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INTRODUCTION |
Cyclin-dependent kinases
(Cdks)1 are key regulatory
enzymes in the eukaryotic cell cycle. The activation of a Cdk depends
on its association with its specific cyclin partners. The activity of
these enzymes is further regulated by an intricate system of protein-protein interactions and phosphorylation (1). Members of the
Cdk family are closely related by sharing a high level of amino acid
sequence identity (40-70%). In contrast, cyclins are a family of
molecules of diverse molecular mass and low sequence identity. Sequence
alignments have shown that cyclins share a somewhat conserved region of
approximately 100 amino acids in the center of the molecule, and this
region is called the cyclin box (2). Recent crystal structures of
cyclin A and cyclin H have shown that the cyclin box sequence forms a
compact 5-helix domain called the cyclin fold (3-5), and that a region
of cyclin A, C-terminal to the cyclin box also forms a cyclin fold.
However, there is virtually no sequence similarity between the two
cyclin fold domains. Theoretical predictions have suggested that other members of the cyclin family also contain two cyclin folds (3, 6).
Unlike other Cdks, Cdk5 activity has been observed only in neuronal and
developing muscle cells although the catalytic subunit of the enzyme is
present in many mammalian tissues and cell extracts (7-11). Recent
experimental evidence has demonstrated that Cdk5 plays important roles
in neurite outgrowth (12), patterning of the cortex and cerebellum
(13), and cytoskeletal dynamics (9, 14, 15). Loss of regulation of Cdk5
has been suggested to be involved in Alzheimer's disease (16). Active
Cdk5 was first purified from brain extracts as a heterodimer with
subunit molecular masses of 33- and 25-kDa, respectively (8, 10). The
33-kDa subunit was later identified as Cdk5, and the 25-kDa activator
(neuronal Cdk5 activator, Nck5a) was a novel protein with no sequence
similarity to any other known proteins. The 25-kDa subunit was later
found to be a proteolytic product of a larger 35-kDa protein (17, 18).
An isoform of Nck5a (Nck5ai) with 57% sequence identity to Nck5a has
also been identified (19). Despite their functional similarity in terms
of binding and activation of a Cdk, the Cdk5 activators share little
sequence similarity to cyclins. Moreover, while the activation of the
well characterized Cdks such as Cdk1 and Cdk2 by cyclins depends on the
phosphorylation of the Cdk at a specific threonine residue, Cdk5
activation by its activator is phosphorylation-independent (20, 21).
Recently, the activation domain of Nck5a was precisely mapped to amino
acid residues from Glu150 to Asn291 (21, 22).
Extensive truncation and site-directed mutation studies of Nck5a,
together with computer modeling, strongly suggested that the
142-residue activation domain of Nck5a adopts a cyclin fold structure
(3, 22).
In this work, we describe the discovery of a 29-residue Cdk inhibitory
peptide which is derived from an internal fragment of Nck5a. This
peptide is able to bind to and hence to inhibit the kinase activities
of Cdk5·Nck5a and Cdk2·cyclin A complexes in a non-competitive
manner. The solution structure of the peptide determined by
two-dimensional NMR spectroscopy showed that a large part of the
peptide adopts an amphipathic
-helical structure, and this helix is
likely to be the main binding surface of the peptide to the enzyme complexes.
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MATERIALS AND METHODS |
Peptide Synthesis--
A 28-residue peptide
(ASTSELLRCLGEFLCRRCYRLKHLSPTD) corresponding to peptide fragment
Ala146 to Asp173 of Nck5a was synthesized on an
Applied Biosystems model A431 automated peptide synthesizer using
Fmoc-based chemistry (Fig. 1). A
31-residue peptide (DYHEDIHTYLREMEVKCKPKVGYMKKQPDIT) corresponding to
the sequence Asp177 to Thr207 of human cyclin A
was synthesized in the same manner (Fig. 1). After cleavage from the
solid support, the peptides were purified by gel filtration and reverse
phase high performance liquid chromatography to a purity greater than
95%. All of the peptide purifications were carried out in 0.1%
trifluoroacetic acid/H2O (v/v) as low pH can prevent the
oxidation of the sulfhydryl group of the Cys residues in the peptides.
Trifluoroacetic acid was removed from the peptide by dissolving the
lyophilized peptide powder in pre-chilled Milli-Q water before
re-lyophilization. The authenticity of the peptides was checked using
mass spectrometry as described earlier (23).

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Fig. 1.
Amino acid sequences of synthetic peptides
derived from the N-terminal end of Nck5a (the
N peptide) and the cyclin A peptide
used in this work. The alignment of the two peptide sequences was
derived by aligning cyclin A and the minimal activation domain of Nck5a
(for details, see Ref. 22). The black bar above
the cyclin A peptide represents the N-terminal -helix of the protein
found in the x-ray structure (3). The dotted region
below the N peptide depicts the predicted
-helix of the peptide.
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Protein Expression and Purification--
GST-Cdk5 and GST-Nck5a
were prepared as described previously (20, 22). The N-terminal
histidine-tagged Cdk5 (H6-Cdk5) was expressed in and purified from
Escherichia coli cells (strain M15 from Qiagen). The host
cells, harboring a H6-Cdk5-containing expression plasmid, were cultured
in 6 liters of tryptone/phosphate-rich medium containing 50 µg/ml of
ampicillin and 25 µg/ml kanamycin to A600
1.0 before induction of protein expression with
isopropyl-1-thio-
-D-galactopyranoside (0.4 mM). The cell culture was subsequently incubated for
10 h at 22 °C. The pelleted cells were washed with 20 mM Tris-HCl, pH 7.5, containing 1 mM EDTA.
Cells were then lysed in the French press in 50 mM Tris-HCl
buffer, pH 7.5, and the lysate was subjected to centrifugation at
38,700 × g for 30 min. The pH of the resulting supernatant was adjusted to 7.9, and this was then incubated with 3 ml
of Ni2+-nitrilotriacetic acid-agarose
(Ni2+-NTA) beads for 1 h with stirring. The resin was
packed onto a column and then washed with 50 ml of binding buffer (20 mM Tris-HCl, pH 7.9, 0.5 M NaCl, 5 mM imidazole) and 30 ml of washing buffer (20 mM Tris-HCl, pH 7.9, 0.5 M NaCl, 60 mM imidazole). H6-Cdk5 was then eluted with about 18 ml of
elution buffer (20 mM Tris-HCl, pH 7.9, 1 M
NaCl, and 1 M imidazole).
The C-terminal histidine-tagged human cyclin A (cyclin A-H6) was
expressed and purified in essentially the same manner as described for
H6-Cdk5. Briefly, the expression plasmid pET21d containing a cyclin A
gene lacking the N-terminal 173 amino acids was transformed into
BL21(DE3) E. coli cells. The host cells were cultured in LB
medium containing 100 µg/ml ampicillin, and cyclin A expression was
induced by adding isopropyl-1-thio-
-D-galactopyranoside to a final concentration of 0.1 mM. The induction of cyclin
A expression was for 3 h at 30 °C. The pelleted cells were
resuspended in 50 mM Tris-HCl, pH 7.5, 300 mM
NaCl, and 0.05% Triton X-100, and then lysed in the French press. The
subsequent purification of cyclin A using a Ni2+-NTA column
was carried out in a manner identical to that described for the
purification of H6-Cdk5. The monomeric, active form of cyclin A-H6 was
further purified by passing the cyclin A-H6-containing eluent through a
Sephacryl S-200 gel filtration column (Amersham Pharmacia Biotech).
Peptide/Cdk Binding Assay--
H6-Cdk5 (about 3 µg) and 15 µg of GST-p25 were premixed in 300 µl of 1 × phosphate-buffered saline with 0.5 mg/ml bovine serum albumin. Some
50-µl quantities of the mixture was taken out to mix with various
concentrations of the
N peptide (from serial dilutions
made from a 2.0 mM stock solution), and the total volume of
the mixture was adjusted to 150 µl using Buffer R (1 × phosphate-buffered saline containing 1 mM EDTA, 1 mM dithiothreitol, 0.6 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml antipain, 5 mg/ml bovine serum
albumin). The reaction mixture was incubated at 4 °C for 15 h.
GST-Nck5a·H6-Cdk5 complex was then precipitated by the addition of 40 µl of GSH-Sepharose beads pre-equilibrated with 1 × phosphate-buffered saline (50%, v/v). The GSH-Sepharose beads were
washed three times with 1 × phosphate-buffered saline buffer, and
subsequently resuspended in 20 µl of water and 20 µl of 2 × protein sample treatment buffer. The co-precipitated H6-Cdk5 was
detected by SDS-PAGE followed by Western blot using a monoclonal
antibody against Cdk5 (22).
The binding of the
N peptide to the GST-Cdk2·cyclin
A-H6 complex was studied in a similar manner to that described above for H6-Cdk5. Briefly, 3 µg of GST-Cdk2 and 10 µg of cyclin A-H6 were reconstituted in Buffer R with various concentrations of the
N peptide at 4 °C for 15 h. GST-Cdk2 was then
precipitated by the addition of 20 µl of GSH-Sepharose beads. After
washing, the co-precipitated cyclin A-H6 was detected by SDS-PAGE
followed by Western blot using a monoclonal antibody against cyclin A
(Santa Cruz).
Peptide Inhibition of Cdk5 and Cdk2 Kinase Activity--
Cdk5
kinase activity was assayed essentially as described previously (20,
22), except that for each Cdk5 kinase reaction 1 µg of GST-Cdk5 was
reconstituted with 2 µg of GST-Nck5a, and 1 µg of GST-Cdk2 was
mixed with 3 µg of cyclin A-H6 in Cdk2 assays. The reconstituted
complexes were added, in duplicate, to an assay mixture containing 30 mM MOPS, pH 7.4, 10 mM MgCl2, 40 µM of the histone H1 peptide, 50 µM
[
-32P]ATP, and various concentrations of various
peptides at 30 °C for 30 min before measuring the Cdk5 and Cdk2
kinase activities.
Steady State Kinetic Experiments--
All assay conditions were
the same as in the inhibition assay described above, except that the
reaction time was kept at 15 min so that the product formed was less
than 5% of the total substrate concentration used. To determine
suitable concentrations of the substrate and the inhibitor for the
steady state kinetic experiments, the Km value of
GST-Cdk5·GST-Nck5a complex and a concentration-dependent inhibition profile of the enzyme by the
N peptide were
determined. Four different concentrations of the histone H1 peptide
(7.5, 10, 15, and 30 µM) were used in the kinetic
analysis of the enzyme inhibition. For each substrate concentration,
four concentrations of the
N peptide (0, 10, 20, and 30 µM) were used in the inhibition assay.
CD Experiments--
Concentrations of the
N
peptide stock solutions were determined by the UV absorption of the
single Tyr residue at 280 nm. For CD measurement, the
N
peptide was dissolved in a 20 mM sodium acetate buffer, pH
4.0, containing various concentrations of 2,2,2-trifluoroethanol (TFE).
The concentration of the peptide was fixed at 50 µM
throughout the experiment. CD spectra were collected at 35 °C on a
JASCO J-720 CD spectropolarimeter equipped with a Neslab temperature controller using a cell path length of 1 mm.
NMR Experiments--
For NMR studies, the
N
peptide was dissolved in unbuffered 90% H2O, 10%
D2O, or 99.99% D2O containing various
concentrations of deuterated TFE-d3 (0~30%,
v/v), and 2 mM deuterated
dithiothreitol-d10 at pH (or pD) 4.0. The
concentration of the
N peptide was approximately 2.0 mM. All 1H NMR data were recorded on a Varian
INOVA 500 spectrometer at a 1H frequency of 500.11 MHz.
Two-dimensional TOCSY and NOESY spectra were acquired with a spectral
width of 6000 Hz in both dimensions (24). The "WET" pulse sequence
was employed for solvent suppression (25) of the peptide samples in
H2O. FID data matrices were composed of 512 × 2048 (t1 × t2) data points.
The mixing times used in NOESY experiments were 150 and 300 ms. TOCSY
spectra were recorded with a mixing time of 75 ms using the MLEV17 spin
lock sequence (26). All NMR data were processed and displayed using
the nmrPipe software package (27).
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RESULTS |
A Peptide Derived from the N-terminal Region of the Activation
Domain of Nck5a Inhibits the Kinase Activity of Cdk5 and Cdk2--
In
the course of studying the structure and function relationship of the
activation domain of Nck5a, we created a series of truncated forms of
GST-Nck5a mutants. One such GST fusion mutant, which contains a
29-residue peptide fragment corresponding to residues
Gln145 to Asp173 of Nck5a (termed
N as it represents the N-terminal
-helix of Nck5a,
see "Discussion") was found to inhibit Cdk5 kinase activity in a
dose-dependent manner (Fig.
2A). About 50% kinase
activity was inhibited at a GST-
N concentration of 5 µM. The inhibition of Cdk5 activity originated solely
from the peptide fragment as GST did not have any effect on the kinase
activity of the enzyme (Fig. 2B). Release of the peptide
from the fusion protein by thrombin had no effect on the inhibitory
activity (Fig. 2B). Furthermore, the addition of
GST-
N before or after the reconstitution of GST-Cdk5 with GST-Nck5a gave rise to the same inhibition profiles (Fig. 2C), suggesting that the peptide does not compete with Nck5a
for Cdk5 (see below for more details). We also tested the inhibitory effect of GST-
N toward the GST-Cdk2·cyclin A-H6
complex, and found that GST-
N also inhibited the kinase
activity of Cdk2. As expected, the inhibition of the Cdk2·cyclin A
complex originated from the
N peptide portion of the
fusion protein, as seen in the case of Cdk5 (data not shown, see also
below for results obtained with the synthetic peptide).

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Fig. 2.
Discovery and inhibitory properties of the
GST- N fusion peptide.
A, dose-dependent inhibition of
GST-Cdk5·GST-Nck5a by the bacterially expressed GST- N.
The inset shows SDS-PAGE analysis of the purified
GST- N, its thrombin digestion product, and GST as a
control. The minor bands seen in the GST- N lane are due
to proteolytic degradation of GST- N, as cleavage of the
fusion peptide by thrombin resulted only one band corresponding to GST.
B, reconstituted GST-Cdk5·GST-Nck5a kinase activity
(B1), and its inhibition by GST- N
(B2), by the N peptide released from thrombin
cleavage (B3), and not by GST (negative control,
B4). C, inhibition of GST-Cdk5 activity by
GST- N both before and after GST-Cdk5 reconstitution with
Nck5a. The addition of the GST- N to GST-Cdk5 before
reconstitution with GST-Nck5a (GST-Cdk5 + GST-Nck5a, C2) or
after reconstitution with GST-Nck5a (GST-Cdk5/GST-Nck5a, C3)
results in the same inhibition of kinase activity. C1 serves
as a positive control (no GST- N addition).
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Low expression level (about 1 mg/ml of soluble GST-
N)
and poor homogeneity of the GST fusion product prevented us from a detailed characterization of this inhibitory peptide. To overcome these
problems, we decided to use a synthetic peptide instead of the GST
fusion protein. A 28-residue peptide corresponding to amino acid
residues Ala146 to Asp173 of Nck5a (abbreviated
as the
N peptide) was synthesized and purified to
homogeneity. The N-terminal Gln residue (Gln145) was
deleted from the synthetic peptide sequence to avoid complication from
its cyclization to form gyroglutamate. The titration curves shown in
Figs. 2 and 3A revealed that
both the recombinant and synthetic inhibitory peptides inhibit
GST-Cdk5·GST-Nck5a complex with similar potencies.

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Fig. 3.
Dose-dependent inhibition of
(A) GST-Cdk5·GST-Nck5a and (B)
GST-Cdk2/cyclin A-H6 activities by various concentrations of the
N peptide. The negative control
peptide was derived from residues 6 to 20 of Cdc2 with an alanine to
serine substitution at position 14. For comparison, the inhibition
profiles of the enzymes by the cyclin A peptide, and the MLCK peptide,
are also included. , N peptide; , MLCK peptide;
, cyclin A peptide; , control.
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In order to assess whether the
N peptide was a specific
inhibitor of Cdk5 and Cdk2, we synthesized a 31-residue peptide
corresponding to Asp177 to Thr207 of cyclin A
(the cyclin A peptide, Fig. 1), and tested for its ability to inhibit
GST-Cdk5·GST-Nck5a. This region of cyclin A was previously shown to
align with the
N peptide based on the sequences, the
secondary structures, and the functions of the two proteins (22).
Despite the fact that the cyclin A peptide could adopt an
-helical
conformation similar to that seen in its crystal structure (28), the
cyclin A peptide inhibited neither GST-Cdk5·GST-Nck5a nor
GST-Cdk2·cyclin A-H6 (Fig. 3). Therefore, it is likely that the
unique amino acid sequence of the
N peptide entails its
inhibition of Cdk5 and Cdk2.
In addition, the
N peptide was shown to adopt an
amphipathic
-helical structure in solution (see below), we tested
the Cdk inhibitory effect of another amphipathic peptide, a 26-residue peptide fragment comprising the calmodulin-binding domain of myosin light kinase (the MLCK peptide) (29, 30). Although MLCK peptide was
indeed able to inhibit the activities of both GST-Cdk5·GST-Nck5a and
GST-Cdk2·cyclin A-H6, its inhibitory efficiency is significantly lower than that of the
N peptide (Fig. 3). Therefore, it
is suggested that the potent and efficient inhibitory activity of the
N peptide is due to the unique amino acid sequence.
The Binding of the
N Peptide to Cdk5·Nck5a and
Cdk2·Cyclin A Complexes Does Not Lead to a Dissociation of Nck5a and
Cyclin A--
The inhibition of Cdk5 and Cdk2 by the
N
peptide shown in Fig. 3 may result from a direct competition of the
peptide with GST-Nck5a and cyclin A-H6 for GST-Cdk5 and GST-Cdk2,
respectively, or from the binding of the peptide to the binary
complexes of GST-Cdk5·GST-Nck5a and GST-Cdk2·cyclin A-H6. To
discriminate between these possibilities, we performed direct binding
competition experiments. Various concentrations of the peptides were
added to a H6-Cdk5·GST-Nck5a mixture, and GST-Nck5a was then
precipitated using GSH-agarose beads. The amount of H6-Cdk5 in complex
with GST-Nck5a was determined by Western blotting of the enzyme
co-precipitated by GSH-agarose beads. The amount of H6-Cdk5 and cyclin
A-H6 used in the experiment described in Fig.
4, A and B, were
chosen to ensure that their respective antibodies would be in excess.
The addition of various amounts of the
N peptide
(corresponding to approximately 10, 50, and 90% inhibition of the Cdk5
activity) did not lead to a dissociation of GST-Nck5a from H6-Cdk5
(Fig. 4A), indicating that the
N peptide was
able to bind to and hence inhibit the binary complex of
H6-Cdk5·GST-Nck5a. Similarly, the inhibition of GST-Cdk2·cyclin A-H6 activity by the
N peptide also resulted from the
formation of a ternary complex between the
N peptide and
GST-Cdk2·cyclin A-H6 rather than from a direct competition between
the
N peptide and cyclin A for Cdk2 (Fig.
4B). The activity of the Cdk5·Nck5a complex could be
inhibited immediately upon the addition of the
N peptide
(Fig. 2C), further indicating that the
N
peptide can bind to and inhibit Cdk5 without the dissociation of
its activator.

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Fig. 4.
The N
peptide acts as a noncompetitive inhibitor with respect to the Cdk
activators. A, Increasing amounts of the
N peptide (lanes 1-4: 0, 5, 20, 50 µM, which correspond to 0, 10, 50, and 90% inhibition of
the Cdk5 activity, respectively) were incubated with
H6-Cdk5·GST-Nck5a complex. The H6-Cdk5·GST-Nck5a complex was
precipitated by GSH-agarose beads, and the amount of complex remaining
was assayed by immunoblotting of H6-Cdk5. Lane 5, negative
control. GST was substituted for GST-Nck5a. B, increasing
amounts of the N peptide (lanes 1-4, 0, 0.5, 2, 10 µM, which correspond to 0, 10, 50, and 90%
inhibition of Cdk2 activity, respectively) were incubated with the
GST-Cdk2·cyclin A-H6 complex. The GST-Cdk2·cyclin A-H6 complex was
precipitated with GSH-agarose beads. The remaining amount of complex
was measured by Western blot of cyclin A-H6. Again, GST instead of
GST-Cdk2 was used in the negative control for binding to cyclin A-H6
(lane 5).
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To further prove that the
N peptide does not compete
with Nck5a for Cdk5, we performed a direct competition experiment.
First, GST-Cdk5 was reconstituted with various concentrations of its activator. Then, we assayed the inhibition of the reconstituted GST-Cdk5·GST-Nck5a complexes by the
N peptide (50 µM, a concentration which leads to about 90% inhibition
of the enzyme, see Fig. 3). If the inhibitory peptide were to compete
with Nck5a for Cdk5, the large excess of Nck5a would mask the
inhibition of the kinase by the peptide at low concentrations. However,
data in Fig. 5 show that the presence of
a large excess of GST-Nck5a has no significant effect on the enzyme
inhibition profile by the
N peptide, further supporting
the contention that the inhibitory peptide does not compete with Nck5a
for Cdk5.

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Fig. 5.
High concentrations of Nck5a cannot mask Cdk5
inhibition by the N peptide.
Various quantities of GST-Nck5a (to attain final concentrations of
approximately 1, 10, and 20 µM) were reconstituted with 1 µg of GST-Cdk5. The kinase activities were assayed in the absence
( ) or presence ( ) of the N peptide (50 µM). The concentration of the N peptide
used gave rise to approximately 90% inhibition of the enzyme
activity.
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Kinetic Analysis of Cdk5 Inhibition by the
N
Peptide--
We also analyzed the kinetic properties of the
N peptide with respect to the kinase substrate, the
histone H1 peptide. The double-reciprocal plot shown in Fig.
6A demonstrates that the peptide acts as a noncompetitive inhibitor of the enzyme complex with
respect to its substrate. Dixon plot analysis of the data reveals that
the Ki for the
N peptide inhibition
of the GST-Cdk5·GST-Nck5a complex is approximately 25 µM (Fig. 6B). Linearity of the Dixon plots
indicates that the
N peptide is a dead end inhibitor
(i.e. the kinase-inhibitor complex is catalytically inactive) (31). This notion is further supported by the near complete
inhibition of the kinase by an excess amount (i.e. 400 µM) of the
N peptide (data not shown).

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Fig. 6.
Steady state kinetic analysis of the
inhibition of Cdk5·Nck5a complex by the
N peptide. A,
double-reciprocal plot of the inhibition of the Cdk5·Nck5a complex by
the N peptide. Four different concentrations of the
N peptide were used in the assay (0 µM,
; 10 µM, ; 20 µM, ; 30 µM, ). The data were fitted by the standard linear
least square fitting. B, Dixon plot of the inhibition of
Cdk5·Nck5a complex by the N peptide. The
concentrations of the histone H1 peptide used were: 7.5 µM ( ), 10 µM ( ), 15 µM
( ), 30 µM ( ).
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Structure of the
N Peptide Determined by CD and
NMR--
In order to understand the structural basis of Cdk5 and Cdk2
inhibition by the
N peptide, we determined the structure
of the peptide by NMR spectroscopy. The 1H NMR spectra of
the
N peptide in aqueous solution showed reasonable chemical shift dispersion (data not shown). However, careful inspection of the spectra revealed that the majority of resonance in both the
amide and aliphatic regions had exceptionally broad line widths for a
peptide of only 28 amino acid residues. These results indicated that
the peptide might be in equilibrium between multiple conformers or in
an aggregated state. A number of amide protons throughout the peptide
displayed more than one cross-peak to their
protons in a TOCSY
spectrum of the peptide in 90% H2O, 10% D2O,
pH 4.0, at 30 °C (data not shown). When the pH of the sample was
raised to 4.5 or above, the line widths of the NMR signals broadened further, and the TOCSY spectra were more complicated. Changing the
sample temperature (from 8 to 35 °C) or concentration (from 0.5 to
3.5 mM) did not improve the quality of the NMR spectra. CD
studies also showed that the molar ellipticity of the peptide at 222 nm
remained constant when the concentration of the peptide was varied from
8 µm to 0.2 mM (data not shown). These results indicated
that the
N peptide in aqueous solution has multiple conformational states that are exchanging at slow to intermediate rates. Such multi-conformational equilibrium prevented us from a
detailed structural characterization of the peptide in aqueous solution, although we were still able to obtain nearly complete backbone assignment of the peptide at pH 4, 35 °C.
To overcome the complications encountered in aqueous solution, we used
TFE as a co-solvent for the structural characterization of the peptide.
Fig. 7 shows CD spectra of the
N peptide at various concentrations of TFE. The CD
spectrum of the peptide in the absence of TFE did not show the well
defined double minima at 222 and 208 nm which are characteristics of an
ordered
-helix in aqueous solution. However, the shape of the CD
curve does suggest a measurable population of
-helix (32). For the
samples dissolved in 5, 10, 15, 20, and 30% (v/v)
TFE/H2O mixtures, the CD spectra showed increasingly
clearer double minima at 222 and 208 nm, indicating an increasing
amount of ordered
-helix. The CD spectra of the peptide in 5 to 30%
TFE (v/v) solution had a common intersection at 204 nm, indicating that
the peptide was undergoing a two-state conformational transition (Fig.
7), whereas the CD curve of the peptide in aqueous solution did not
join this intersection (Fig. 7). This result further supports the
suggestion that the peptide in aqueous solution adopts
multiconformational states. The structural transition induced by TFE
was effectively complete at a TFE concentration of 30% (v/v).
Consequently, detailed structural characterization of the
N peptide was carried out at a TFE concentration of
30%.

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Fig. 7.
Ultraviolet CD spectra of the
N peptide (50 µM) in various concentrations of TFE (0, 5, 10, 15, 20, and 30%) at 35 °C, pH 4.0. The inset
shows the change of molar ellipticity at 222 nm as a function of TFE
concentration.
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The complete assignment of the
N peptide in 30% TFE (pH
4.0, 35 °C) was achieved using standard two-dimensional
1H NMR techniques (24). Fig.
8 shows the amide-amide region of the
NOESY spectrum of the
N peptide in 30% TFE. A number of
well resolved, intense dNN cross-peaks
throughout the residues Thr148 to Arg162 were
observed, suggesting the existence of
-helical structure within this
stretch of the peptide. Fig. 9 summarizes
some of the NOE connectivities observed for the
N
peptide in 30% TFE. The data were extracted from a number of NOESY
spectra of the peptide recorded both in D2O/TFE and
H2O/TFE mixtures. Measured
H chemical shifts (presented
as the chemical shift index (33) are also included (Fig. 9). Based on
the data in Fig. 9, we conclude that the
N peptide
adopts an
-helical structure from Ser149 to
Arg162. The location of the
-helix of the peptide was
determined based on two criteria: (i) the upshifted
H chemical
shifts (chemical shift index value of
1), and (ii) a number of
intense dNN connectivities and continuous medium
range NOEs (d
N(i, i+3) and
d
(i, i+3)). Fig.
10 is a helical wheel presentation of
the
-helical region of the
N peptide. It is obvious
that the
-helix of the
N peptide is highly
amphipathic with the hydrophobic face consisting of 1 Phe and 4 Leu
residues. Nearly identical dNN cross-peaks, albeit with lower intensity, were also observed in the NOESY spectrum of the peptide in aqueous solution under the same pH at room
temperature (data not shown), suggesting that the same
-helical
structure also exists. The population and stability of such
-helical
conformation is, however, significantly lower in aqueous solution than
in the presence of TFE.

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Fig. 8.
Amide-amide region of the NOESY spectrum
(mixing time = 150 ms) of the
N peptide dissolved in 30%
(v/v) TFE/H2O at 35 °C.
A number of strong and continuous (i, i+1) NOEs are
observed, and correspondingly labeled in the spectrum.
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Fig. 9.
Summary of the NOE connectivities of the
N peptide in 30% (v/v) TFE aqueous
solution. The height of the boxes indicates
the relative intensities of the NOE cross-peaks. The dashed
lines indicate the NOEs that are ambiguous due to resonance
overlap. Chemical shift index (CSI) data of the protons
are also included (33).
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Fig. 10.
Helical wheel presentation of the
N peptide structure in 30%
TFE/H2O derived from NMR data. The
hydrophobic and hydrophilic faces of the peptide are separated by a
dashed line for clarity.
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DISCUSSION |
The minimal activation domain of Nck5a has previously been mapped
to contain 142 amino acid residues spanning residues Asp150
to Asn291 (21, 22). A number of theoretical and
experimental studies have suggested that this minimal activation domain
of Nck5a adopts a cyclin-fold (3, 6, 22). In this work, we have
identified a 29-residue peptide, residues Gln145 to
Asp173 of Nck5a, that can inhibit the kinase activities of
the Cdk5·Nck5a and Cdk2·cyclin A complexes. Based on our earlier
prediction, the sequence of this peptide encompasses the N-terminal
-helix of the cyclin fold (thus the peptide is termed the
N peptide) as well as some flanking amino acid residues
at both ends of the helix (3, 4, 22). The inhibition of Cdk5 by the
N peptide supports an earlier study that a 50-amino acid
fragment spanning residues 109 to 159 of Nck5a retains partial binding
capability to Cdk5 (21). Knowing that Nck5a only weakly activates Cdk2 to the basal level, i.e. the activity observed for a
Cdk2·cyclin A complex without Thr160 phosphorylation (20,
21), it is surprising that the
N peptide inhibits
Cdk2·cyclin A activity with an even higher potency than in the case
with Cdk5·Nck5a inhibition (Fig. 3). In contrast, the corresponding
peptide encompassing the N-terminal
-helix of cyclin A inhibits
neither Cdk2 nor Cdk5 (Fig. 3). In this work, we have investigated the
inhibition of Cdk5 and Cdk2 by the
N peptide, and it
would be interesting to know whether the
N peptide can
also inhibit other members of the Cdk family. Further work is in
progress on this matter in our laboratories.
Since the
N peptide was derived from an internal
fragment of Nck5a, it is expected that it might act as a noncompetitive inhibitor with respect to the substrate of Cdk5 (Fig. 6). However, it
is unusual that the
N peptide also functions as a
noncompetitive inhibitor with respect to Nck5a (Figs. 4 and 5). Our
results indicate that the inhibition of Cdk5 by the
N
peptide results from the formation of a ternary complex between the
N peptide and the Cdk5·Nck5a complex. Presumably, the
N peptide competes with the corresponding fragment in
Nck5a for Cdk5 binding. This suggestion is in agreement with an earlier
observation that the removal of 4 amino acid residues from the helical
part of the peptide fragment from Nck5a completely abolished the
ability of Nck5a to activate Cdk5 (22). Comparison of the crystal
structures of cyclin A in complex with Cdk2, and cyclin H, has
indicated that the N-terminal helix of various cyclins may function as
a relatively independent structural unit with respect to the tightly
packed cyclin folds (4, 5, 28). However, this N-terminal helix is
indispensable for the activity of cyclins (22, 32, 33-35), although
the contacts between the helix and the kinase are not extensive (4).
Therefore, we hypothesize that the binding of the
N
peptide dislodges the corresponding N-terminal
-helix of Nck5a from
Cdk5, thereby inhibiting the activity of the enzyme. The dislocation of
the N-terminal
-helix does not lead to dissociation of the whole
activator. Unlike the
N peptide, the control peptide
derived from cyclin A inhibits neither Cdk2·cyclin A nor Cdk5·Nck5a
(Fig. 3), suggesting a significant difference between the binding and
activation of Cdk2 by cyclin A, on the one hand, and Cdk5 by Nck5a, on
the other.
The
-helical structure detected by CD spectroscopy for the
N peptide in aqueous solution (Fig. 6) qualitatively
agrees with our earlier prediction that part of the
N
peptide could adopt an
-helical conformation (22). The existence of
multiconformational states of the peptide prevented us from a detailed
structural determination of the peptide in aqueous solution. Hence, TFE
and water were used as a co-solvent to study the structure of the
N peptide. The peptide segment from Ser149
to Arg162 was found to adopt a stable
-helical
conformation in aqueous TFE solution. Similar NOE patterns (especially
dNN NOE connectivities that were relatively well
resolved) have also been observed for the
N peptide in
pure water solution (data not shown), suggesting that the same
-helical conformation exists in this solution. It has been observed
in numerous cases that TFE can either stabilize unordered
-helices
in various peptide fragments in aqueous solution or promote the
formation of
-helices in peptide fragments that have intrinsic
propensities to form
-helix, but not induce new
-helical
conformation (for example, see Refs. 30, 32, 36, and 37). Therefore, we
suggest that the
-helical region observed in the
N
peptide would probably adopt a similar
-helical structure in Nck5a.
The peptide region found to adopt an
-helical conformation has also
been predicted to be an
-helix in the protein, and this
-helix
aligns well with the N-terminal
-helix of the first cyclin-fold of
cyclin A (Refs. 3, 4, and 22, also see Fig. 1). The above notion is
further underscored by the fact that the same
-helical structure was
observed for the cyclin A peptide in solution as the corresponding
N-terminal helix in the full-length cyclin A structures (28).
A helical wheel presentation of the
-helix found in the
N peptide shows that the peptide is amphipathic with 4 Leu and 1 Phe on the hydrophobic face (Fig. 10). Indeed, deletion of
part of the N-terminal end of the
-helix completely abolished the inhibitory effect of the
peptide.2 In an earlier
study, we have also shown that mutations of the hydrophobic amino acid
residues in the
-helix (Leu151, Leu152) to a
polar amino acid residue (Asn) greatly reduced the Cdk5 activation
ability of Nck5a (22). In the crystal structure of the Cdk2·cyclin A
complex, the corresponding N-terminal
-helix of cyclin A makes a
significant amount of contacts with various regions (e.g.
T-loop and
3 helix) of Cdk2 via hydrophobic interactions (4). It is
likely that the hydrophobic face of the peptide forms the major binding
area between the
N peptide and Cdks. This hypothesis was
supported by the result shown in Fig. 3 that an unrelated amphipathic
MLCK peptide was able to inhibit both Cdk5 and Cdk2. Like the
N peptide, the
-helical structure of the MLCK peptide
in solution can be promoted by TFE, and the MLCK peptide binds to
calmodulin in an
-helical conformation with its hydrophobic face
forming the main contact area with calmodulin (30, 38, 39). The lower
extent and potency of inhibitory activity observed with the MLCK
peptide may originate from a large sequence difference in the
-helical region as well as the C-terminal random coil region between
the MLCK peptide and the
N peptide.
The structure of the
N peptide determined here and the
interaction observed between the N-terminal
-helix of cyclin A and Cdk2 (4) suggest that systematic alterations of the amino acid residues
in the hydrophobic face of the
-helix and the C-terminal end of the
N peptide may enable us to find peptide inhibitors with
higher specificity and/or potency toward various Cdks. We note that the
present Cdk5 inhibitory peptide was discovered based on the unique
regulatory property of the enzyme by its activator. It is, therefore,
promising to develop the peptide into a Cdk5 specific inhibitor in
contrast to the majority of ATP analog derived compounds, which acts as
general kinase inhibitors. Also, the peptide in its present form can be
used to screen for chemical compounds that can inhibit the activity of
the Cdk5·Nck5a complex.