Identification of Substrate Binding Site of
Cyclin-dependent Kinase 5*
Pushkar
Sharma,
Peter J.
Steinbach
,
Monica
Sharma,
Niranjana D.
Amin,
Joseph J.
Barchi Jr.§, and
Harish C.
Pant¶
From the Laboratory of Neurochemistry NINDS, the
Center for Molecular Modeling, Center for Information
Technology, and the § Laboratory of Medicinal Chemistry,
NCI, National Institutes of Health, Bethesda, Maryland 20892
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ABSTRACT |
Cyclin-dependent kinase 5 (CDK5),
unlike other CDKs, is active only in neuronal cells where its
neuron-specific activator p35 is present. However, it phosphorylates
serines/threonines in S/TPXK/R-type motifs like other CDKs.
The tail portion of neurofilament-H contains more than 50 KSP repeats,
and CDK5 has been shown to phosphorylate S/T specifically only in
KS/TPXK motifs, indicating highly specific interactions in
substrate recognition. CDKs have been shown to have a high preference
for a basic residue (lysine or arginine) as the n+3
residue, n being the location in the primary sequence of a
phosphoacceptor serine or threonine. Because of the lack of a crystal
structure of a CDK-substrate complex, the structural basis for this
specific interaction is unknown. We have used site-directed mutagenesis
("charged to alanine") and molecular modeling techniques to probe
the recognition interactions for substrate peptide (PKTPKKAKKL) derived
from histone H1 docked in the active site of CDK5. The experimental
data and computer simulations suggest that Asp86 and
Asp91 are key residues that interact with the lysines at
positions n+2 and/or n+3 of the substrates.
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INTRODUCTION |
Cyclin-dependent kinases
(CDKs)1 phosphorylate
proline-directed serine and threonine residues in peptide/protein
substrates. These kinases are active only when bound to their
regulatory partners, cyclins. However, maximal kinase activation is
achieved only after phosphorylation of these kinases by CDK-activating
kinase (1 and references therein). CDK5 is slightly different from
other CDKs; it is active only in neuronal cells because of the presence
of its cyclin-like neuronal activator p35 and does not seem to require phosphorylation for its catalytic activity (1-3). However, like other
CDKs, it phosphorylates S/T in S/TPXK/R motifs unique to the
CDK family of kinases (4, 5). The specificity of this interaction is
demonstrated by the phosphorylation of the tail portion of high
molecular weight neurofilament-H (NF-H) by CDK5. The tail portion of
this molecule contains 52 KSP repeats that can be classified as either
KSPXK or KSPXXX. CDK5 specifically phosphorylates only KSPXK motifs and does not
phosphorylate any other KSP site (4, 5). Histone H1 is phosphorylated
in vitro at a KTPKK motif (6). These studies indicate that
the CDKs have high affinity for a basic residue like lysine or arginine at the n+3 position of the sequence, where n is
the position of the phosphoacceptor serine or threonine. The mechanism
of activation of CDK2 by cyclin A was revealed by the crystal
structures of CDK2 (7) and its complex with cyclin (8). A recently
published structure of a cyclin A-CDK2 complex phosphorylated by
CDK-activating kinase demonstrated the structural basis for further
activation by phosphorylation (9). However, because of the lack of a
crystal structure of a CDK-peptide substrate complex, the highly
specific interactions addressed above are not well understood.
Alignment of more than 60 protein kinases (PKs) revealed the minimal
catalytic domain to be ~ 260 amino acids; this group exhibited
20-60% sequence homology (10). The crystal structures of the PKs
solved to date reveal that the sequence homology and secondary
structural elements are conserved at the functionally important sites,
such as those involved in ATP binding and catalysis. When these amino
acids are used to align the PKs, the major differences occur in the
regions coding for loops (10). Despite their homologous sequences and
similar global structures, PKs differ surprisingly in the substrate
sequences they recognize, indicating highly specific interactions
between the active sites of the enzyme and the substrates.
cAMP-dependent protein kinase (PKA) was the first kinase to
be crystallized without (11) and with (12) a RRXS/A pseudo substrate peptide (PKI). Consequently, the crystal structure of the
PKA·PKI complex has been used as a model kinase-substrate system. The
recently published structure of phosphorylase kinase with a peptide
substrate further confirms the specificity of these interactions (13).
These structures reveal a highly specific network of interactions
between the charged residues in the substrate with their oppositely
charged partners in the enzyme. The differences in amino acid
composition in the catalytic clefts of PKs account for differences in
substrate specificity for these kinases (14, 15).
To understand the key interactions involved in this highly specific
enzyme-substrate interaction, we have used site-directed mutagenesis,
reaction kinetics, and molecular modeling. We constructed a homology
model of CDK5 using the CDK2 coordinates from the cyclin-CDK2 crystal
structure (9) and docked a peptide derived from histone H1, which is an
in vitro substrate for CDK5 and other CDKs (6). Site-directed mutagenesis was used to confirm the interactions suggested by the computer modeling. Further refinement of the model
included inter-residue restraints suggested by the enzyme kinetics
measurements of the designed CDK5 mutants.
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EXPERIMENTAL PROCEDURES |
All fine chemicals were purchased from Sigma. The peptide
substrates were synthesized by either Research Genetics or QCB
Chemicals. [
-32P]ATP was purchased from NEN Life
Science Products.
Expression and Purification of Proteins--
GST-CDK5 in pGEX
2TK was constructed by putting the BamHI fragment of His-tag
CDK5 (3) into BamHI-cut pGEX 2TK. GST-p25 in pGEX 4T-2 was
constructed by a polymerase chain reaction method using the
oligonucleotides 5'-GTCCGGATCCGCCCAGCCCCCGCCG-3' as a forward primer,
5'-GTGATGAATTCTGGATCACCGATC-3' as a reverse primer, and DNA from the
His-tag p35 construct as a template (3). The polymerase chain reaction
products were digested with BamHI and EcoRI, and
the resulting fragments were cloned into
BamHI-EcoRI-cut pGEX 4T-2. The proteins were
expressed as described earlier (4, 5). Purification of GST fusion
proteins was carried out using glutathione-agarose chromatography by
standard procedures (Amersham Pharmacia Biotech) and as described
earlier (5). The purity of the proteins was assessed by
SDS-polyacrylamide gel electrophoresis and Coomassie Blue staining of
the gels. The protein concentration was estimated by densitometric
analysis of these gels.
Site-directed Mutagenesis--
The "charged to alanine"
mutations were made using a commercial kit (Quick
ChangeTM, Stratagene). In brief, a pair of
complementary primers of 30-40 bases was designed with desired
mutations placed in the middle of the sequence. Parental cDNA
inserted in pGEX 2TK was amplified using Pfu DNA polymerase
with these primers for 15 cycles in a DNA thermal cycler
(Perkin-Elmer). After digestion of the parental DNA with
DpnI, the mutants were transformed into Escherichia
coli (DH5
strain, Life Technologies, Inc.). The mutations were
confirmed by DNA sequencing. The GST fusion mutant proteins were
expressed and purified as described above.
Phosphorylation Assay and Enzyme Kinetics--
Standard assay
mixtures contained 50 mM Tris, pH 7.4, 2.5 mM
MgCl2, 1 mM EGTA, 150-165 ng of wild-type CDK5
or different mutants complexed with a molar excess of p25 (preincubated
enzyme preparation), 100 µM [
-32P]ATP,
and 0.1-10 mM peptide substrate in a total volume of 30 µl. Reactions were initiated by adding [
-32P]ATP and
were carried out at 30 °C for 60-80 min. Under these conditions,
the rate of phosphorylation was constant for more than 2 h and
proportional to the amount of enzyme. Reactions were terminated by
adsorption of the assay mixture on phosphocellulose paper P81(Whatman).
Phosphopeptide formation was measured by counting the radiolabeled
32P incorporation after several washings of the
phosphocellulose paper with 75 mM phosphoric acid. Kinetic
constants were derived by fitting the Michaelis-Menten equation to the
data using the Kaleidagraph program. Each experiment was done at least
twice. Representative kinetic constants are shown in Tables I and II, and the variation in these constants from one experiment to another was
typically 10-15%. The apparent peptide affinities are compared on the
basis of the Km and
Vmax/Km values.
Molecular Modeling--
Using the program LOOK (16), CDK5 was
aligned to CDK2 (9), and a model of CDK5 was built exploiting its high
homology to CDK2 (62% identity). Atoms of CDK5 were assigned
coordinates using the SegMod algorithm (16). An ATP molecule and
magnesium were docked to CDK5 by copying the corresponding coordinates
from the CDK2 complex.
The CDK5 model was then superimposed on the crystal structure of PKA
(pdb file 2CPK) by best-fitting only the backbone (N, C
, C) atoms in
the 13-residue "catalytic loops." Of particular interest was the
close proximity (1.0 Å separating C
atoms) of Asp86 in
CDK5 to Glu127 of PKA. The Glu127 of PKA forms
a salt bridge with Arg18 of PKI (2cpk), and
Arg18 of PKI corresponds to Lys6 of the H1
peptide in the antiparallel alignment (Fig.
1B). This structural alignment
and subsequent modeling of CDK5 complexed with the H1 peptide were
performed with the program CHARMM (17) and an all-atom parameter set
(18, 19) running on Hewlett-Packard workstations.

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Fig. 1.
Panel A, sequence alignment and
comparison of CDK2, CDK5, and ERK2. The residues mutated in CDK5 are
underlined. Panel B, alignment of H1 peptide in
parallel and antiparallel orientations. Thr3 of H1 and
Ala21 of PKI are the phosphorylation sites. Basic residues
that are aligned are boxed.
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In modeling the CDK5·H1·peptide complex, several approximations
were employed. A distance-dependent dielectric (
= r) coefficient was used to screen electrostatic interactions
with a potential energy "shifted" to zero at 12 Å. The shifted
potential monotonically damps the electrostatic forces for this
dielectric model (20). The distance-dependent dielectric is
a rather crude approximation to an implicit solvent, but it maintains
reasonable hydrogen bond distances.
Entropic effects were ignored, and a limited (non-ergodic) search of
conformations was performed. In other words, we did not attempt to
solve the ab initio docking problem, which would require a
more accurate energy function and more exhaustive conformational sampling. Rather, the goal of the modeling was to aid in the choice and
interpretation of experiments. Although our electrostatic approximations overly stabilize salt bridges, our intent was to enforce
these interactions, not to predict them from first principles. The
details of the peptide backbone conformation and side chain packing
should not be overinterpreted, but our model can be used to test
"lower resolution" hypotheses. For example, we can predict whether
the H1 peptide is long enough and flexible enough to interact simultaneously with the ATP and a specific subset of CDK5 residues. Thus, prospective interactions between CDK5 and the peptides identified in early stages of the modeling led us to perform specific mutation experiments. Similarly, the measured enzyme kinetics were incorporated into later stages of modeling through the addition of inter-residue distance restraints favoring specific salt bridges between CDK5 and the
H1 peptide.
Multiple families of short Monte Carlo (MC) simulations were run in
succession, differing in the moves attempted and the restraints employed. In the latter stages of the modeling, protein residues near
the peptide were free to move, but in each MC simulation the CDK5
molecule was held fixed while random rigid body translations and
rotations of the entire H1 peptide were explored as well as random
rotations of the peptide dihedral angles. Configurations were energy
minimized somewhat to relieve bad contacts before applying the
Metropolis criterion (21) for accepting conformations at a temperature
of 600 K. A subset of torsions was included in the MC move set, but all
peptide degrees of freedom were allowed to relax upon minimization.
Positional restraints were used to guide the docking of the H1 peptide
in a configuration antiparallel to that of PKI binding to PKA. Again,
this configuration was investigated because it allows interaction
between Asp86 of CDK5 and Lys6 of the peptide.
In each MC simulation, a restraint was used to keep the gamma oxygen of
the peptide Thr3 close to the third phosphate group of the
ATP molecule.
H1 Peptide Docked in the Antiparallel Orientation--
The goal
of the first family of MC simulations was to dock only the first
several residues of the H1 peptide. 100 runs were started with the
peptide in an extended conformation near the ATP molecule.
Inter-residue distance restraints were used to favor energetically four
interactions between a CDK5 residue and a peptide residue:
Asp86 and Lys5, Asp86 and
Lys6, Asp125 and Lys2, and
Asp143 and Lys2. Only 11
and
torsions
(excluding proline
angles) of the first seven residues were
eligible for rotation during MC moves.
The lowest energy conformation obtained from these simulations was used
to initiate the second family of 50 MC runs. The goal here was to guide
the peptide toward an orientation like that of PKI bound to PKA. Two
additional inter-residue restraints were added: Asp91 and
Lys8, Glu193 and Lys9. Only the 11
and
angles not involving the first four residues of the peptide
were moved by the MC procedure.
Next, MC simulations were performed with both backbone and side chain
dihedral angles included in the move set, but the fixed CDK5 molecule
resulted in the acceptance of very few moves. Therefore, the protein
was made increasingly flexible in the neighborhood of the peptide as
follows. First, simulated annealing was employed during which the
peptide and all atoms within 5 Å of it were free to move. The system
was heated to 600 K in 20 ps and cooled to 100 K over 230 ps and energy
minimized. Next, 100 ps of molecular dynamics at 300 K was simulated
during which all residues with any atom within 5 Å of the peptide were
in motion. The 100 structures saved (1/ps) were energy minimized to a
root mean square gradient of 0.01 kcal/mol/Å. Finally, all residues in
the lowest energy structure with any atom within 10 Å of the peptide
were freed of constraints and energy minimized to a root mean square
gradient of 0.001 kcal/mol/Å. The resulting structure is
depicted in Fig. 2.

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Fig. 2.
Model of CDK5 after H1 peptide (not shown)
was docked as described under "Experimental Procedures." The
ATP (red) and magnesium ion (blue) are shown.
Side chains of acidic residues potentially involved with the peptide
binding in the antiparallel (cyan) and parallel
(yellow) configuration are represented as balls
and sticks. The PSSALRE helix
(Pro45-Leu55) and the loop
(Phe150-Asp170) analogous to the T-loop of
CDK2 are colored purple. Glycines 11, 13, and 16 of the
glycine-rich motif are colored green. This figure was
produced using the programs Molscript (30) and Raster 3D (29).
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H1 Peptide Docked in the Parallel Orientation--
Docking of
the H1 peptide in an orientation parallel to that of PKI relative to
PKA was also investigated (Fig. 1B). Analogous to the first
family of MC runs described above, 40 simulations were performed during
which inter-residue restraints were applied between Asp86
and Lys2, Asp143 and Lys5, and the
ATP and Thr3. Again, 11
and
torsions of the first
seven residues were rotated during MC moves. The lowest energy
structure was inspected visually. Aside from Asp143 and
Asp125, the only acidic residues within reach of the
terminus of the peptide were observed to be in two clusters. The first
includes Asp38, Asp39, Asp40,
Asp41, and Glu42. Based on the cyclin A-CDK2
structure, it seems likely that these residues are buried in the
CDK5·p35 complex. The other cluster includes Glu160,
Asp206, Asp208, Asp209, and
Asp234 (9).
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RESULTS |
Homology Model of CDK5--
It is well known that CDKs are not
active on their own; they require binding to regulatory cyclins to be
activated (1). However, maximal activation is achieved after
phosphorylation at Thr/Ser in the T-loop of most CDKs. In the case of
CDK2, it is Thr160 in the cyclin A-CDK2 complex. This
complex has been crystallized in both phosphorylated (9) and
unphosphorylated (8) forms. These crystal structures help elucidate the
mechanism of activation of CDKs. Briefly, the binding of cyclin A to
CDK2 causes the T-loop (146-166) to move 21 Å from its position in
the CDK2 structure, thereby opening the catalytic cleft, allowing
access to the substrate, and orienting ATP in a position that favors
phosphotransfer (8, 9). The main regulator of CDK5 is a neuron-specific
protein p35 or its truncated form p25 (2, 3), accounting for the activity of this enzyme mainly in neurons. p35 does not have any sequence similarity to cyclins. Sequence homology among cyclins is
rare; even two structurally similar cyclin motifs typically have very
different sequences (22, 23). Recent studies by two groups have
suggested that p35 consists of a cyclin-like fold (24, 25); however,
unlike the regulators of other CDKs, p35 seems to achieve maximal
activation of CDK5. Other CDKs seem to be catalytically active only
upon phosphorylation in the T-loop subsequent to cyclin binding (1, 2).
A recent crystal structure of cyclin A-CDK2 in this phosphorylated
state has revealed that the T-loop moves further away from the
catalytic cleft upon phosphorylation at Thr160 in the
T-loop (9). There seems to be no difference in substrate specificity
caused by this effect; it only increases the rate of phosphorylation.
Therefore, we assume that the active conformation of CDK5 is similar to
that of CDK2 in the cyclin A-CDK2 (phosphorylated) complex (9). Thus,
we modeled CDK5 in its active form using coordinates from the cyclin
A-CDK2 (1jst.pdb) complex using the program LOOK (as described under
"Experimental Procedures") taking advantage of the 62% sequence
identity and a high sequence homology between these two kinases (Fig.
1A). The CDK5 model exhibits a bilobal structure (Fig. 2), a
conformation representative of protein kinases. The small lobe is rich
in
-sheets and contains the glycine-rich motif important for ATP
binding. The ATP binding site is common to the entire PK family.
The N-lobe of CDK5 also contains the PSSALRE helix, which corresponds
to the PSTAIRE helix in CDK2. This helix is another feature found in
all CDKs and is important for interaction with cyclins. The PSTAIRE
helix rotates by several Å into the catalytic cleft (7- 9) upon cyclin
A binding to CDK2. Residues 150-170 in the model of CDK5 are analogous
to the T-loop in CDK2. Three conserved residues, Lys33,
Glu51, and Asp143, form a catalytic triad that
helps orient ATP and facilitate catalysis. These interactions are
similar to those found for PKA and other PKs (10). Mutation of
Lys33 to Ala in case of CDK5 abolishes the kinase activity,
implicating the importance of this residue in catalysis since the salt
bridge between Lys33 and Glu51 is broken (3).
The bigger C-lobe is predominantly helical but contains loops that are
important for interaction with suc family proteins (26) and
also the T-loop, which is important for the kinase activity as
described above. The catalytic site of the enzyme lies between the two
lobes, enabling the ATP to transfer phosphate to Ser/Thr residues of
the peptide substrate. The residues important for substrate binding
have not been identified; but based on the PKA·PKI structure, some
residues in the C-lobe have been suggested in substrate binding
(9).
Peptides Bound to the Active Site of CDK5--
Both of the
peptides studied here (H1 and NF-H, Table
I) contain at least one S/TPXK
motif, which is a consensus sequence for CDK family kinases. The H1
peptide used for docking was derived from histone H1 and contains the
TPKK motif, which is the in vitro site phosphorylated by
CDK5 in histone H1 (6). It is possible that this peptide has a higher
affinity because of the presence of 5 lysines (out of 10 residues) that
interact with acidic residues in the catalytic cleft and with the ATP.
We also used an NF-H-derived peptide for our enzyme kinetics studies
with different mutants because NF-H is phosphorylated at similar
SPXK motifs by CDKs. We showed recently that CDK5 has a
higher affinity for the first KSPXK repeat
(X = A) than for the second repeat (X = E) (5), consistent with the work of Beaudette et al. (6),
who showed that CDK5 prefers X = K or R over
X = neutral amino acid. When X =D or E, the
peptides make very poor substrates.
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Table I
Kinetic parameters of CDK5 mutants for peptide substrates
Phosphorylation assays and calculation of kinetic constants were
performed as described under "Experimental Procedures." H1, histone
peptide (PKTPKKAKKL); NF, neurofilament peptide (VKSPAKEKAKSPEK).
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It is likely that the substrate in CDK5 or other CDKs binds to a site
analogous to that observed in the PKA·PKI complex (12) because CDKs
and PKA have similar overall structures and almost superimposable
catalytic clefts that include the glycine-rich ATP binding domains and
the catalytic base, Lys33. When the peptide substrate
sequences are aligned with the PKI sequence in a parallel
(conventional) fashion with the Ser/Thr matching the corresponding
residue in PKI, none of the lysines of the CDK5 substrate peptides
corresponds to any arginine of PKI (Fig. 1B). However, if
the substrate sequences are aligned with PKI in an antiparallel
fashion, the n+3 lysine of either NF or H1 peptide aligns
with the n
3 arginine in the RRXS motif of PKI.
For the H1 peptide, two other lysines align with PKI arginines in the
antiparallel alignment (Fig. 1B).
Effect of Site-directed Mutagenesis of Residues Implicated in
Substrate Recognition--
Charged to alanine scanning mutagenesis of
CDK5, combined with kinetic analyses of the mutant enzymes, was used to
investigate interactions involving acidic residues near the ATP binding
pocket in our homology model of CDK5. As described above,
Asp86 and Asp91 were implicated in binding to
the peptide substrates. Independent mutation of these residues to
alanine caused a 2-fold decrease in apparent affinity for the NF
peptide (Table I). However, there was no significant difference
measured in the Km for ATP binding to the D86A
(Km = 140 µM) and D91A
(Km = 210 µM) mutants compared with
CDK5 (Km = 160 µM).
For the higher affinity H1 substrate, the decrease in apparent affinity
was 5-fold for both CDK5 mutants (Table I). Assuming that the Asp to
Ala mutations do not greatly affect the structure of CDK5, it would
appear that both Asp86 and Asp91 form
stabilizing interactions with both peptides, arguably salt bridges to
one or more lysines. To identify the alleged salt bridge interactions
between CDK5 and the H1 substrate, the D86A and D91A mutants were
subjected to kinetic analyses with Ala-substituted H1 peptides in which
either the n+2 or n+3 residue (Lys5
or Lys6) was replaced by alanine (Table
II). We report
Vmax/Km in Table II to
represent the catalytic efficiency of the enzyme. Because, in general
Vmax/Km decreases as
Km increases, we interpret our results in terms of
Km, which reflects apparent binding affinity.
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Table II
Kinetic studies of CDK5 mutants with Ala-substituted H1 peptide
Phosphorylation assays and calculation of kinetic constants were
performed as described under "Experimental Procedures." Alanine
substitutions in H1 peptide are shown in bold. n represents
threonine, the phosphoacceptor residue.
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In H1 peptide binding to wild-type CDK5, the n+2 alanine
substitution increased Km by a factor of 5, whereas
the n+3 alanine substitution increased Km
by a factor of 7 (Table II). These results are in agreement with
reports that the presence of non-basic residues in the n+2
or n+3 position makes for a very poor substrate for CDKs (6,
14).
The Km values for H1 peptide binding to CDK5 and to
the D86A mutant differ by a factor of 5. For the n+2 alanine peptide, these Km values differ by a factor of only
1.5. Thus, the n+2 alanine peptide is considerably less
sensitive to changes in Asp86 than is the H1 peptide,
suggesting that Lys5 of H1 probably interacts with
Asp86 of CDK5. The weakening of the
Lys5-Asp86 interaction was essentially
independent of whether it was Lys5 or Asp86
(factor of 5) that was replaced by Ala (Table II).
For the n+3 alanine peptide, the Km
values for binding to CDK5 and to D86A differ by a factor of 2.5 compared with the factor of 5 for the H1 peptide. The effects observed
for Lys6 (n+3) are less dramatic than those
discussed above for Lys5 (n+2). Still, the data
suggest that Lys6 also interacts with Asp86.
The proximity of Lys6 to Lys5 certainly makes
this conclusion plausible.
When Asp91 was mutated to Ala, the reduction in apparent H1
affinity was comparable to that resulting from the D86A mutation, implicating Asp91 as another residue that interacts
favorably with H1. D91A binds both the n+2 alanine and
n+3 alanine peptides very poorly compared with wild type,
suggesting that a second interaction has been weakened, i.e.
that Asp91 interacts with an H1 residue other than
Lys5 or Lys6. The data in Table II suggest that
disruption of any one of the interactions between Asp86 or
Asp91 and the peptide substrate results in a reduced
apparent affinity but not a complete loss of binding. Multiple
interactions stabilize the binding of the substrates to CDK5.
The simultaneous disruption of Asp86 and Asp91
interactions with the peptide was studied using a CDK5 double mutant
(D86A/D91A). Relative to wild-type CDK5, this mutant showed an ~18
fold decrease in apparent affinity (Km = 0.7 mM) for the H1 peptide, and relative to either D86A or
D91A, it showed a 3.5-fold decrease. These results are further evidence
that these residues are important for peptide binding.
The modeled complex (Fig. 3) highlights
the interactions implicated by the measured enzyme kinetics while
suggesting additional interactions that may stabilize the complex. Our
model suggests that Asp91 may interact with
Lys8 and/or Lys9 of the substrate, which could
account for the large Km values for the binding of
the n+2 alanine and n+3 alanine peptides to D91A.
To identify any interactions between Asp91 and
Lys8 or Lys9, we measured enzyme kinetics with
two additional Ala-substituted H1 peptides, n+5 alanine and
n+6 alanine. The results indicate no increase in
Km for the binding of these peptides to D91A (Table
II). In comparison, the binding to wild-type CDK5 and D86A showed a
3-5-fold and 2-fold increase in Km, respectively.
Taken together, the kinetic data for the n+5 alanine and
n+6 alanine peptides support the possibility of interactions between Lys8 and Lys9 of the H1 peptide and
Asp91 of CDK5, as was suggested originally by the molecular
modeling.

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Fig. 3.
Panel A, modeled binding site of CDK5
with H1 peptide docked. The CDK5 surface is colored according to the
electrostatic potential calculated with the program GRASP (31). Regions
of negative potential are red; positive regions are
blue. Selected residues of CDK5 visible at the surface are
labeled in white; selected peptide residues are labeled in
green. Panel B, stereo view of the H1 peptide
residues from Lys5 to Lys9, as docked to CDK5.
Asp86 (top) and Asp91
(bottom) are also shown. Each of the four N -O
distances shown (Lys5- Asp86,
Lys6-Asp86,
Lys8-Asp91,
Lys9-Asp91) is between 2.5 and 2.6 Å.
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In the model of the H1 peptide bound to CDK5 (Fig. 3), each of the
peptide's five lysines interacts with one or more negatively charged
groups. Additional salt bridges identified in the model involve the
ATP, Asp143, and Lys88 (Table
III).
Effect of Mutation of Residues Implicated in Substrate Binding in
the Parallel Orientation--
The crystal structure of the PKA·PKI
complex can serve as a template for substrate binding. We modeled the
H1 substrate peptide in an orientation comparable to that of PKI bound
to PKA (Fig. 1B) with a distance restraint that kept the
phosphoacceptor threonine within hydrogen bonding distance of the ATP.
It appeared from the modeling (not shown) that the only likely salt
bridge partners for Lys5 and Lys6 were
Asp143 and Asp125. Other acidic residues in the
vicinity of the substrate included Asp206,
Asp208, Asp209, Asp234, and
Glu160 (Fig. 2). We mutated each of these six residues
(except Asp208) to Ala. The D143A and E160A mutants were
not active. None of the other three Asp
Ala mutations resulted in a
significantly altered Km value for H1 peptide (Table
I). These data strongly support the conclusions based on the D86A and
D91A data, namely that the peptide substrate binds in the antiparallel orientation.
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DISCUSSION |
There is no available crystal structure of a CDK-substrate
complex. Thus, the mechanism of this interaction is unknown. Because of
the very high specificity of CDKs for the S/TPXK motifs, it is important to probe the mode of binding of these substrates. Although
unambiguous interpretation of mutagenesis experiments is difficult, we
assume that non-local conformational changes induced by mutations and
many-body interactions can be neglected to a first approximation. We
therefore interpret our mutagenesis data in terms of pairwise
interactions, specifically salt bridges between CDK5 and the peptide substrates.
Based on the crystal structure of the PKA·PKI complex, it has been
speculated that some of the acidic residues (e.g.
Glu160, Glu206, Glu209) in the
C-lobe of CDKs might interact with the essential lysines of the peptide
substrate (9). The homology model and mutagenesis of CDK5 have
implicated several acidic residues in the binding of peptide
substrates: Asp86, Asp91, Asp125,
Asp143, and Glu193 (Fig. 2A). The
consensus sequence for PKA is RRXS/A. Therefore, Lys5 and Lys6 of the CDK5 substrates align well
with the n
2 and n
3 arginines in an
antiparallel alignment (Fig. 1B). Furthermore,
Lys9 of the H1 peptide corresponds to the n
6 arginine.
The parallel alignment of H1 peptide to PKI is not as compelling
because no lysines line up with the arginines of PKI (Fig. 1B). Preliminary docking of the peptide in this parallel
orientation suggested the possibility of salt bridges involving
residues Asp143, Asp206, Asp208,
Asp209, Asp234, and Glu160. These
acidic residues have been considered important in substrate binding by
other reports (9). We mutated each of them (except Asp208)
to Ala. There was no significant change in Km values for peptide interaction with these mutants except for D143A and E160A.
For these mutants, there was no kinase activity, and determining the
apparent peptide affinity was not possible. Along with
Lys33, Asp143 orients the ATP in a conformation
that facilitates phosphotransfer (7-9). It is thus not surprising that
the D143A mutant is inactive. Glu160 is part of the
flexible T-loop, and it may be that the conformation of CDK5 is
especially sensitive to mutations in this region (Fig. 2).
The pairwise combinations of the D86A and D91A mutants (each defective
in substrate binding) with Ala-substituted peptides suggest that
n+2 and n+3 lysines interact with
Asp86 in the KTPKK peptide substrate. Presumably, when
binding to the D86A mutant, Lys5 and Lys6
interact with the ATP or Asp91. The first family of MC
simulations suggested that Asp91 is within reach of
Lys8 and Lys9 when the N2-terminal
half of the peptide is docked to the ATP. Although these lysines are
not part of the TPKK consensus CDK motif, the D91A mutant data suggest
that a stabilizing interaction outside the TPKK region is present in
the native system. Interestingly, in the case of other major
proline-directed kinases, extracellular signal-regulated kinases
(ERKs), the residue corresponding to Asp91 is a neutral
residue (Fig. 1A). These kinases do not have any known
preferences for charged residues following the proline (15). Recently,
it was shown that the NF peptide and other related peptides containing
n+3 as basic residues were very poorly recognized by ERKs
(27) compared with the peptides with n+3 as nonbasic
residues. A proline at the n+1 position is a common
essential feature in substrates for both CDKs and ERKs. These kinases
do not phosphorylate substrates without proline. It is possible that
orientation of the serine hydroxyls for hydrogen bonding with ATP
requires proline at the n+1 position. CDKs seem to require
additional interactions with the substrate lysines at the
n+2 and n+3 positions to stabilize the
substrate-enzyme complex. Replacement of Lys5 or
Lys6 with alanine decreases the apparent affinity for the
peptide substrate (Table II). However, when both of these lysines are replaced by a neutral or acidic residue, the peptide is not
phosphorylated by CDK5 (27). Our data and model also suggest additional
stabilization of the complex by salt bridges involving Lys8
and Lys9 of the substrate and Asp91 of CDK5.
PKA and phosphorylase kinase have been the only two serine/threonine
kinases to be crystallized as ternary complexes with ATP and their
peptide substrates (12, 13). These two kinases show a high degree of
structural similarity in their ternary complexes. Because the cyclin
A-CDK2 (phosphorylated) crystal structure differs from PKA and
phosphorylase kinase in several ways, it has been speculated that CDKs
might have a different catalytic mechanism (10). A recently reported
crystal structure of CDK2 complexed with staurosporine, an inhibitor of
CDKs and other protein kinases, which binds at the ATP binding pocket,
revealed that staurosporine interacts with Asp86 (28). This
interaction suggested that the peptide substrate binding site of CDKs
is very close to the ATP pocket, consistent with our observation that
mutation of Asp86 results in a decreased apparent affinity
for the peptide. Also, recent work in our laboratory has shown that a
pseudo substrate analogue of the NF peptide was surprisingly
competitive with ATP.2 Given
the close proximity of the ATP and peptide binding sites, peptide
binding at or near Asp86 could affect ATP binding.
Interestingly, in the same study we showed that peptide binding
promoted ATP binding.2 Taken together, these data suggest
that a conformational change resulting from peptide binding could
trigger events important for catalysis.
Because of the lack of a crystal structure of a CDK-substrate complex,
the peptide substrate binding site has not been identified. Therefore,
design of specific inhibitors for CDK family kinases has not been
possible. Most of the available inhibitors bind in the ATP pocket,
which makes them less specific since the ATP pockets of most PKs are
structurally very similar. But in the case of CDKs, the interactions
with the substrate are very specific; CDKs specifically target
SPXK-type motifs. The enzyme kinetics and molecular modeling
reported here indicate that the H1 peptide most likely binds to CDK5 in
an orientation that is antiparallel to that of PKI bound to PKA.
The binding site implicated by this study should help guide the
design of specific inhibitors for CDKs.
 |
ACKNOWLEDGEMENTS |
We thank Jim Neagle for assistance with DNA
sequencing, Robert A. Pearlstein and R. W. Albers for thoughtful
discussions, and Bernard R. Brooks for computational resources.
 |
FOOTNOTES |
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Laboratory of
Neurochemistry, NINDS, National Institutes of Health, Bldg. 36, Rm.
4D20, Bethesda, MD 20892. Tel.: 301-402-2124; Fax: 301-496-1339; E-mail: hcp{at}codon.nih.gov.
2
P. Sharma, N. D. Amir, R. W. Albers, and H. C. Pant, manuscript in preparation.
 |
ABBREVIATIONS |
The abbreviations used are:
CDK(s), cyclin-dependent kinase(s);
NF, neurofilament(s);
NF-H, high molecular weight NF protein;
PK, protein kinase;
PKA, cAMP-dependent protein kinase;
PKI, cAMP-dependent protein kinase inhibitor;
GST, glutathione
S-transferase;
MC, Monte Carlo;
ERK, extracellular
signal-regulated kinase.
 |
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