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; and cStructural 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- ligase activity (10). Structurally related to MDM2, MDMX of tivity and stability of the tumor suppressor protein p53—a cellular 490-aa residues possesses domain structures arranged similarly process initiated by MDM2 and/or MDMX binding to the N- to MDM2, except that MDMX lacks ubiquitin-ligase function terminal transactivation domain of p53. MDM2 and MDMX in many (11, 12). Growing evidence supports that in unstressed cells tumors confer p53 inactivation and tumor survival, and are impor- MDM2 primarily controls p53 stability through ubiquitylation to tant molecular targets for anticancer therapy. We screened a target the tumor suppressor protein for constitutive degradation duodecimal peptide phage library against site-specifically biotin- by the proteasome (13, 14), whereas MDMX mainly functions as ylated p53-binding domains of human MDM2 and MDMX chemi- a significant p53 transcriptional antagonist independently of cally synthesized via native chemical ligation, and identified sev- MDM2 (15, 16). Under stress conditions, MDM2 and MDMX eral peptide inhibitors of the p53-MDM2/MDMX interactions. The cooperate to activate p53 through mechanisms involving both most potent inhibitor (TSFAEYWNLLSP), termed PMI, bound to MDM2 autodegradation (autoubiquitylation) and MDM2- MDM2 and MDMX at low nanomolar affinities—approximately 2 depedent degradation of MDMX (17–20). orders of magnitude stronger than the wild-type p53 peptide of In many tumors, p53 is present in its wild-type form. The the same length (ETFSDLWKLLPE). We solved the crystal structures presence of wild-type p53 strongly correlates to amplification of synthetic MDM2 and MDMX, both in complex with PMI, at 1.6 and/or over-expression of MDM2/MDMX, resulting directly in Å resolution. Comparative structural analysis identified an exten- p53 suppression and malignant progression (8, 9). Inhibition of sive, tightened intramolecular H-bonding network in bound PMI the p53-MDM2 interactions by MDM2 antagonists has been that contributed to its conformational stability, thus enhanced shown both in vitro and in vivo to reactivate the p53 pathway and binding to the 2 oncogenic proteins. Importantly, the C-terminal selectively kill tumor cells in a p53-dependent manner. Acting residue Pro of PMI induced formation of a hydrophobic cleft in synergistically in tumor cells, MDM2 and MDMX have become MDMX previously unseen in the structures of p53-bound MDM2 or 2 of the most attractive molecular targets for anticancer therapy. MDMX. Our findings deciphered the structural basis for high- Toward this end, much of the current efforts have been focused affinity peptide inhibition of p53 interactions with MDM2 and on combinatorial library search for and structure-based rational MDMX, shedding new light on structure-based rational design of design of low molecular weight inhibitors that target the N- different classes of p53 activators for potential therapeutic use. terminal p53-binding domains of MDM2 and MDMX (21). Successful examples include, but are not limited to, cis- 53 is best known as a tumor suppressor that transcriptionally imidazoline analogs termed Nutlins and, more recently, a spiro- pregulates, in response to cellular stresses such as DNA oxindole-derived compound termed MI-219 (22, 23). damage or oncogene activation, the expression of various target Peptides, because of their large interacting surfaces, offer the genes that mediate cell-cycle arrest, DNA repair, senescence or prospect of enhanced potency, high specificity and low toxicity. apoptosis—all of these cellular responses are designed to prevent However, most of the peptidic and peptidomimetic inhibitors damaged cells from proliferating and passing mutations on to the examined to date bind MDM2 at affinities ranging from high next generation (1–3). In 50% of human cancers, p53 is defective nanomolar to low micromolar concentrations, and none is nearly due usually to somatic mutations or deletions primarily in its as effective as Nutlins and MI-219 in tumor killing in vitro (21). DNA-binding domain and, to a lesser extent, to posttranslational Further, because the structural basis for MDMX inhibition is modifications such as phosphorylation, acetylation and methyl- much less understood than that for MDM2 inhibition, antago- ation that affect p53 function and stability. Altered p53 fails to nists designed for MDM2 are, in general, significantly less regulate growth arrest and cell death upon DNA damage, inhibitory toward MDMX. Potent peptide inhibitors against BIOCHEMISTRY directly contributing to tumor development, malignant progres- MDM2 and/or MDMX are needed as important cellular probes sion, poor prognosis and resistance to treatment (4). Conversely, of the p53 pathway in cancer biology and as useful templates for restoring endogenous p53 activity can halt the growth of can- structure-based rational design of different classes of p53 acti- cerous tumors in vivo by inducing apoptosis, senescence, and vators for potential therapeutic use. Here, we report identifica- innate inflammatory responses (5–7). tion and functional and structural characterizations of a high- As p53 mediates growth arrest and apoptosis, it is essential to keep its activity in check during normal development (2). Author contributions: M.P., M.L., and W.L. designed research; M.P., M.L., G.Z., W.Y., Multiple mechanisms exist to negatively regulate p53 activity, Changqing Li, Chong Li, J.L., J.M., D.Z., and S.G.T. performed research; M.P., D.Z., S.G.T., and among which the E3 ubiquitin ligase MDM2 and its homolog W.L. analyzed data; and M.P. and W.L. wrote the paper. MDMX (also known as MDM4) play a central regulatory role in The authors declare no conflict of interest. the developing embryo and in mature differentiated cells (8, 9). Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, MDM2 consists of 491-aa residues, comprising an N-terminal www.pdb.org (PDB ID codes 3EQS and 3EQY). p53-binding domain, a central domain preceded by nuclear 1M.P. and M.L. contributed equally to this work. export and localization signals essential for nuclear-cytoplasmic 2To whom correspondence should be addressed. E-mail: [email protected]. trafficking of MDM2, a zinc finger domain, and a C-terminal This article contains supporting information online at www.pnas.org/cgi/content/full/ zinc-dependent RING finger domain that confers E3 ubiquitin 0900947106/DCSupplemental. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0900947106 PNAS ͉ March 24, 2009 ͉ vol. 106 ͉ no. 12 ͉ 4665–4670 Downloaded by guest on September 30, 2021 Fig. 1. Quantification of the interactions of synMDM2 (50 nM) and synMDMX (100 nM) with varying concentrations of PMI, pDI, (15–29)p53, (17–28)p53, or Nutlin-3 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 for synMDMX 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 for synMDM2-Nutlin-3 and synMDMX-pDI. affinity peptide inhibitor, termed PMI (p53-MDM2/MDMX regression analyses yielded for PMI a Kd value of 3.4 nM for inhibitor), of p53 interactions with both MDM2 and MDMX. synMDM2 and of 4.2 nM for synMDMX. By contrast, (15–29)p53 bound to synMDM2 and synMDMX at affinities of 140 nM and Results 270 nM, respectively. Although Nutlin-3 showed little binding to PMI—a Potent Inhibitor of the p53-MDM2/MDMX Interactions Se- MDMX, its Kd value of 263 nM for MDM2 was largely in line lected from a Phage Displayed Peptide Library. The p53-binding with the published result (IC50: 90 nM for Nutlin-3a and 13.6 ␮M domains of MDM2 and MDMX (25–109MDM2 and for Nutlin-3b) (23). Importantly, compared with (17–28)p53, PMI 24–108MDMX, referred to thereafter as synMDM2 and bound to synMDM2 135-fold stronger and to synMDMX 156-fold synMDMX) and their site-specifically biotinylated forms were tighter. Our data suggest that PMI, with low nanomolar affinities chemically synthesized using native chemical ligation (SI Text). for both MDM2 and MDMX, is one of the strongest peptidic Using biotin-synMDM2 and biotin-synMDMX as bait, we inhibitors of the p53-MDM2/MDMX interactions ever reported. screened a duodecimal peptide library displayed on M13 phage. Shown in Fig. S1 are the amino acid sequences from 15 binding PMI Binds to MDM2 and MDMX in a Canonical Mode Previously clones obtained after 4 rounds of selection. Two consensus Described for p53 Peptides. The overall structures of human sequences emerged for both MDM2 and MDMX: LTFEHY- synMDM2 and synMDMX are similar, as evidenced by a root WAQLTS, also termed pDI (24), and PMI–TSFAEYWNLLSP. mean square deviation (rmsd) of 1.2 Å between superimposed The 3 most critical residues involved in p53-MDM2/MDMX C␣ atoms. synMDM2 and synMDMX share the basic structural recognition, i.e., Phe-19, Trp-23 and Leu-26 (p53 numbering), elements and conserved global fold reported for human MDM2 were all present in the phage-selected consensus sequences. pDI (23, 25–28) and zebra fish MDMX (29).