After loading, the column
was washed with five column volumes of buffer A
followed by five column volumes of buffer A with only
10 mM DTT, and finally five column volumes of buffer
B (50 mM Hepes (pH 7.2), 10% glycerol, 3 mM DTT,
150 mM NaCl). The protein was cleaved on the
column by adding half a column volume of buffer B
and Prescission enzyme (30 ml/ml of resin; Amersham
Biosciences Biosciences) for 16 hours at 4 8C. After
cleavage, the protein was eluted with 4 £ 1/2 column
volumes of buffer B and fractions containing GSK-3b, as
analyzed by SDS-PAGE, were pooled and dialyzed
against 50 mM Hepes (pH 7.2), 10% glycerol, 3 mM
DTT, 80 mM NaCl. After dialysis, GSK-3b was loaded
onto Mono S column (Amersham Biosciences) and
the column was washed for 15 column volumes. Next, a
linear gradient was applied from 0 to 100% of elution
buffer (50 mM Hepes (pH 7.2), 10% glycerol, 3 mM DTT,
500 mM NaCl) for 100 column volumes. GSK-3b was
thereby separated into three peaks corresponding to the
different phosphorylation forms (unphosphorylated,
monophosphorylated and diphosphorylated). Fractions
containing the different phosphorylated forms of GSK3b, as analyzed by SDS-PAGE, Western blot and ES-MS,
were pooled and concentrated to approximately 12 mg/
ml in 10% glycerol, 185 mM NaCl and 100 mM Hepes
(pH 7.2). A typical final yield for each liter of High
Fivee culture was 1 – 2 mg of purified GSK-3b.
ATP-mimetic inhibitors
The inhibitors AMP-PNP, staurosporine, indirubin
and alsterpaullone were obtained commercially. I-5, the
3-anilino-4-arylmaleimide inhibitor, was synthesized as
described in the protocols reported by Smith and
co-workers.31
crystals. A seed stock solution was prepared by crushing
a single GSK-3b crystal in 20 ml of reservoir solution. At
the time of crystallization, the seed stock solution was
vortexed and 0.1 ml was added to the 1 ml of protein
and reservoir solutions.
X-ray data were collected at the ESRF beamlines ID141 and ID14-4 (Grenoble, France). Data were processed
using DENZO and SCALEPACK.35 Bound ligands in the
active site were identified by the difference Fourier
method using the phases of the apoenzyme GSK-3b
structure.11 Model building of the protein and inhibitors
were performed with O36 and the structures were refined
with CNX37 using non-crystallographic symmetry
restraints for residues of the C-terminal lobes in the two
monomers. Graphic Figures were made using MolScript
version 2.138 and Raster3D version 2.4.39 Surface areas
were calculated by the Lee and Richards algorithm40
using the software package CNX.37 A summary of the
diffraction data and the refinement statistics are shown
in Table 1.
Activity assay
FGFR1, GSK-3b and VEGFR2 were expressed as GSTfusion proteins and purified using a Glutathione
Sepharose column. Each kinase was assayed for its
ability to phosphorylate the appropriate biotin labeled
peptide/protein substrate using radioactively labeled
ATP. Reactions were stopped by the addition of a
concentrated EDTA solution þ Streptavidin coated
Scintillation Proximity Assay beads. The beads were
then captured by CsCl flotation, counted by liquid
scintillation and IC50 values were generated by fitting
data using a sigmoidal dose-response (variable slope)
model.
Protein Data Bank accession numbers
Atomic coordinates for the GSK-3b complexes with
AMP-PNP, staurosporine, indirubin, alsterpaullone and
I-5 have been deposited with the RSCB Protein Data
Bank (accession codes 1PYX, 1Q3D, 1Q4I, 1Q3W, and
1Q4L, respectively).
Crystallographic studies
Acknowledgements
All crystals of unphosphorylated GSK-3b were grown
at 20 8C by vapor diffusion in hanging drops. Inhibitor
concentrations of 1 mM in the crystallization drops were
used for all crystallization trials. Crystals of the AMPPNP/Mg complex were grown by mixing 1 ml of protein
solution with 1 ml of reservoir solution (16– 24% (w/v)
PEG 3350 monodisperse, 5% glycerol, 20 mM magnesium chloride and 100 mM Hepes (pH 7.0)). These
crystals are isomorphous to the apo GSK-3b
crystals,11,12,34 with two molecules in the asymmetric
b ¼ 85:2 A
and
unit and cell dimensions of a ¼ 82:7 A;
Prior to data collection, a single crystal of
c ¼ 178:1 A:
GSK-3b-AMP-PNP was serially transferred into cryoprotectant solutions containing increasing levels of
glycerol (final concentration 25%) and frozen by
plunging into liquid nitrogen.
In order to avoid the serial transfer step, subsequent
crystal complexes were grown using 10% glycerol in the
reservoir solution. However, under these conditions
seeding techniques were necessary to obtain high-quality
We thank Elspeth Gordon and Stéphanie
Monaco for generous help during data collection
at the ESRF, Paolo Pevarello and Angelo Casalino
for the I-5 synthesis, Paola Magnaghi for providing
the initial GSK-3b clone, Fulvia Roletto and the
KSS team for the activity tests and Laurence Pearl
for advanced access to the apoenzyme coordinates.
We gratefully acknowledge Alex Cameron, Elena
Casale, Marina Marconi, Irimpan Mathews and
Nikolaus Schiering for support and encouragement over the course of this work.
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Edited by I. Wilson
(Received 3 April 2003; received in revised form 7 April 2003; accepted 15 August 2003)