Structural basis for high-affinity peptide inhibition of p53 interactions with MDM2 and MDMX.
ABSTRACT The oncoproteins MDM2 and MDMX negatively regulate the activity and stability of the tumor suppressor protein p53--a cellular process initiated by MDM2 and/or MDMX binding to the N-terminal transactivation domain of p53. MDM2 and MDMX in many tumors confer p53 inactivation and tumor survival, and are important molecular targets for anticancer therapy. We screened a duodecimal peptide phage library against site-specifically biotinylated p53-binding domains of human MDM2 and MDMX chemically synthesized via native chemical ligation, and identified several peptide inhibitors of the p53-MDM2/MDMX interactions. The most potent inhibitor (TSFAEYWNLLSP), termed PMI, bound to MDM2 and MDMX at low nanomolar affinities--approximately 2 orders of magnitude stronger than the wild-type p53 peptide of the same length (ETFSDLWKLLPE). We solved the crystal structures of synthetic MDM2 and MDMX, both in complex with PMI, at 1.6 A resolution. Comparative structural analysis identified an extensive, tightened intramolecular H-bonding network in bound PMI that contributed to its conformational stability, thus enhanced binding to the 2 oncogenic proteins. Importantly, the C-terminal residue Pro of PMI induced formation of a hydrophobic cleft in MDMX previously unseen in the structures of p53-bound MDM2 or MDMX. Our findings deciphered the structural basis for high-affinity peptide inhibition of p53 interactions with MDM2 and MDMX, shedding new light on structure-based rational design of different classes of p53 activators for potential therapeutic use.
- SourceAvailable from: Mark Bycroft[show abstract] [hide abstract]
ABSTRACT: We have investigated the kinetic and thermodynamic basis of the p53-MDM2 interaction using a set of peptides based on residues 15-29 of p53. Wild-type p53 peptide bound MDM2 with a dissociation constant of 580nM. Phosphorylation of S15 and S20 did not affect binding, but T18 phosphorylation weakened binding tenfold, indicating that phosphorylation of only T18 is responsible for abrogating p53-MDM2 binding. Truncation to residues 17-26 increased affinity 13-fold, but further truncation to 19-26 abolished binding. NMR studies of the binding of the p53-derived peptides revealed global conformational changes of the overall structure of MDM2, stretching far beyond the binding cleft, indicating significant changes in the domain dynamics of MDM2 upon ligand binding.Journal of Molecular Biology 11/2002; 323(3):491-501. · 3.91 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The tumor suppressor p53 is degraded by the ubiquitin-proteasome system. p53 was polyubiquitinated in the presence of E1, UbcH5 as E2 and MDM2 oncoprotein. A ubiquitin molecule bound MDM2 through sulfhydroxy bond which is characteristic of ubiquitin ligase (E3)-ubiquitin binding. The cysteine residue in the carboxyl terminus of MDM2 was essential for the activity. These data suggest that the MDM2 protein, which is induced by p53, functions as a ubiquitin ligase, E3, in human papillomavirus-uninfected cells which do not have E6 protein.FEBS Letters 01/1998; 420(1):25-7. · 3.58 Impact Factor
Article: Surfing the p53 network.Nature 12/2000; 408(6810):307-10. · 38.60 Impact Factor
Structural basis for high-affinity peptide inhibition of
p53 interactions with MDM2 and MDMX
Marzena Pazgiera,1, Min Liua,b,1, Guozhang Zoua, Weirong Yuana, Changqing Lia, Chong Lia, Jing Lia, Juahdi Monboa,
Davide Zellaa, Sergey G. Tarasovc, and Wuyuan Lua,2
aInstitute of Human Virology, University of Maryland School of Medicine, 725 West Lombard Street, Baltimore, MD 21201;bThe First Affiliated Hospital,
School of Medicine, Xi’an Jiaotong University, Shaanxi Province 710061, China; andcStructural Biophysics Laboratory, National Cancer Institute at Frederick,
Frederick, MD 21702
Communicated by Robert C. Gallo, University of Maryland, Baltimore, MD, January 28, 2009 (received for review September 29, 2008)
The oncoproteins MDM2 and MDMX negatively regulate the ac-
tivity and stability of the tumor suppressor protein p53—a cellular
process initiated by MDM2 and/or MDMX binding to the N-
tumors confer p53 inactivation and tumor survival, and are impor-
tant molecular targets for anticancer therapy. We screened a
duodecimal peptide phage library against site-specifically biotin-
ylated p53-binding domains of human MDM2 and MDMX chemi-
cally synthesized via native chemical ligation, and identified sev-
eral peptide inhibitors of the p53-MDM2/MDMX interactions. The
most potent inhibitor (TSFAEYWNLLSP), termed PMI, bound to
MDM2 and MDMX at low nanomolar affinities—approximately 2
orders of magnitude stronger than the wild-type p53 peptide of
the same length (ETFSDLWKLLPE). We solved the crystal structures
of synthetic MDM2 and MDMX, both in complex with PMI, at 1.6
Å resolution. Comparative structural analysis identified an exten-
sive, tightened intramolecular H-bonding network in bound PMI
that contributed to its conformational stability, thus enhanced
binding to the 2 oncogenic proteins. Importantly, the C-terminal
residue Pro of PMI induced formation of a hydrophobic cleft in
MDMX previously unseen in the structures of p53-bound MDM2 or
MDMX. Our findings deciphered the structural basis for high-
affinity peptide inhibition of p53 interactions with MDM2 and
MDMX, shedding new light on structure-based rational design of
different classes of p53 activators for potential therapeutic use.
damage or oncogene activation, the expression of various target
genes that mediate cell-cycle arrest, DNA repair, senescence or
next generation (1–3). In 50% of human cancers, p53 is defective
due usually to somatic mutations or deletions primarily in its
DNA-binding domain and, to a lesser extent, to posttranslational
modifications such as phosphorylation, acetylation and methyl-
ation that affect p53 function and stability. Altered p53 fails to
regulate growth arrest and cell death upon DNA damage,
directly contributing to tumor development, malignant progres-
sion, poor prognosis and resistance to treatment (4). Conversely,
restoring endogenous p53 activity can halt the growth of can-
cerous tumors in vivo by inducing apoptosis, senescence, and
innate inflammatory responses (5–7).
As p53 mediates growth arrest and apoptosis, it is essential to
keep its activity in check during normal development (2).
Multiple mechanisms exist to negatively regulate p53 activity,
among which the E3 ubiquitin ligase MDM2 and its homolog
MDMX (also known as MDM4) play a central regulatory role in
the developing embryo and in mature differentiated cells (8, 9).
MDM2 consists of 491-aa residues, comprising an N-terminal
p53-binding domain, a central domain preceded by nuclear
export and localization signals essential for nuclear-cytoplasmic
trafficking of MDM2, a zinc finger domain, and a C-terminal
zinc-dependent RING finger domain that confers E3 ubiquitin
53 is best known as a tumor suppressor that transcriptionally
regulates, in response to cellular stresses such as DNA
ligase activity (10). Structurally related to MDM2, MDMX of
490-aa residues possesses domain structures arranged similarly
to MDM2, except that MDMX lacks ubiquitin-ligase function
(11, 12). Growing evidence supports that in unstressed cells
MDM2 primarily controls p53 stability through ubiquitylation to
target the tumor suppressor protein for constitutive degradation
by the proteasome (13, 14), whereas MDMX mainly functions as
a significant p53 transcriptional antagonist independently of
MDM2 (15, 16). Under stress conditions, MDM2 and MDMX
cooperate to activate p53 through mechanisms involving both
MDM2 autodegradation (autoubiquitylation) and MDM2-
depedent degradation of MDMX (17–20).
In many tumors, p53 is present in its wild-type form. The
presence of wild-type p53 strongly correlates to amplification
and/or over-expression of MDM2/MDMX, resulting directly in
p53 suppression and malignant progression (8, 9). Inhibition of
the p53-MDM2 interactions by MDM2 antagonists has been
shown both in vitro and in vivo to reactivate the p53 pathway and
selectively kill tumor cells in a p53-dependent manner. Acting
synergistically in tumor cells, MDM2 and MDMX have become
2 of the most attractive molecular targets for anticancer therapy.
Toward this end, much of the current efforts have been focused
on combinatorial library search for and structure-based rational
design of low molecular weight inhibitors that target the N-
terminal p53-binding domains of MDM2 and MDMX (21).
Successful examples include, but are not limited to, cis-
imidazoline analogs termed Nutlins and, more recently, a spiro-
oxindole-derived compound termed MI-219 (22, 23).
Peptides, because of their large interacting surfaces, offer the
prospect of enhanced potency, high specificity and low toxicity.
However, most of the peptidic and peptidomimetic inhibitors
examined to date bind MDM2 at affinities ranging from high
nanomolar to low micromolar concentrations, and none is nearly
as effective as Nutlins and MI-219 in tumor killing in vitro (21).
Further, because the structural basis for MDMX inhibition is
much less understood than that for MDM2 inhibition, antago-
nists designed for MDM2 are, in general, significantly less
inhibitory toward MDMX. Potent peptide inhibitors against
MDM2 and/or MDMX are needed as important cellular probes
of the p53 pathway in cancer biology and as useful templates for
structure-based rational design of different classes of p53 acti-
vators for potential therapeutic use. Here, we report identifica-
tion and functional and structural characterizations of a high-
Author contributions: M.P., M.L., and W.L. designed research; M.P., M.L., G.Z., W.Y.,
W.L. analyzed data; and M.P. and W.L. wrote the paper.
The authors declare no conflict of interest.
Data deposition: The atomic coordinates have been deposited in the Protein Data Bank,
www.pdb.org (PDB ID codes 3EQS and 3EQY).
1M.P. and M.L. contributed equally to this work.
2To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/cgi/content/full/
March 24, 2009 ?
vol. 106 ?
no. 12 ?
affinity peptide inhibitor, termed PMI (p53-MDM2/MDMX
inhibitor), of p53 interactions with both MDM2 and MDMX.
PMI—a Potent Inhibitor of the p53-MDM2/MDMX Interactions Se-
lected from a Phage Displayed Peptide Library. The p53-binding
domains of MDM2and
24–108MDMX, referred to thereafter as
synMDMX) and their site-specifically biotinylated forms were
chemically synthesized using native chemical ligation (SI Text).
Using biotin-synMDM2 and biotin-synMDMX as bait, we
screened a duodecimal peptide library displayed on M13 phage.
Shown in Fig. S1 are the amino acid sequences from 15 binding
clones obtained after 4 rounds of selection. Two consensus
sequences emerged for both MDM2 and MDMX: LTFEHY-
WAQLTS, also termed pDI (24), and PMI–TSFAEYWNLLSP.
The 3 most critical residues involved in p53-MDM2/MDMX
recognition, i.e., Phe-19, Trp-23 and Leu-26 (p53 numbering),
were all present in the phage-selected consensus sequences. pDI
was recently identified from the same Ph.D.-12TMphage library,
using GST-tagged recombinant MDM2 and MDMX immobi-
lized on glutathione-agarose beads (24). However, because of
low solubility and relatively weak activity of pDI, this work
focused only on PMI, which is highly soluble in aqueous solution
(ETFSDLWKLLPE), were used for comparison.
We quantified direct interactions between
synMDMX and the 3 peptide inhibitors, using isothermal titra-
tion calorimetry (ITC), and the results are tabulated in Table S1
(see Fig. S2 for additional data). PMI bound tosynMDM2 and
synMDMX with Kd values of 3.3 and 8.9 nM, respectively,
?20-fold stronger than(15–29)p53 for either protein. Compared
with that of(17–28)p53—the wild type sequence of the same
length, the binding affinity of PMI increased by a factor of 89 for
MDM2 and of 43 for MDMX. In both cases, favorable enthalpy
changes overcame unfavorable entropy changes, contributing to
a dramatically enhanced binding of PMI to
To verify the ITC results, we devised a surface plasmon
resonance (SPR)-based competition assay, in which(15–29)p53
was immobilized on a CM5 sensor chip for kinetic analysis of a
fixed concentration ofsynMDM2 (50 nM) orsynMDMX (100 nM)
preincubated with varying concentrations of PMI,(15–29)p53, or
(17–28)p53. Nutlin-3—a racemic mixture of Nutlin-3a and Nut-
lin-3b (23), was used as a control. As shown in Fig. 1, all 4
inhibitors competed, in a dose-dependent manner, with immo-
bilized(15–29)p53 forsynMDM2 orsynMDMX binding. Nonlinear
(15–29)p53 (SQETFSDLWKLLPEN) and
regression analyses yielded for PMI a Kdvalue of 3.4 nM for
synMDM2 and of 4.2 nM forsynMDMX. By contrast,(15–29)p53
bound tosynMDM2 andsynMDMX at affinities of 140 nM and
270 nM, respectively. Although Nutlin-3 showed little binding to
MDMX, its Kdvalue of 263 nM for MDM2 was largely in line
with the published result (IC50: 90 nM for Nutlin-3a and 13.6 ?M
for Nutlin-3b) (23). Importantly, compared with(17–28)p53, PMI
bound tosynMDM2 135-fold stronger and tosynMDMX 156-fold
tighter. Our data suggest that PMI, with low nanomolar affinities
for both MDM2 and MDMX, is one of the strongest peptidic
inhibitors of the p53-MDM2/MDMX interactions ever reported.
PMI Binds to MDM2 and MDMX in a Canonical Mode Previously
Described for p53 Peptides. The overall structures of human
synMDM2 andsynMDMX are similar, as evidenced by a root
mean square deviation (rmsd) of 1.2 Å between superimposed
C?atoms.synMDM2 andsynMDMX share the basic structural
elements and conserved global fold reported for human MDM2
(23, 25–28) and zebra fish MDMX (29). Both molecules are
characterized by structural repetition of 2 assemblies of a ?????
topology, which are related by an approximate dyad axis of
pseudosymmetry (Fig. 2). Superposition of
synMDMX points toward a large structural variation in the
C-terminal half of the molecules, whereas the N-terminal halves
are nearly identical. Except for the loop regions, the largest
conformational changes are located in the ?2? helix, stemming
from a change of His-96 in MDM2 to Pro-95 in MDMX.
by SPR-based competition assays. Each curve is the mean of 4 independent measurements at 25 °C in 10 mM Hepes, 150 mM NaCl, 0.005% surfactant P20, pH
7.4. Nutlin-3 was too weak forsynMDMX and is not reported. Solubility of both Nutlin-3 and pDI decreased at the highest concentrations used, attributing to
an upward curvature of the inhibition curves forsynMDM2-Nutlin-3 andsynMDMX-pDI.
Quantification of the interactions ofsynMDM2 (50 nM) andsynMDMX (100 nM) with varying concentrations of PMI, pDI,(15–29)p53,(17–28)p53, or Nutlin-3
green) andsynMDMX-PMI (blue/yellow) in a ribbon diagram.
Stereoview of superimposed structures ofsynMDM2-PMI (orange/
www.pnas.org?cgi?doi?10.1073?pnas.0900947106 Pazgier et al.
Interestingly, a crystallographic 2-fold axis generates a dimer of
the PMI-synMDM2 complex (Fig. S3), although no evidence for
dimerization has been found using native gel electrophoresis and
size exclusion chromatography.
The N-terminal transactivation domain of p53 encompasses
T18F19S20D21L22W23K24L25L26minimally required for effective
MDM2 binding (30, 31). The side chains of Phe-19, Trp-23 and
Leu-26, involved in p53 transactivation (32–34), dock, in an
amphipathic ?-helix, inside a hydrophobic cavity of the oncop-
rotein (25). Not surprisingly, PMI retains the functionally con-
served hydrophobic triad, Phe-3/Trp-7/Leu-10. Structural anal-
ysis indicates that the Phe-3-binding pockets are nearly identical
in MDM2 and in MDMX. However, the Trp-7-binding pockets
slightly differ in geometry (Leu-54 and Ile-99 in MDM2 versus
Met-53 and Leu-98 in MDMX). Nevertheless, the Phe/Trp dyads
of PMI and p53 appear indistinguishable in MDM2/MDMX
binding, as indicated by calculations of buried surface area
(BSA) and binding energy (Table S2). In contrast, the Leu-10-
binding pocket in MDM2 (lined by Leu-54, Val-93, His-96,
Ile-99, Tyr-100) differs from that in MDMX (lined by Met-53,
Val-92, Pro-95, Leu-98, Tyr-99), mainly because of the shift of
the ?2? helix caused by the His-96 to Pro-95 change. Because
Leu-10 of PMI is buried in the peptide-protein complexes to a
different extent from Leu-26 of p53, the energetic contribution
of that position may be context-dependent.
In addition to the hydrophobic interactions, 3 intermolecular
H-bonds contribute to PMI binding to MDM2 and MDMX. The
same number of intermolecular H-bonds is also present in the
p53-MDM2 complex between Phe-19 N, Trp-23 N?1, and Asn-29
OXT of the peptide and Gln-72 O?1, Leu-54 O, and Tyr-100 O?
of the protein, respectively (25). However, the H-bonding pat-
terns involving Tyr-100 in PMI-synMDM2 and Tyr-99 in PMI-
synMDMX differ. Tyr-100 O?donates an H-bond to Leu-10 O of
PMI, whereas Tyr-99 O?forms an H-bond with Ser-11 N of PMI,
reflecting structural differences in the C-terminal region of PMI
and in the vicinity of Tyr-100/Tyr-99 between different protein-
PMI Differs from p53. A secondary-structure analysis, using the
Kabsch–Sander algorithm (35), reveals that the ?-helix of PMI
is more extended than that of p53. In PMI, the regular ?-helix
starts at Phe-3 and ends at Asn-8 (6 residues), followed by a
helical turn comprising Leu-9 and Leu-10 in MDM2 and only
Leu-9 in MDMX. By contrast, the regular ?-helix in p53, starting
at Phe-19 and ending at Trp-23, is shortened by 1 residue. Two
(i, i ? 3) ?CAO???HN? H-bonds involving Trp-7-Leu-10 (3.0 Å)
and Asn-8-Ser-11 (3.3 Å) in PMI-synMDM2, or the Trp-7-Leu-10
H-bond (3.1 Å) in PMI-synMDMX, are missing in the helical turn
region of p53 (Fig. 3). Further, Ser-2 of PMI participates in a
more extensive and stronger H-bonding network than does
Thr-18 of p53. In addition to Ser-2 O-Glu-5 N and Ser-2 O-Tyr-6
N—two backbone H-bonds also found for Thr-18 of p53, PMI
possesses 2 additional main chain-side chain H-bonds, i.e., Ser-2
N-Glu-5 O?1(2.8 Å in MDM2 and 2.9 Å in MDMX) and Ser-2
O?-Glu-5 N (3.1 Å in both MDM2 and MDMX) (Fig. 3).
Optimally aligned, these two H-bonds likely contribute to the
conformational stability of PMI in the MDM2/MDMX complex.
By contrast, the topologically equivalent main chain-side chain
H-bonds involving Thr-18 of p53, i.e., Thr-18 N-Asp-21 O?2(3.5
Å) and Thr-18 O?1-Asp-21 N (3.6 Å) (25), appear too long to be
energetically significant. It is worth noting that a side chain-side
chain H-bond was thought to exist between Thr-18 O?1and
Asp-21 O?2as part of the Thr-18-Asp-21 H-bonding network
important for p53 stability (25). Similar interactions also exist
between Ser-2 O?and Glu-5 O?1in PMI-MDM2 and PMI-
Tyr-6 of PMI or Leu-22 of p53 makes van der Waals contacts
with Val-93 of MDM2 or Val-92 of MDMX. As reported in refs.
24 and 36, Tyr is strongly selected by phage display over a Leu
residue at the same position. This selection probably can be
rationalized by the tyrosyl side chain capable of making addi-
tional hydrophobic, ?-cation, and electrostatic interactions with
residues inside the PMI-binding pocket. Tyr-6 forms cation-?
interactions with Lys-94 of MDM2 or possibly Lys-93 of MDMX.
Further, the buried surface area of Tyr-6 of PMI is significantly
group of Tyr-6 participates in an elaborate, water-mediated
H-bonding network comprising the side chain(s) of Gln-72 and
Lys-94 of MDM2 or Gln-71 of MDMX (Fig. S4).
synMDM2-PMI Differs fromsynMDMX-PMI. The most profound struc-
tural difference between the 2 complexes centers on the 2
C-terminal residues (Ser-11 and Pro-12) of PMI in the vicinity
of Tyr-100/Tyr-99 (Fig. 4). In the
Pro-12 of PMI is fully disordered (refer to the electron density
map, Fig. S5), and the side chain of Ser-11 does not contribute
to MDM2 binding. This finding is consistent with the previous
observation that C-terminal residues flanking Leu-26 of p53
(equivalent to Leu-10 of PMI) do not make direct contact with
MDM2 (25). The aromatic ring of a protruding Tyr-100, H-
bonded to the carbonyl O of Leu-10 of PMI, appears to ‘‘squeeze
out’’ Ser-11 and Pro-12 to point away from the protein. By
contrast, structurally ordered Ser-11 and Pro-12 of PMI fit
snugly in thesynMDMX-PMI complex, where Tyr-99 recesses to
form an H-bond with Ser-11 N of the peptide.
The ?2? helix of MDMX moves outward in relation to the ?2?
helix of MDM2, coinciding with the C?atoms of His-96 of MDM2
and Pro-95 of MDMX moving apart by 2.4 Å. This change
(Left, green) and to MDMX (Right, yellow). In PMI, Ser-2 to Ala-4 make 3
regular (i, i ? 4) ?CAO???HN? H-bonds in the ?-helix. Ser-2 and residues from
also make 4 or 3 (i, i ? 3) ?CAO???HN? H-bonds. In addition, Ser-2 also forms
2 main chain-side chain, and 1 side chain-side chain H-bonds with Glu-5.
However, only the (energetically significant) H-bonds unique to PMI are
shown in dashes. (B) Superposition of PMI (green) and p53 bound to MDM2
(Left), and of PMI (yellow) and p53 bound to MDMX (Right). Residues 17–29
of p53 (gray) bound to human MDM2 (PDB entry 1YCR) and 17–27 of p53
(pink) bound to zebra fish MDMX (PDB entry 2Z5T) are shown.
Structures of PMI and p53 in bound state. (A) PMI bound to MDM2
Pazgier et al.PNAS ?
March 24, 2009 ?
vol. 106 ?
no. 12 ?
propagates to Tyr-99 of MDMX. A resultant conformational
adjustment surrounding Tyr-99, characterized by a shift of its C?
Met-53, Tyr-99 and Leu-102 (a BSA of Pro-12: 99 Å2). Notably,
Leu-10 of PMI is largely shielded from the bulk solvent by His-96
and Tyr-100 of MDM2 (a BSA of Leu-10: 122 Å2). However, it
helix region. Pro-12 interacting with its newly formed hydrophobic
cleft insynMDMX-PMI likely compensates, at least in part, for the
lost binding energy by a partially exposed Leu-10.
Inhibition of p53 interactions with the oncogenic proteins MDM2
and MDMX is of important therapeutic value in cancer treatment.
However, potent inhibitors active at low nanomolar concentrations
against both MDM2 and MDMX are nonexistent. Using phage
display coupled with chemical protein synthesis via native chemical
ligation (37, 38), we identified PMI—the strongest peptide ligand
ever reported for MDM2/MDMX. Further, high-resolution crystal
structures ofsynMDM2 andsynMDMX in complex with PMI have
been determined, unveiling not only the structural differences
between MDM2 and MDMX but also the molecular determinants
for high-affinity peptide inhibition of the 2 oncogenic proteins.
PMI binds MDM2 and MDMX similarly to p53 peptides. How-
ever, the improvement in binding affinity of PMI over(17–28)p53 by
2 orders of magnitude was surprising, partly because both peptides
contained the same hydrophobic triad, Phe/Trp/Leu, known to be
most critical for p53 transactivation and for MDM2 interactions
(32–34, 39). Structural analyses of the PMI complexes and of
p53-bound MDM2/MDMX structures suggest that an extensive,
tightened intramolecular H-bonding network found in PMI likely
plays an important role in stabilizing its more extended ?-helical
conformation in the complex, thus contributing to high-affinity
PMI binding to the oncoproteins. Central to the N-terminal H-
bonding network in PMI is Ser-2, which donates and accepts up to
5 H-bonds as judged by their geometry. Not surprisingly, mutation
of Ser-2 to Ala reduced the binding affinity of PMI for MDM2 by
1 order of magnitude, underscoring the importance of intramolec-
ular hydrogen bonding in PMI-MDM2/MDMX interactions. It has
been shown that Thr-18, highly conserved in p53 and important for
the structural findings, the results from ITC measurements showed
MDM2/MDMX came entirely from a favorable enthalpy change
counteracted by an unfavorable entropy change. A greater entropy
loss for PMI (Table S1) was indicative of a more stable peptide
conformation in the complex than(17–28)p53.
lecular H-bonding network seen in the MDM2 and MDMX
complexes likely result, at least in part, from the selection of Ser-11
(as opposed to Pro-27 in p53). Zondlo et al. recently showed that
mutation of the highly conserved Pro-27 in
increased its binding affinity for MDM2 by 50-fold (a decrease in
Kdfrom 229 to 4.7 nM) (40). The authors attributed the improve-
complex—a thesis largely confirmed in a molecular dynamic sim-
ulation study by Dastidar et al. (41).
One of the most interesting structural findings entails the C-
unique to MDMX. Pro-12 is the third most buried residue at the
PMI-MDMX interface with the second highest solvation energy
effect (Table S2). By contrast, residues flanking Leu-26 of p53 do
not make energetically meaningful contact with either human
Pro-12 in PMI, while exhibiting little effect on MDM2 binding,
weakened MDMX binding by 1 order of magnitude (Kdjumped
from 3.6 to 29 nM), indicative of the importance of Pro-12 in
of MDM2 and zebra fish MDMX (23, 25–29), the geometry of the
binding pocket for the C-terminal residues of p53 hinges on the
conformation of Tyr-100 of MDM2 or Tyr-99 of MDMX. The
orientation of Tyr-100 or Tyr-99 seen in different MDM2 and
MDMX complex structures (Fig. 5) is clearly determined not only
nature of the C-terminal residues of the ligand, supporting the
(12–30)p53 to Ser
(A) Electrostatic potential distribution (negative in red, positive in blue, and
PMI is shown in a ribbon and stick diagram. (B) Close-up view of the binding
pockets for Leu-10 and Pro-12 of PMI, where only the residues lining the
binding pockets are shown in sticks. The side chain of Tyr-100 of MDM2 is
H-bonded to Leu-10 O of PMI (Left), whereas Tyr-99 O?of MDMX forms an
H-bond with Ser-11 N of PMI (Right).
Structural differences in PMI binding between MDM2 and MDMX.
protein complexes: PMI-synMDM2, green; p53-MDM2, gray (PDB entry 1YCR);
PMI-synMDMX, yellow; and p53-MDMX, pink (PDB entry 1Z5T).
Conformational changes of Tyr-100/Tyr-99 seen in different peptide-
www.pnas.org?cgi?doi?10.1073?pnas.0900947106 Pazgier et al.
Finally, it is worth noting that PMI, despite its high binding
killing of p53?/?HCT116 cells (Fig. S6). The attenuated cytotoxic
activity in vitro may reflect a low intracellular concentration of
PMI, likely resulting from a combination of proteolytic degrada-
tion, inefficient cellular uptake, and endosomal sequestration (42).
are generally considered ‘‘undruggable’’ compared with traditional
low molecular weight compounds. Importantly, however, the dis-
covery of small molecule drugs depends on high-resolution crystal
ligands (22, 23). The information obtained from high-affinity
peptide inhibition of MDM2/MDMX is equally valuable for the
design of miniature proteins that potently activate the p53 pathway
and effectively kill p53?/?tumor cells in vitro (24, 43, 44). Our
structural work reported here should aid in silico library screening
and structure-based rational design of different classes of p53
activators for anticancer therapy.
resolution was reported (45). The complex structure is very similar
to zebra fish MDMX-(15–29)p53 used for comparison in our article.
Superposition of human MDMX-(15–29)p53 andsynMDMX-PMI
yielded a RMSD of 0.8 Å. As observed in zebra fish MDMX-
(15–29)p53 (29), the C-terminal residues of p53 (Pro-27 and Glu-28)
are loosely bound to human MDMX with a single stabilizing
H-bond between Tyr-99 O?and Pro-27 O. The hydrophobic cleft
for Pro-12 of PMI seen insynMDMX-PMI is not formed in the
human MDMX-(15–29)p53 complex.
(23–111)MDMX in complex with
(15–29)p53 at 1.9 Å
Materials and Methods
Phage Display. Ph.D.-12—a combinatorial library of random peptide 12-mers
fused, via a short spacer GlyGlyGlySer, to the N terminus of a minor coat protein
(pIII) of M13 phage—was purchased from New England Biolabs, Inc. The basic
procedures for library screening are as follows: (i) incubate input phage (10 ?L)
phage-target solution to 50 ?L of streptavidin-agarose resin (Pierce) for affinity
steps 1–4; (vi) sequence selected binding clones according to the procedures
recommended by the manufacturer.
Surface Plasmon Resonance (SPR) Spectroscopy. Competition binding kinetics
was carried out at 25 °C on a Biacore T100 SPR instrument, using a(15–29)p53-
immobilized CM5 sensor chip (17 RUs for MDM2 and 36 RUs for MDMX). The
buffer was 10 mM Hepes, 150 mM NaCl, 0.005% surfactant P20, pH 7.4. 50 nM
synMDM2 or 100 nMsynMDMX was incubated at room temperature for 30 min
for 2 min, followed by 4 min dissociation. The concentration of unboundsyn-
from a calibration curve established by RU measurements of different
concentrations ofsynMDM2 orsynMDMX injected alone. Nonlinear regres-
sion analysis was performed using GraphPad Prism 4 to give rise to Kd
values. Protein and peptide solutions were quantified by UV absorbance
measurements at 280 nm, using molar extinction coefficients calculated
from an algorithm published in ref. 46.
collection are described in SI Text. The structures of both complexes were solved
by molecular replacement, using Phaser (47) and search models based on the
previously solved and refined structures of(17–125)MDM2-(15–29)p53 (PDB entry
1YCR) and zebra fish(15–129)MDMX-(15–29)p53 (PDB entry 2Z5T) (25, 29). The
and rebuilt using the program COOT (49). Parameters for data collection and
results of refinement are summarized in Table S3. The atomic coordinates of
synMDM2-PMI (3EQS) andsynMDMX-PMI (3EQY) have been deposited in the
Protein Data Bank.
Note Added in Proof. Several new MDMX structures have been determined in
complexes with a single-domain antibody (PDB ID code 2VYR) (50), peptido-
mimetic inhibitors (PDB ID codes 3FE7 and 3FEA) (51), and pDI (PDB ID code
ACKNOWLEDGMENTS. This work was supported by American Cancer Society
Research Scholar Grant CDD112858, National Institutes of Health Grants
AI056264 and AI061482 (to W.L.), and the Intramural Research Program of the
National Institutes of Health (S.G.T.).
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