Ren DeBrosse '18 and Katie Kaestner '16
Glycogen synthase kinase 3-beta (GSK-3beta) is a
serine-threonine protein kinase involved in several signaling
pathways regulating cell proliferation, differentiation, and
apotosis. Although first discovered in glycogen metabolism and
insulin response pathways, GSK-3beta plays a critical role in Wnt,
Hedgehog, and Reelin signaling pathways as well. GSK-3beta's
wide-ranging regulatory effects make it a focus of study for many
diseases, including diabetes, Alzheimer's Disease, Parkinson's
Disease, schizophrenia, and some types of cancers, among
In the search for pharmaceuticals that can inhibit this
kinase, the major targets for inhibition are the kinase active sites
where its targets get phosphorylated and the ATP activation site.
The problem with many compounds that can bind these sites is that
they also bind to other necessary signaling molecules, such as
cyclin-dependent kinases. In this tutorial, we present a molecule
designed by the pharmaceutical company Astra-Zeneca that inhibits
GSK-3beta at an ATP-binding site specific to the protein.
II. General Structure
Here, we see the homodimer GSK-3beta in its
form. Each monomer consists of a beta-strand,
N-terminal domain and a ?-helical,
C-terminal domain. A short
helix interrupts the beta-barrel. The activation
segment is responsible for the protein kinase's catalytic
All protein kinases
share a conserved overall fold but differ in the core sequences of
their activation segments, and phosphorylation of these activation
segments serve to activate many kinases.
, and a
, in order from N to C terminus. The segment is flanked by
at the N-terminal and C-terminal ends, respectively.
III. Dimer Interface
monomers of the homodimeric GSK-3beta protein align in an
intermolecular head-to-tail fashion, where the residues Asp
260 to Val 263 at the N terminus of one monomer interacts
with the residues
Tyr 216 to Arg 220 through a series of
at the end of the activation segment on the
C terminal domain
of the other monomer. Residues Ile
Phe 229, Gly
263, and Leu
266 on the one monomer form a
in which the hydrophobic side chain of Tyr
216 of the other monomer interacts.
Although the monomers at the
visibly possesses shape complementarity overall, direct interactions
between them are few. These
interactions include hydrogen bonding between the peptide carbonyls
and nitrogens of
IV. Phosphorylation Sites
Kinase activity is typically regulated by the
phosphorylation of a particular residue of the activation loop,
called the primary phosphorylation site. The interaction between the
basic residues of the N-terminal domain and the phosphorylated
activation loop drives the conformational change of the loop into a
substrate binding pocket. GSK-3beta, however, lacks a primary
phosphorylation site and has Val
214 in the place of the phosphorylatable residue
. Some protein kinases like GSK-3beta have instead what are
called secondary phosphorylation sites, which do not interact with
the substrate binding pocket. The residue Tyr 216 is one such site.
216 is an important secondary phosphorylation site to
GSK-3beta catalytic activity, but
buries much of the protein’s accessible surface.
Phosphorylation of Tyr 216 rotates its side chain from the “down”
conformation in the dimer interface to the “up” conformation,
thereby opening up the substrate binding pocket previously
obstructed by the dimerization.
The above is an illustration of an unphosporylated
GSK-3beta and a phosphorylated
GSK-3beta, with AR-A014418 (represented as the
space-filling molecule) bound to the substrate groove on the
phosphorylated GSK-3beta (Bhat et
al.) Our interactive model comes from a file that codes for
unphosphorylated GSK-3beta, but here it is apparent how Tyr 216
moves out of the way to allow the ligand to bind.
V. Substrate Binding Pocket
GSK-3beta has a
made up of 3 basic N-terminal residues - Arg
96 from helix beta C, Arg
180 from the activation loop, and Lys
205 from beta9 – and the peptide nitrogen of Val
214. An anion fills the pocket opened by secondary
phosphorylation, neutralizing and stabilizing the basic pocket,
and marks it for a residue in the substrate phosphorylated by
another kinase. Note how the substrate binding pocket is
located on the periphery of the molecule for easier substrate
access and recognition.
Thus, GSK-3beta uses the pocket not as a site of regulatory
control like other protein kinases with primary phosphorylation
sites, but exclusively as a substrate recognition site for primed,
pre-phosphorylated substrates, which allows GSK-3beta to work more
VI. ATP-mimetic Inhibitor Interaction
Here, the inhibitor designed by Astra-Zeneca is
shown bound at Ser 396 (not shown due to instability of crystal
structure past reside 384). The inhibitor
N-(4-methoxybenzyl)-N'-(5-nitro-1,3-thiazol-2-yl)urea, or AR-A014418
as its manufacturer calls it, binds in competition with ATP to the
. The inhibitor interacts with the ATP binding pocket primarily through
with residues Lys
Asp 200 and hydrogen bonding with residues
. The residue Asp 200 is a conserved
catalytic residue in the
of the magnesium binding loop that binds a magnesium ion, which will
position the ATP phosphates for transfer to serine side chains.
Some additional residues are important in differentiating the
selectivity of the GSK-3beta ATP binding pocket for AR-A014418
from cdk2, another protein kinase that has an ATP binding pocket
similar to that of GSK-3beta.
acts as a "selectivity residue",
and a Glu-Arg salt bridge defines the edge of the binding pocket
(Ghat et al.).
Bhat, Ratan, Yafeng Xue, Stefan Berg, Sven Hellberg, Mats Ormö,
Yvonne Nilsson, Ann-Cathrin Radesäter, Eva Jerning, Per-Olof
Markgren, Thomas Borgegård, Martin Nylöf, Alfredo Giménez-Cassina,
Félix Hernández, Jose J. Lucas, Javier Díaz-Nido, and Jesús Avila.
2003. Structural Insights and Biological Effects of Glycogen
Synthase Kinase 3-specific Inhibitor AR-A014418. J.
Biol. Chem. 278:45937-45945.
Dajani, Rana, Elizabeth Fraser, S. Mark Roe, Neville YOung,
Valerie Good, Trevor C. Dale, and Laurence H. Pearl. Crystal
Structure of Glycogen Synthase Kinase 3b: Structural Basis for
Phosphate-Primed Substrate Specificity and Autoinhibition. 2001. Cell
Nolen, Brad, Susan Taylor, and Gourisankar Ghosh. 2004.
Regulation of Protein Kinases: Controlling Activity through
Activation Segment Conformation. Molecular
Cell 15: 661-675.
ter Haar, Ernst, Joyce T. Coll, Douglas A. Austen, Hsun-Mei Hsiao,
Lora Swenson, and Jugnu Jain. 2001. Structure of GSK3b reveals a
primed phosphorylation mechanism. Nature
Publishing Group 8:593-596.
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