Structure of the human Mdmx protein bound to the p53 tumor suppressor transactivation domain.
ABSTRACT The Mdmx oncoprotein has only recently emerged as a critical-independent to Mdm2-regulator of p53 activation. We have determined the crystal structure of the N-terminal domain of human Mdmx bound to a 15-residue transactivation domain peptide of human p53. The structure shows why antagonists of the Mdm2 binding to p53 are ineffective in the Mdmx-p53 interaction.
- SourceAvailable from: Mu-Shui Dai[show abstract] [hide abstract]
ABSTRACT: Within the past decade, there has been a revolution in the types of drugs developed to treat cancer. Therapies that selectively target cancer-specific aberrations, such as kinase inhibitors, have made a dramatic impact on a subset of patients. In spite of these successes, there is still a dearth of treatment options for the vast majority of patients. Therefore, there is a need to design therapies with broader efficacy. The p53 tumor suppressor pathway is one of the most frequently altered in human cancers. However, about half of all cancers retain wild-type p53, yet through various mechanisms, the p53 pathway is otherwiseinactivated. Targeting this pathway forreactivation truly represents the "holy grail" in cancer treatment. Most commonly, destabilization of p53 by various components of ubiquitin-proteasome system, notably the ubiquitin ligase MDM2 and its partner MDMX as well as various deubiquitinating enzymes (DUBs), render p53 inert and unresponsive to stress signals. Reinstating its function in cancer has been a long sought-after goal. Towards this end, a great deal of work has been devoted to the development of compounds thateither interfere with the p53-MDM2 and p53-MDMX interactions, inhibit MDM2 E3 activity, or target individual DUBs. Here we review the current progress that has been made in the field, with a special emphasis on both MDM2 and DUB inhibitors. Developing inhibitors targeting the upstream of the p53 ubiquitination pathway will likely also be a valuable option.Current pharmaceutical design 11/2012; · 4.41 Impact Factor
Article: The MDM2-p53 pathway revisited.[show abstract] [hide abstract]
ABSTRACT: The p53 tumor suppressor is a key transcription factor regulating cellular pathways such as DNA repair, cell cycle, apoptosis, angiogenesis, and senescence. It acts as an important defense mechanism against cancer onset and progression, and is negatively regulated by interaction with the oncoprotein MDM2. In human cancers, the TP53 gene is frequently mutated or deleted, or the wild-type p53 function is inhibited by high levels of MDM2, leading to downregulation of tumor suppressive p53 pathways. Thus, the inhibition of MDM2-p53 interaction presents an appealing therapeutic strategy for the treatment of cancer. However, recent studies have revealed the MDM2-p53 interaction to be more complex involving multiple levels of regulation by numerous cellular proteins and epigenetic mechanisms, making it imperative to reexamine this intricate interplay from a holistic viewpoint. This review aims to highlight the multifaceted network of molecules regulating the MDM2-p53 axis to better understand the pathway and exploit it for anticancer therapy.Journal of biomedical research. 07/2013; 27(4):254-71.
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ABSTRACT: MDM2 and MDMX are homologous proteins that bind to p53 and regulate its activity. Both contain three folded domains and ∼70% intrinsically disordered regions. Previous detailed structural and biophysical studies have concentrated on the isolated folded domains. The N-terminal domains of both exhibit high affinity for the disordered N-terminal of p53 (p53TAD) and inhibit its transactivation function. Here, we have studied full-length MDMX and found a ∼100-fold weaker affinity for p53TAD than does its isolated N-terminal domain. We found from NMR spectroscopy and binding studies that MDMX (but not MDM2) contains a conserved, disordered self-inhibitory element that competes intramolecularly for binding with p53TAD. This motif, which we call the WWW element, is centered around residues Trp200 and Trp201. Deletion or mutation of the element increased binding affinity of MDMX to that of the isolated N-terminal domain level. The self-inhibition of MDMX implies a regulatory, allosteric mechanism of its activity. MDMX rests in a latent state in which its binding activity with p53TAD is masked by autoinhibition. Activation of MDMX would require binding to a regulatory protein. The inhibitory function of the WWW element may explain the oncogenic effects of an alternative splicing variant of MDMX that does not contain the WWW element and is found in some aggressive cancers.Proceedings of the National Academy of Sciences 10/2013; · 9.74 Impact Factor
©2008 LANDES BIOSCIENCE. DO NOT DISTRIBUTE.
(residues 15–29); the second is for the analogous complex involving
a longer p53 peptide, between residues 17–37, and a variant of
zebrafish Mdmx (residues 15–129) in which the p53 binding site has
[Cell Cycle 7:15, 2441-2443; 1 August 2008]; ©2008 Landes Bioscience
The Mdmx oncoprotein has only recently emerged as a critical—
independent to Mdm2—regulator of p53 activation. We have
determined the crystal structure of the N-terminal domain of human
Mdmx bound to a 15-residue transactivation domain peptide of
human p53. The structure shows why antagonists of the Mdm2
binding to p53 are ineffective in the Mdmx-p53 interaction.
The tumor suppressor p53 protein, “the guardian of the genome”,
has an overarching role in protecting the organism from cancer.1,2 In
order to escape the “safeguard” system mediated by p53 nearly all
human cancers have either mutated the p53 itself (50% all cancers) or
compromised the effectiveness of the p53 pathway.1-10 In tumors that
retain the wildtype p53, the p53 pathway is mostly inactivated by its
negative regulators, the Mdm2 and Mdmx proteins.1-10 The rescue of
the impaired p53 function by disrupting the Mdm2-p53/Mdmx-p53
interaction offers a fundamentally new principle for anticancer thera-
peutics.2-16 Three independent teams have very recently verified that
restoring the antitumor activity of p53 can indeed halt tumor growth in
a broad spectrum of cancers.6 Mdm2, the first to gain the distinction of
a principal antagonist of p53, promotes ubiquitination of p53 followed
by degradation in proteasome.2-16 Mdmx has only recently emerged
as a critical, independent regulator of p53 activation3-5,8,10 it strongly
inhibits p53 transactivation capacity, but not its stability.3-5,8,10
Several lead compounds have recently been reported to inhibit the
Mdm2-p53 interaction7,11-16—for example, nutlins, the most potent
drug-like compounds developed so far, induce apoptosis in p53 wild-
type cells and show in vivo efficacy in mice xenograft models.11-13
Regarding Mdmx, our own recent study17 agree with other reports that
nutlins do not inhibit Mdmx-p53 complexes.3-5,14
In this study, we determined two crystal structures of the p53-binding
domain of Mdmx in complex with peptides derived from the transactiva-
tion domain of human p53 (Fig. 1, Suppl. Fig. 1, Suppl. Table 1 and
Suppl. Methods online). The first structure is between the N-terminal
domain of human Mdm2 (residues 23–109) and the p53 peptide
been mutated to that of human Mdmx (L46V and V95L). These structures
show that although the principle features of the Mdm2-p53 interaction
are preserved in the Mdmx-p53 structures, the central hydrophobic cleft
of Mdmx on which the p53 peptide binds is smaller and differently
shaped than that of Mdm2. This is because the sidechains of Met53 and
Tyr99 in human Mdmx (and Met50 and Tyr96 in the zebrafish variant)
protrude into the binding pocket thus blocking a part of the cleft on its
opposite sides. The Met50 sidechain is in the same orientation as the
corresponding Leu46 of human Mdm2, but the larger Met50 sidechain
makes the p53-binding cleft smaller (Figs. 1A and 2). For Tyr99 (Tyr96
in zebrafish variant), the position of the aromatic ring is nearly identical
in all Mdmx molecules present in our structures (χ1 = -72.3 ± 2.5) (Suppl.
Fig. 2). We designate this configuration as the “closed conformation” of
Tyr99 (Tyr96). This conformation is different to that of the corresponding
Hdm2 Tyr100 in the structure of wt-p53/Hdm2 (χ1 = +197.7) (ref. 18).
In this Hdm2 structure the Tyr100 sidechain is flipped away from the
p53 binding pocket (the “open conformation of the Tyr).16 The PDB Data
Bank contains five additional structures of complexes of Mdm2 with
short peptidic analogues: 1T4F (ref. 14), 2GV2 (ref. 19), 2AXI (ref. 20)
and two small molecular weight compounds: nutlin (1RV1; ref. 18) and
a benzodiazepinedione derivative (1T4E; ref. 16). In all these cases
the Tyr100 ring collapses into the p53 packet assuming “the closed
conformation” (χ1 = -83.3 ± 3.2), thus filling the empty space that
was occupied by the p53 residue Pro27. However, Tyr100 in human
Mdm2’s never fully acquire the conformation seen in Mdmx structures.
Our present zebrafish variant of Mdmx includes a C-terminally extended
p53 peptide to check whether such an extension could flip the tyrosine
into the “open” conformation. This has not happened, and therefore we
believe that the “closed” Tyr99 arrangement is the intrinsic property of
Mdmx’s that most probably arises from the differences between Mdmx
and Mdm2 that are located in helix α2’ (Fig. 1). This helix is moved by
3.0 Å at Pro95 and 1.5 Å at Lys104 in Mdmx towards its C-terminus
and to the center of molecule compared to human Mdm2. It also starts
later, all because of the Pro95-Ser96-Pro97 sequence unique to Mdmx
(although with the φ,ψ angles still in the α-helical range). Significant
changes in α2’ start already at the Lys93 (human Mdm2, Lys94) and the
α2’ helices of Mdmx and Mdm2 get successively out of the register to
each other until Asn105 (human Mdm2: Asn106), without large single
differences in φ,ψ angles. Different position of helix α2’ additionally
changes the shape of the p53 binding pocket.
The primary contacts of the α-helical p53 peptide to Mdmx are
made by its hydrophobic Phe19, Trp23 and Leu26 that form together
an interface that is complementary to and fills up a hydrophobic pocket
of Mdmx (Fig. 1). When compared to previously solved structure of
zebrafish Mdmx complexed to the human p53 peptide, the structures
are similar with rmsd of 0.66 Å, however human Mdmx surrounds
peptide much tighter and overall fit of this two molecules is much better.
The surface area occupied by aminoacids 17–27 of the p53 peptide has
440 Å2 for human Mdmx and 407 Å2 for zebrafish Mdmx. This trend is
in agreement with the strength of the binding of the p53 peptide to these
proteins; the KD’s of the p53-Mdmx binding are 2.20 ± 0.30 μM and
0.21 ± 0.05 μM, for zebrafish and human Mdmx, respectively.15
The structures presented here show clearly that Mdmx must be
targeted independently from Mdm2 for drug development. The structure
of human Mdmx should provide a base for rational anticancer drug
design against the p53-Mdmx interaction.
Accession Codes. Protein data bank. Coordinates have been depos-
ited with accession codes 3DAB and 3DAC.
Supplementary materials can be found at:
*Correspondence to: Tad A. Holak; Max Planck Institute for Biochemistry; Martinsried
82152 Germany; Tel.: +49.89.8578.2673; Fax: +49.89.8578.3777; Email:
Submitted: 05/21/08; Accepted: 05/27/08
Previously published online as a Cell Cycle E-publication:
Letter to the Editor
Structure of the human Mdmx
protein bound to the p53 tumor
suppressor transactivation domain
Grzegorz M. Popowicz, Anna Czarna and Tad A. Holak*
Max Planck Institute for Biochemistry; Martinsried, Germany
Key words: Mdmx, Mdm4, p53, cancer, nutlin, structure
©2008 LANDES BIOSCIENCE. DO NOT DISTRIBUTE.
Figure 1. Structure of the Mdmx/p53 complex. (A) Stereoview of the complex of human Mdmx (dark-blue) and human p53 (light-blue). The p53 peptide
is shown additionally as a stick-plot. p53 residues important for binding are labeled. (B) Stereoplot of differences in binding mode between p53 peptides
(stick plot, light-blue for Mdmx, and light-gray for Mdm2) with part of helix α2’ of Mdmx (dark-blue) and Mdm2 (dark-grey).
2442Cell Cycle2008; Vol. 7 Issue 15
Structure of human Mdmx
©2008 LANDES BIOSCIENCE. DO NOT DISTRIBUTE.
Structure of human Mdmx
Figure 2. Surface representations of human Mdmx (the left side) and two human Mdm2 structures (the right side).
Significant differences in the shape of p53 binding pocket are clearly visible. Residues responsible for differences in the
binding groove are labeled.
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