Proc. Natl. Acad. Sci.
USA
Vol. 93, pp. 11504-11509, October 1996
Biochemistry
Structural studies of p2lWa1CiPl/Sdil in the free and Cdk2-bound
state: Conformational disorder mediates binding diversity
(cell cycle regulation/NMR spectroscopy/protein folding/kinase inhibitor/MALDI mass spectrometry)
RiCHARD W. KRIWACKI, LUDGER HENGST, LINDA TENNANT, STEVEN I. REED, AND PETER E. WRIGHT*
Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037
Communicated by Carl Freiden, Washington University, St. Louis, MO, July 30, 1996 (received for review May 6, 1996)
ABSTRACT The cyclin-dependent kinase (Cdk) inhibitor
p2lWan/Cipl/sdil, important for p53-dependent cell cycle con-
trol, mediates G1/S arrest through inhibition of Cdks and
possibly through inhibition of DNA replication. Cdk inhibi-
tion requires a sequence of approximately 60 amino acids
within the p21 NH2 terminus. We show, using proteolytic
mapping, circular dichroism spectropolarimetry, and nuclear
magnetic resonance spectroscopy, that p21 and NH2-terminal
fragments that are active as Cdk inhibitors lack stable sec-
ondary or tertiary structure in the free solution state. In sharp
contrast to the disordered free state, however, the p21 NH2
terminus adopts an ordered stable conformation when bound
to Cdk2, as shown directly by NMR spectroscopy. We have,
thus, identified a striking disorder-order transition for p21
upon binding to one of its biological targets, Cdk2. This
structural transition has profound implications in light of the
ability of p21 to bind and inhibit a diverse family of cyclin-Cdk
complexes, including cyclin A-Cdk2, cyclin E-Cdk2, and
cyclin D-Cdk4. Our findings suggest that the flexibility, or
disorder, of free p21 is associated with binding diversity and
offer insights into the role for structural disorder in mediating
binding specificity in biological systems. Further, these ob-
servations challenge the generally accepted view of proteins
that stable secondary and tertiary structure are prerequisites
for biological activity and suggest that a broader view of
protein structure should be considered in the context of
structure-activity relationships.
The cyclin-dependent kinase (Cdk) inhibitor p2jWaf1/CiP1/Sdi1
(1-3), an important component of cell-cycle control mecha-
nisms (4), is transcriptionally regulated in a p53-dependent (2)
and -independent (5, 6) manner and, in the former case, is
directly involved in DNA-damage-induced cell cycle arrest at
the G1/S checkpoint (4, 7). p21 mediates G1/S arrest through
association with and inhibition of Cdks (1), and possibly
through inhibition of DNA replication (8). Cdk binding re-
quires a sequence of approximately 60 amino acids within the
p21 NH2 terminus (9-11) (Fig. 1A), a region that is homolo-
gous with the NH2-terminal domains of three other Cdk
inhibitors, p27 (12, 13), p28 (14, 15), and p57 (16, 17). The
conservation of this novel Cdk inhibitory domain within this
protein family, coupled with its putative biological role in
p53-mediated tumor suppression, identifies p21 as a potential
structural prototype for novel therapeutic agents designed for
the treatment of cancer through inhibition of cell proliferation.
We report herein the results of biophysical studies aimed at
understanding the relationship between the structural prop-
erties of p21 and its activity as a Cdk inhibitor.
EXPERIMENTAL METHODS
The gene for human p2jWafl/Cip1/sdil was obtained as reported
(7) and cloned into the bacterial expression vector pET8c
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payment. This article must therefore be hereby marked "advertisement" in
accordance with 18 U.S.C. §1734 solely to indicate this fact.
11504
(Novagen) to give pET8c-p2WaflI/CiPlISdil. Several bacterial
expression cassettes tagged with His6 at the COOH terminus
were obtained using the polymerase chain reaction, pET8c-
p2lwafl/cipl/sdil as template, and the following oligonucleo-
tides: for His6 tagged full-length p21 (p21-F), 5'-GCGAAG-
CTTGGATCCTCAGAGGAGGCGCCATGTCAGAA-3'
and 5'-GGATCCTTAATGGTGATGGTGATGGTGGGG-
CTTCCTCTTGGAGAA-3' (note: this combination gives
NcoI and BamHI restriction sites at the NH2 and COOH
termini of the p21-F expression cassette, respectively, and
forces the first amino acid after the Met codon to be the
nonnative Ala, rather than Ser); for p21 amino acids 1-94
(p21-A), 5'-CGGCGGTCAGTTCATATGTCAGAACCG-
GCTGGGGATGTCCG-3' and 5 '-CCACGCCACTCCG-
GATCCTTATTACCGCCTGCCTCCTCCC-3'; and for p21
amino acids 9-84 (p21-B), 5'-CGGCGGTCAGTTCATAT-
GCGTCAGAACCCATGCGGCAGCAAGGCC-3' and 5'-
CCACGCCACTCCGGATCCTTATTATCGCCGGG-
GCCCCGTGGG-3'. The p21-F cassette was cleaved with
NcoI and BamHI and cloned into pET24d (Novagen), and the
cassettes for p21-A and -B were cleaved with NdeI and BamHI
and cloned into pET24a (Novagen).
p21-F, -A, and -B were overexpressed in Escherichia coli
[BL21(DE3), Novagen] using standard techniques (18). Cells
were grown at 37°C in 2xYT medium [or in minimal medium
containing 15NH4CI (Isotec) for NMR samples (19)] followed
by induction with 0.5 mM isopropyl ,B-D-thiogalactopyrano-
side. p21 proteins were purified using Ni2+-affinity chroma-
tography (Chelating Sepharose, Pharmacia), anion-exchange
chromatography (Q-Sepharose, Pharmacia), and, in some
cases, C4 high performance liquid chromatography (HPLC,
Vydac). The purified proteins were analyzed by analytical C4
HPLC (Vydac), sodium dodecyl sulfate/polyacrylamide gel
electrophoresis (SDS/PAGE), NH2-terminal amino acid se-
quence analysis, and electrospray mass spectrometry. At pH
values less than 6.5, lyophilized protein was dissolved directly
in aqueous buffer solutions; at- more than pH 6.5, lyophilized
protein was dissolved in aqueous buffer solutions containing
6.0 M urea, followed by buffer exchange through repeated
ultrafiltration (YM-3 membrane, Amicon) using urea-free
buffer. Cdk2 was overexpressed and purified as described (20).
The p21-B/Cdk2 complex was prepared by addition of p21-B
to Cdk2 with both components at <100 ,ug/ml, followed by
ultrafiltration to a final concentration of 100 ,M.
The cyclin A kinase activity assay was performed as de-
scribed (21). The equilibration of cyclin A-Cdk2 with the p21
proteins was carried out at 30°C for 30 min under the following
conditions: (i) 10 mM Tris HCl, pH 7.0/500 mM NaCl/10 mM
Abbreviations: Cdk, cyclin-dependent kinase; HSQC, heteronuclear
single quantum correlation; MALDI, matrix-assisted laser desorp-
tion/ionization.
*To whom reprint requests should be addressed at: Department of
Molecular Biology/MB2, The Scripps Research Institute, 10550
North Torrey Pines Road, La Jolla, CA 92037. e-mail: wright@
scripps.edu.
 Biochemistry: Kriwacki et aL
A) p21 domain structure
Cdk Inhibitory domain PCNA binding domain
p21 Z Z I I
B) p21 trypsin cleavage sites
19.20 83,84,86
46.48 67,69 75 ; 93.94 121
9 16;
Z1
11
32
11 11 111 11
13/17, 76% 14/29, 48%
C) p21 proteins over-expressed in E. coli
1 9 84 94
p21-F a..
p21-A H6
p21-B _ H6
FIG. 1. p21 domain structure (A), trypsin cleavage sites (B), and
p21-related proteins overexpressed in E. coli (C). (B) The boxed
regions I and II represent p21 residues 17-33 and 49-77, respectively,
which show 76% and 48% identity, with p27 (12, 13). Exact cleavage
sites were determined using MALDI mass analysis of crude digests
(22). (C) p21 proteins overexpressed in E. coli, each with a COOH-
terminal His6 affinity tag; p21-F, full-length human p21, amino acids
1-164; p21-A, amino acids 1-94; p21-B, amino acids 9-84.
dithiothreitol (DTT)/1 mM EDTA, (ii) 10 mM Tris HCl, pH
7.0/330 mM NaSCN/200 mM NaCl/10 mM DTT/1 mM
EDTA, and (iii) 10 mM acetate, pH 5.0/50 mM NaCl/10 mM
DTT/1 mM EDTA.
Proteolysis was performed with 10 ,ug to 50 ,ug of p21-F
dissolved in 50 mM Tris HCl (pH 8.0) containing 0.5 or 1.0 M
NaCl, 10 mM DTT, and 1 mM EDTA at 4°C or 22°C.
Proteolytic digests were also performed under these conditions
with the addition of 2.0 or 4.0 M urea. The reaction mixture
contained trypsin (Sigma), chymotrypsin (Sigma), subtilisin
(Boehringer Mannheim), or thermolysin (Boehringer Mann-
heim), each at between 4 ng and 600 ng, in a total volume of
20 ,ul to 300 Al. Reaction products were analyzed using
SDS/PAGE, C4-HPLC, and matrix-assisted laser desorption/
ionization (MALDI) mass spectrometry. Cleavage sites were
identified on the basis of MALDI mass analysis of crude
proteolytic fragment mixtures (22).
Circular dichroism (CD) spectra were recorded using an
AVIV 61DS spectropolarimeter. Samples of p21-F, p21-A, and
p21-B were prepared in 1 mM Tris-HCl, pH 7.0/500 mM
NaCl/5 mM DTT/0.5 mM EDTA. Also, protein samples were
prepared as above in the presence of 1.0-6.0 M urea. Protein
concentrations were determined by quantitative amino acid
analysis.
Two-dimensional 1H-'5N heteronuclear single quantum cor-
relation (HSQC) nuclear magnetic resonance (NMR) spectra
were acquired at 27°C using pulsed-field gradients and water
flip-back pulses (23) using a Bruker DMX750 NMR spectrom-
eter. Data processing was performed using the software pack-
age FELIX 2.30 (Biosym Technologies, San Diego).
RESULTS
The conformational properties of p21 and NH2-terminal frag-
ments of p21 were probed using a variety of methods, including
protease mapping, CD spectropolarimetry, and NMR spec-
troscopy. At first, we overexpressed human p21 with a COOH-
terminal His6 affinity tag (p21-F). Proteolytic mapping using
trypsin, chymotrypsin, subtilisin, and thermolysin showed mul-
tiple cleavages throughout the entire primary amino acid
sequence. Detailed analysis of trypsin cleavage products using
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MALDI mass spectrometry (22) showed that p21-F is cleaved
at all predicted trypsin sites (Fig. 1B) and reveals that globular
protease-resistant domains do not exist. Further, no difference
in the protease maps was observed in the absence or presence
Proc. Natl. Acad. Sci. USA 93 (1996) 11505
of urea (2.0 M and 4.0 M), as judged using SDS/PAGE and
HPLC (data not shown), providing further evidence that p21-F
lacks stable structured domains. Based on the homology
between p21 and two other Cdk inhibitors, p27 (12,13) and p57
(16, 17), within the NH2 terminus (Fig. iB), we overexpressed
two shorter fragments of p21, p21-A and -B (Fig. 1C). These
140-143 162,163 two fragments show behavior similar to that for p21-F when
4 154-1856
subjected to proteolytic mapping (i.e., all trypsin and chymo-
trypsin cleavage sites are accessible) (22).
The inhibitory activity of p21-F, -A, and -B toward Cdks was
tested in vitro using cyclin A-Cdk2 purified from HeLa cells by
immunoprecipitation and using histone Hi as the kinase
164
substrate. The concentration producing 50% inhibition for all
H6
three p21 proteins is between 32 pM and 320 pM (Fig. 2A).
Cdk2 inhibitory activity was tested at 32 nM under the solution
conditions used for structural studies (vide infra), including (i)
high ionic strength, pH 7.0 (Fig. 2 A, B Left, and C Left), (ii)
high ionic strength with sodium thiocyanate, pH 7.0 (Fig. 2 A,
B Middle, and C Middle), and (iii) low ionic strength, pH 5.0
(Fig. 2 A, B Right, and C Right). Cdk2 kinase activity is
diminished under conditions ii and iii compared with condition
i (Fig. 2B); significantly, however, p21-F, -A, and -B are potent
Cdk2 inhibitors under all solution conditions (Fig. 2C). Since
full-length p21 and NH2-terminal p21 fragments are active as
Cdk2 inhibitors at similar concentrations, structural studies of
the p21 NH2 terminus will be useful in understanding the
structural properties of full-length p21 pertaining to Cdk2
inhibition. Similar inhibitory behavior is observed for p21-F
purified under denaturing (including HPLC and protein ly-
ophilization) and entirely nondenaturing aqueous conditions
(without HPLC and lyophilization). Previous studies have
shown that His-tagged and untagged p21 have similar Cdk
inhibitory activity (1).
CD spectropolarimetry was used to investigate the second-
ary structure of the p21 NH2 terminus. CD spectra for p21-F,
-A, and -B are very similar (Fig. 3A) and indicate that (i)
despite the clear demonstration of Cdk inhibitory activity, p21
and its fragments possess very little regular secondary struc-
ture, and (ii) the lack of secondary structure within the
NH2-terminal fragments is not due to a requirement that
COOH-terminal domains be present to promote a stable fold.
The magnitude of [01222 in Fig. 3A (-6 to -8 x 103 deg cm2
dmol-1) is consistent with the existence of an a-helical con-
formation for 15-20% of the residues. The absence of an
inflection or local minimum in the CD spectra at 217 nm
indicates that the population of (3-sheet secondary structure is
relatively small. CD spectra were examined as a function of
temperature and urea concentration to determine the degree
to which these denaturants induce conformational changes.
Fig. 3B shows that for p21-A the degree of helicity is virtually
unchanged from 5°C to 90°C (similar behavior is seen for p21-F
and p21-B) while Fig. 3C shows that helicity is reduced, in a
noncooperative manner, over the concentration range from 0
M to 6.0 M urea (Fig. 3C). The temperature invariance of
p21-A CD spectra is consistent with the reported insensitivity
of p21-dependent Cdk inhibitory activity to high-temperature
treatment (7). The CD data thus indicate that p21 does not
exhibit the characteristic cooperative unfolding behavior ex-
pected of a globular protein but rather provide further evi-
dence that the protein is highly flexible and disordered in the
free solution state. Similar results are obtained under a wide
variety of solution conditions.t
To obtain further insights into structure, two-dimensional
'H-'5N chemical shift correlated NMR spectra (25) of 15N-
tAt less than pH 5.0, p21-F, -A, and -B aggregate, as shown by dynamic
light scattering and gel filtration chromatography measurements
(data not shown). The aggregation phenomenon leads to subtle
changes in CD spectra (data not shown) -but does not change the
generally disordered conformational state.
 11506 Biochemistry: Kriwacki et al. Proc. Natl. Acad. Sci. USA 93 (1996)
A 5
00
I"
'a>
E 5
U N
E -10
3 so
;15&
C - * 15

o 25 o
-
,-M
-20
OD
-25
21 260
10~1 1 10 102 *C I 105 lo pM Ip2l-XJ
wavelength (nm)
B 1 2 3 4
.....
5_6 7 8 9 10 11 12 0
"W_
...._ Histone Hi
-5 -1
E
'B
1 2 3 4 5 6 7 8

lEa -2
N

-3
-4

0 -5
I- 0 20 40 60 80 10
IL t X g
temperature ("C)
_ _ _
0)
0
0
FIG. 2. p21-F, -A, and -B inhibit cyclin A-Cdk2 under a variety of
solution conditions. (A) Inhibition of cyclin A-Cdk2 by p21-F (solid
squares), -A (shaded diamonds), and -B (open circles) as a function of N
-1
C
inhibitor concentration at high ionic strength, pH 7.0, as discussed in loE
the text. Cyclin A-Cdk2 kinase activity is shown as a percentage of the
activity in the absence of the inhibitors. (B and C) Comparison of cyclin -2
A-Cdk2 inhibitory activity for p21-F, -A, and -B at 32 nM under
several solution conditions, including (i) high ionic strength, pH 7.0
(Left); (ii) high ionic strength with sodium thiocyanate, pH 7.0 -o -3
(Center); and (iii) low ionic strength, pH 5.0 (Right). (B) SDS/PAGE
autoradiograph showing levels of [32P]phosphate incorporated into
histone Hi by cyclin A-Cdk2. (C) Normalized levels of cyclin A-Cdk2
kinase activity derived from B.
N -4
N

labeled p21-A and -B were acquired. Two-dimensional NMR -5

spectra, however, could not be obtained for p21-F due to its 0.0 1.0 2.0 3.0 4.0 5.0 6.0
limited solubility (<10 ,uM). The IH-15N correlation spectrum
of p21-A (Fig. 4A)t reveals resonances that are clustered
[ureal (M)
within a narrow chemical shift range (total 1H chemical shift FIG. 3. CD spectra for p21 protein series. (A) Spectra for p21-F
dispersion of backbone amides 0.80 ppm) and indicates that (soldi circles), p21-A (solid squares), and p21-B (solid triangles) in 1
the chemical environment of each amide group, as reported by mM Tris-HCI, pH 7.0/500 mM NaCl/5 mM DTT/0.5 mM EDTA at
1H and 15N chemical shifts, is influenced predominantly by the 5°C. Estimates for a-helical content are based on [01222 = -40 x 103
chemical nature of its own amino acid side chain and not by deg cm2 dmol-1 for 100% a-helix (24). (B) [01222 versus temperature
and (C) [01222 versus urea concentration for p21-A.
long-range effects due to secondary or tertiary structure. The
spectrum lacks the chemical shift dispersion expected for a
tThe spectrum shown in Fig. 4A was obtained in the presence of 330 folded globular protein. In fact, 16 resonances in the native
mM NaSCN and 200 mM NaCl to improve the solubility of p21-A
Downloaded by guest on June 21, 2021

near pH 7.0. An HSQC spectrum acquired at very low p21-A
concentration (-25 ,uM) in the absence of NaSCN but in the presence
of 500 mM NaCl was very similar to that shown in Fig. 4A, indicating
that the conformational state of p21-A is unchanged by NaSCN.
p21-A spectrum (Fig. 4A) appear within limits that are char-
acteristic of Gly and Ser/Thr residues in proteins denatured by
urea (26, 27). All 12 glycine resonances appear within the
expected limits and four resonances appear within the unique
 Biochemistry: Kriwacki et aL
- GiyA
aP 7 0 9 a1l
8 a
11
12
.~~~15 16
-N,
mp 0MDS1 : 0
4
8.5 8.2
1H chemical shift (ppm)
FIG. 4. 'H-15N heteronuclear single quantum correlation (HSQC) spectra at 750 MHz for 15N-labeled p21-A under "native" (A) and
"denatured" (B) conditions. The regions marked Gly and Ser/Thr show the approximate 1H and 15N chemical shift limits for these residues in
proteins denatured by urea (26, 27). The numbered resonances are referred to in the text. The sample inA contained 300 ,uM 15N-p21-A dissolved
in 10 mM Tris-2H,1, pH 6.9/330 mM NaSCN/200 mM NaCl/10 mM DTT-2HIo/5% 2H20 (vol/vol)/0.02% NaN3, and the sample in B contained
the same components with 1.0 mM 15N-p21-A at pH 7.15 and 6.0 M urea.
portion of the limits for Ser/Thr residues in an unfolded
polypeptide chain (p21-A contains four Ser and three Thr
residues). Further, the 12 Gly residues of p21-A are relatively
evenly distributed throughout the primary sequence§ and,
therefore, report the conformational state of the entire p21
NH2 terminus. Only subtle changes in resonance dispersion
occur in 6.0 M urea (Fig. 4B; 'H chemical shift range 0.77
ppm) showing that the conformational state is changed very
little by denaturant and providing further evidence that, under
native solution conditions, p21-A is largely unstructured and
disordered. Thus, the proteolysis, CD, and NMR experiments
indicate that p21 and its NH2-terminal fragments, in their
biochemically active form, do not possess the conformational
hallmarks of folded globular proteins. Instead, they possess
only limited, and perhaps transiently populated, secondary
structure (15-20% a-helix) within an otherwise disordered
polypeptide.
The conformational state of p21-B, which is 18 amino acids
shorter than p21-A but has similar biochemical activity (Fig. 2),
is dramatically changed upon addition of a stoichiometric
amount of unlabeled Cdk2, as revealed by 'H-15N correlated
spectra (Fig. 5). Upon binding to Cdk2 (Fig. SB), there is a
large increase in resonance dispersion (total 'H chemical shift
dispersion of backbone amides 1.25 ppm). The formation of
the p21-B-Cdk2 complex is supported by other biophysical
data, including results from proteolytic mapping (22) and gel
filtration chromatography (M. Hickey and J. Tainer, personal
communication; the formation of the complex has been con-
firmed by gel filtration chromatography). Representative 15N
line widths at half height for Cdk2-bound p21-B range from 9
Hz to 16 Hz, consistent with a monodisperse 44-kDa protein-
protein complex. The resonances of p21-B in the Cdk2 com-
plex (Fig. SB) can be divided into two groups; those at unique
chemical shifts with respect to Fig. SA (32 red circles) and those
at similar positions in Fig. 5 A and B (29 green squares).
Further, close inspection shows that many of the resonances
marked by green squares have two overlapping components
Downloaded by guest on June 21, 2021
with different line widths (data not shown). It is probable that
§Gly residues occur at positions 6, 14, 23, 40, 61, 70, 72, 81, 85, 90, 91,
and 92 of p21-A.
Proc. Natl. Acad. Sci. USA 93 (1996) 11507
1 Cw=2 GIyB
3 5
78 D a
0 6 10 11 o,Om%
ml 2
q-$
CD
TM
I-l
-
l13ls4
15wD, qgI6
co
Ser/Thr
O!
'V
-
cod
z
_LO
_
o
o7 v
8H5 8st2
'H chemical shift (ppm)
one component arises from p21-B in the complex and the other
arises from residual free p21-B. However, the p21-B/Cdk2
stoichiometry is estimated to be close to 1:1 based on quan-
titation using SDS/PAGE, suggesting that free p21-B is a
relatively minor component. The fact that two sets of reso-
nances appear in Fig. SB indicates that free p21-B and Cdk2-
bound p21-B are in slow exchange on the NMR time scale (28),
meaning that Cdk2-bound p21-B is temporally stable rather
than rapidly exchanging between the free and bound states.
This observation, coupled with the increase in dispersion for
a large number of resonances, indicates that a substantial
portion of the NH2-terminal region of p21 adopts a defined
and stable structure in the complex with Cdk2.
DISCUSSION
Limited structural information is available on the physical
nature of the interaction between p21 and its biological targets,
cyclin-Cdk complexes. The results of proteolysis, CD, and
NMR experiments reported herein show that both full-length
p21 and the kinase inhibitory domain of p21 are highly
disordered in the absence of cyclin-Cdk complexes. Direct
NMR evidence, however, shows that the kinase inhibitory
domain adopts an ordered conformation when bound to Cdk2.
The p21 kinase inhibitory domain, therefore, undergoes a
dramatic disorder-order transition upon binding to Cdk2.
What is the biological role for the disorder-order transition
experienced by the p21 NH2 terminus upon binding to Cdk2?
First, from a thermodynamic perspective, the change in con-
formational entropy for p21 upon binding to Cdk2 is probably
large and negative. This term, however, will be opposed by
other entropic and enthalpic terms associated with p21-Cdk2
binding, leading to the net free energy change favoring bind-
ing. We thus expect that a Gibbs free energy penalty will be
associated with the requirement for folding of p21 on binding.
What, then, is the biological advantage of the disorder-order
transition upon binding? As has been suggested for binding-
induced protein folding transitions associated with protein-
DNA interactions (29), the Cdk2 binding-induced disorder-
order transition for p21 may be associated with increased
specificity for this protein-protein interaction at the expense
 11508 Biochemistry: Kriwacki et al. Proc. Natl. Acad. Sci. USA 93 (1996)

A) free p21 B) Cdk2-bound p21

E3 i 0 0
'o
-(6
0
.0
0 E
TM
*CO
T-C0 0
nowo1! ep _q a._

.A'A'~~~i.,
.

m
A0aE
- C4
o
TM 0
0 co

0
lb4
-o 90 @~~~~ cd
TM

"b': CM CM
o
10
o 7- z
La
CM
TM

8.5 8.0 8:5 8.0

IH chemical shift (ppm) 1H chemical shift (ppm)
FIG. 5. 'H-15N HSQC spectra for 15N-labeled p21-B in the free state (A) and in the Cdk2-bound state (B) under low ionic strength conditions
at pH 5.0. The resonances enclosed by green boxes are found at the same chemical shift values in both A and B and resonances enclosed by red
circles in B are unique to the Cdk2-bound state of p21-B. The sample inA contained 200 ,uM 15N-p21-B dissolved in 10 mM acetate-2H3, pH 5.0/10
mM DTT-2Hio/5% 2H20/0.02% NaN3 while the sample in B contained 100 ,tM 15N-p21-B, 100 ,uM Cdk2, 50 mM NaCl, and the other components
in A. In B, resonances for unlabeled Cdk2 were eliminated through isotope filtering.

of absolute thermodynamic binding affinity. The implication is
that, if p21 existed in a more ordered free state, its affinity for
Cdk2 might be greater, albeit at the expense of specificity.
Second, from a structural perspective, the existence of
multiple conformations for p21 may be related to its biological
role as an inhibitor of multiple cyclin-Cdk complexes that are
involved in regulation of the G1/S checkpoint (including cyclin
A-Cdk2, cyclin E-Cdk2, and cyclin D-Cdk4) (30). While
sequence alignments of the cyclins and of the Cdks show a high
degree of homology within certain regions of their primary
amino acid sequences (on the order of 40-50% identity), the
sequence variability is such that the structural details of the
protein-protein interactions between p21 and each cyclin-Cdk
complex may be subtly different. The ability of p21 to recog-
nize this diverse group of protein complexes derives not from
the complementarity of preformed protein surfaces but rather
from p21's ability to adopt multiple conformations that me-
diate diverse binding events. This proposal challenges the
generally accepted view of proteins that stable secondary and
tertiary structure are prerequisites for biological activity and
suggests that a broader view of structure should be considered
in the context of protein structure-activity relationships.
Finally, while p21 acts in vivo as an inhibitor of Cdks within
binary cyclin-Cdk complexes, our NMR spectroscopy results
provide direct evidence for the formation of a 1:1 p21-B-Cdk2
complex, in the absence of a cyclin. The Kd (- 10 j.M) for this
binary complex (M. Hickey and J. Tainer, personal commu-
nication), however, is much larger than that for the ternary
p21-cyclin A-Cdk2 complex (0.5 nM) (30), suggesting that
direct interactions between p21 and the cyclin component of
cyclin-Cdk complexes might exist. The amino acids within the
p21 NH2 terminus that remain disordered in the p21-B-Cdk2
complex reported herein may be involved in direct interactions
with the cyclin component of cyclin-Cdk complexes. This idea
Downloaded by guest on June 21, 2021
of protease mapping and mutagenesis studies. In a separate set
of experiments (22), we have shown that a 22- to 36-amino acid
segment of p21-B, bracketing positions 46, 48, and 67, is
protected from trypsin cleavage in the presence of an equimo-
lar amount of Cdk2, identifying this segment of p21-B as the
Cdk2 binding site. Further, a previous report (31) shows that
alanine-scanning mutations within homology region I of p21
(Fig. 1B) do not significantly decrease affinity for Cdk2 while
mutations within homology region II do (specifically, at posi-
tions 44, 46, 47, 48, 52, 56, 60, and 62), suggesting that region
I may contact the cyclin component of cyclin-Cdk complexes
and showing that region II contacts the Cdk component, in
agreement with protease mapping. Finally, a recent report (32)
strongly suggests, through the use of a series of deletion
mutants of p21, that homology region I binds the cyclin
component and homology region II binds the Cdk component
of cyclin-Cdk complexes, in direct support of our proposal.
Note Added in Proof. While this manuscript was under review, the
crystal structure at high resolution was reported for the p27Kipl-cyclin
A-Cdk2 ternary complex (33). The details of the cyclin A-Cdk2-bound
p27 structure support our predictions about the interactions between
p21 and cyclin A-Cdk2.
R.W.K. thanks Dr. Tom Steitz of Yale University for critical
discussion of structural studies during the course of this work, mem-
bers of the Wright, Dyson, and Reed laboratories at Scripps for
stimulating discussions, Dr. Mark Watson for Cdk2 samples and advice
on the handling of Cdk2, Dr. Mark Watson and Ms. Alicia Santiago
for the original p21 expression construct, Mr. Michael Hickey and Dr.
John Tainer for communication of results prior to publication and
helpful discussion, Dr. Lisa Bibbs for DNA and protein analysis
services, Dr. Gary Siuzdak for mass spectrometry services, and Ms.
Martine Reymond for technical assistance with HPLC. R.W.K. is a
Leukemia Society Fellow and L.H. is a Leukemia Society Special
Fellow. This work was supported by Grants GM38794 (P.E.W.) and

is supported by the finding that p21 stabilizes cyclin-Cdk GM46006 (S.I.R.) from the National Institutes of Health.
complexes (30). Information about the location of amino acids
of p21 involved in interactions with the cyclin and Cdk 1. Harper, J. W., Adami, G. R., Wei, N., Keyomarsi, K. & Elledge,
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