in Complex with Synthetic Inhibitors
Jennifer L. Howard '09 and Emily K. Staudenmaier '10
Protein kinases belong to the enzyme family of
transferases that catalyze the transfer of a phosphoryl group from a
donor to an acceptor. Cyclin-dependent kinases (CDKs) are
serine/threonine kinases that require association with a cyclin
regulatory protein for activation 
. They are responsible for
progression through the cell division cycle, helping to ensure that the
eukaryotic genome is replicated
only once per cell cycle. CDKs are required for the correct timing and
order of events of cell division [1,2,4,7].
There are four phases in the
cell division cycle: G1, S, G2,
and M. Click
for a schematic
drawing of the cell cycle. CDK2,
a crucial component of
complex, is responsible for the G1/S phase
Cells prepare for divison during the G1 phase
and the actual chromosome
replication occurs during the synthesis or S phase. Each chromosome is
replicated, resulting in two sister chromatids. CDK levels are low
during the G1 phase but
elevated during S, G2, and M phases, limiting
opportunities for cyclin
binding and replication .
retinoblastoma protein is inactivatedthrough phosphorylation by CDK2,
committing the cell to
division during the G1 phase. CDK2
regulated by the A and E forms of cyclin. Cyclin
E binds to CDK2 for progression from the G1
phase to the S phase
and Cyclin A is critical for progression through the S phase of the
division cycle [2,3,4].
Mutations in CDKs and/or their
are associated with overexpression and amplification, leading to
uncontrolled cell growth: tumors. As CDKs play such a huge role in the
cell division cycle, they have become important targets in
drug-discovery programs for cancer, diabetes, and immune diseases.
Certain macrocyclic compounds
have been found to be inhibitors to the Cyclin A and E binding sites to
CDK2 and therefore show great
potential as anti-tumor agents .
II. CDK2 General Structure
CDK2 is a monomer comprised of a
chain of 298 amino acid residues. Structurally, CDK2 is made
up of mainly α-helix
elements as well as a
CDK2 is composed of two
lobes: a smaller amino-terminal
lobe that contains primarily
β-sheets and the PSTAIRE
and a large carboxy-terminal lobe
made up of α-helices.
Two α-helices, the PSTAIRE helix
and the L12 helix ,
are important for cyclin binding and the conformation
changes which occur in CDK2 after cyclin is bound.
also called the activation loop, is located
near the kinase active site. It provides selectivity for substrate
binding by blocking the binding of protein substrates at the entrance
of the active-site cleft .
vitro as a CDK2 inhibitor. This inhibitor binds to the
hinge region of CDK2, which contains the T-loop, PSTAIRE helix,
and L12 helix
III. Cyclin A and E General
A basic knowledge of the
structure of the cyclin forms that bind to CDK2 is useful to fully
understand how synthetic inhibitors act to deactivate CDK2.
Cyclin A and
Cyclin E are
structurally similar but have slight sequential differences,
particularly in the
C-terminal regions of the two forms, which affect the strength of
bonding at the cyclin/CDK2 interface [2,3].
Cyclin E is 395 amino acid
long and consists of two five-helical domains with additional helices
at the N and C terminals.
a schematic drawing clarifying the names of the
helices of Cyclin E, please click
α3 helix forms a hydrophobic core
acts as a point
of organization for the
α1, α2, α4, and α5 helices
arranged around this core.
and Ala184 interact
to allow close
packing of the α2
Cyclin A is 431 amino acid
residues long and consists of two helical domains with identical chain
is a globular structure of 12
a schematic drawing clarifying the names of the helices of Cyclin A,
please click here
Each helical domain consists of
a right-handed 3-helix bundle
with two extra helices
packed against the bundle’s side.
domains are connected by a linker of five amino acids. One domain
contains the cyclin box ,
forming the binding
site for the PSTAIRE helix
and making contacts with the
N-terminal β-sheet of CDK2.
to Cyclin E, the hydrophobic core
of the 5-helix motif is formed by the α3
and α2 helices
pack against the α3 helix
and help form the hydrophobic core
while participating in inter-repeat packing with
α2’ helices .
and α5 helices
pack against the α3 helix and
interact with CDK2. Alanine residues allow close packing
interactions between helices, the most significant being Ala 235 and Ala 264 from the α2 and α3
IV. Cyclin Binding
Understanding how cyclin binding
is essential for successful design of a CDK2 synthetic inhibitor.
The cyclin dependent kinase
includes CDK2, is inactive until complexed with a regulatory protein
called cyclin .
Cyclins share a homologous region of about 100 amino acids
termed the cyclin box but differ in other regions giving them a unique
different kinases bind different forms of cyclin.
interacts with the A and E forms only. Cyclin E associates with CDK2 to
drive cells from the G1 to the S phase through
phosphorylation of certain
targets. Cyclin E is not required for cell cycling since Cyclin A is
structurally similar and can perform the same cell cycle functions.
However, Cyclin E is is required for re-entry of a cell into the cell
cycle from the G0 phase, which is why the
presence of both cyclin
forms is necessary in the cell. On entry into S phase, Cyclin E is
abruptly destroyed by the proteasome that it targets. Cyclin A,
expressed in response to the CDK2/Cyclin E activities, then associates
with CDK2 to drive cells through S phase.
Cyclin E is found in high
most tumors and almost all aggressive cancers. This fact further
stresses the importance of finding an inhibitor to act as an anti-tumor
agent, which will selectively bind CDK2 and prevent binding with Cyclin
Binding of cyclin induces a
change that alters the structure around the active site. The complex
shown to the left is a view of CDK2 unbound to cyclin.
PSTAIRE and L12 helices
are involved in
the conformational changes in CDK2 after it has bound a form of cyclin.
The L12 helix prevents
motion of the PSTAIRE helix in
free CDK2. The
binding of Cyclin A to CDK2 melts the L12 helix into a
nearly 90 degrees about its helical axis, thereby reconfiguring the
The PSTAIRE helix interacts
directly with cyclin by forming a hydrogen-bond between the Glu51
residue and the Lys33
residue on Cyclin E. The PSTAIRE
is virtually parallel to the α5
Cyclin E helix and almost
perpendicular to the C-terminal end of the α3
T-loop is also extremely
correct cyclin binding since accurate orientation of this structural
required for selective recognition of a sequence motif present on the
Cyclin A and E forms. It associates with the
C-terminal lobe of cyclin.
acts as an organizing center and
makes contacts with three Arginine residues (Arg
and 150) also
on the CDK2 molecule.
and Arg150 form hydrogen-bonds
carbonyl groups on Leu187
on Cyclin E.
Cyclin A binds to one side of
interacting with both lobes of CDK2 to form a large, continuous
protein-protein interface. This Cyclin
A-CDK2 interface is formed from
the interlocking of the
PSTAIRE helix , the
T-loop , portions of the
and C-terminal lobe
from CDK2, and helices
α4 , and
α5 from the first
repeat as well as the N-terminal
helix from Cyclin A.
L12 helix of CDK2 is a
crucial component of this
interface. In the Cyclin A-CDK2 complex, this helix rotates and moves
inward toward the catalytic cleft. The cyclin box surrounds
middle part of the PSTAIRE
helix lies parallel to the
PSTAIRE helix on one side and the C terminus
of the α3
helix contacts the other side perpendicularly.
helix is bound by an extended region of hydrophobic
interactions, whereas the N- and C-terminal regions are bound by
networks of hydrogen-bonds. Ile
49 , in particular, fits
tightly into a hydrophobic pocket lined with side chains of Leu 263,
Phe 267 , Leu 299 ,
Leu 306 , and
an aliphatic region
of Lys 266 from
Van der Waals interactions,
such as those between
Ile 52 and Phe
of Cyclin A, are also important to the Cyclin A-CDK2
N-terminal portion of the
bound by helices
α2 , and
α3 from the
first repeat of
Cyclin A, as well as its N-terminal
helix . Van der Waals interactions
occur between Ala 151,
Phe 152 , and Tyr 159
of CDK2 and
Phe 267 , Ile
182 , and Ile 270
of Cyclin A.
link Arg 150
the backbone carbonyl groups
and Ile 270
of Cyclin A.
binding occurs mainly at the PSTAIRE
helix and the T-loop on CDK2. The
CDK2/cyclin interface area is 3252
A°2 in Cyclin E and 2839 A°2 in Cyclin A.
E has a larger area available for interactions
between the two complexes and therefore the potential for stronger
increased binding affinity of CDK2 for Cyclin E has been confirmed
show exciting potential as tumor suppressors. The majority of
kinase inhibitors interact with the kinase backbone motif which is part
of the cyclin binding site. Therefore, the inhibitors act by sterically
inhibiting the binding of the activation molecule that the kinases need
in order to have a physiologically significant activity .
inhibitors are one example of
a compound that acts on CDK2 by the method described above. It forms hydrogen-bonds
Glu81 and Leu83
residues which anchor the
conformationally free inhibitor molecule to the kinase.
sulfonate group on the ligand also interacts with the NH group on the
backbone of Asp86
and the carbonyl group on the backbone of
inhibitor was also designed to
inhibit the CDK2 form over other members of the kinase family. This
target specificity makes thiazolidinone inhibitors an even more
promising discovery in terms of medical applications .
aminopyrimidines are another class of molecules which act
CDK2. These compounds showed potential both in-vitro and in-vivo as
inhibitors and they interact with the same general region of CDK2 that
the thiazolidinone inhibitors do. However, structural differences
between the two inhibitors result in macrocyclic aminopyrimidines
interacting with slightly different amino acid residues. The
aminopyrimidine recognition site was shown to bind to the hinge region
of CDK2 through two hydrogen-bonds
. An oxygen atom from the
meta-sulfonamide forms two
hydrogen-bonds to the backbone nitrogen and
to a side chain oxygen of Asp86
amide nitrogen atom from
this group interacts with the backbone carbonyl group of Ile10 .
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