Structure of free MDM2 N-terminal domain reveals conformational adjustments that accompany p53-binding

Critical to the inhibitory action of the oncogene product, MDM2, on the tumour suppressor, p53, is association of the N-terminal domain of MDM2 (MDM2N) with the transactivation domain of p53. The structure of MDM2N was previously solved with a p53-derived peptide, or small-molecule ligands, occupying its binding cleft, but no structure of the non-liganded MDM2N (i.e. the apo-form) has been reported. Here, we describe the solution structure and dynamics of apo-MDM2N and thus reveal the nature of the conformational changes in MDM2N that accompany binding of p53. The new structure suggests that p53 effects displacement of an N-terminal segment of apo-MDM2N that occludes access to the shallow end of the p53-binding cleft. MDM2N must also undergo an expansion upon binding, achieved through a rearrangement of its two pseudosymetrically related sub-domains resulting in outward displacements of the secondary structural elements that comprise the walls and floor of the p53-binding cleft. MDM2N becomes more rigid and stable upon binding p53. Conformational plasticity of the binding cleft of apo-MDM2N could allow the parent protein to bind specifically to several different partners, although, to date, all the known liganded structures of MDM2N are highly similar to one another. The results indicate that the more open conformation of the binding cleft of MDM2N observed in structures of complexes with small molecules and peptides is a more suitable one for ligand discovery and optimisation.     

The MDM2 RING Domain and Central Acidic Domain Play Distinct Roles in MDM2 Protein Homodimerization and MDM2-MDMX Protein Heterodimerization

The oncoprotein murine double minute 2 (MDM2) is an E3 ligase that plays a prominent role in p53 suppression by promoting its polyubiquitination and proteasomal degradation. In its active form, MDM2 forms homodimers as well as heterodimers with the homologous protein murine double minute 4 (MDMX), both of which are thought to occur through their respective C-terminal RING (really interesting new gene) domains. In this study, using multiple MDM2 mutants, we show evidence suggesting that MDM2 homo- and heterodimerization occur through distinct mechanisms because MDM2 RING domain mutations that inhibit MDM2 interaction with MDMX do not affect MDM2 interaction with WT MDM2. Intriguingly, deletion of a portion of the MDM2 central acidic domain selectively inhibits interaction with MDM2 while leaving intact the ability of MDM2 to interact with MDMX and to ubiquitinate p53. Further analysis of an MDM2 C-terminal deletion mutant reveals that the C-terminal residues of MDM2 are required for both MDM2 and MDMX interaction. Collectively, our results suggest a model in which MDM2-MDMX heterodimerization requires the extreme C terminus and proper RING domain structure of MDM2, whereas MDM2 homodimerization requires the extreme C terminus and the central acidic domain of MDM2, suggesting that MDM2 homo- and heterodimers utilize distinct MDM2 domains. Our study is the first to report mutations capable of separating MDM2 homo- and heterodimerization.     

Identification of a Catalytic Active but Non-Aggregating MDM2 RING Domain Variant

As a key regulator of the tumour suppressor protein p53, MDM2 is involved in various types of cancer and has thus been an attractive drug target. So far, small molecule design has primarily focussed on the N-terminal p53-binding domain although on-target toxicity effects have been reported. Targeting the catalytic RING domain of MDM2 resembles an alternative approach to drug MDM2 with the idea to prevent MDM2-mediated ubiquitination of p53 while retaining MDM2′s ability to bind p53. The design of RING inhibitors has been limited by the extensive aggregation tendency of the RING domain, making it challenging to undertake co-crystallization attempts with potential inhibitors. Here we compare the purification profiles of the MDM2 RING domain from several species and show that the MDM2 RING domain of other species than human is much less prone to aggregate although the overall structure of the RING domain is conserved. Through sequence comparison and mutagenesis analyses, we identify a single point mutation, G443T, which greatly enhances the dimeric fraction of human MDM2 RING domain during purification. Neither does the mutation alter the structure of the RING domain, nor does it affect E2(UbcH5B)–Ub binding and activity. Hence, MDM2-G443T facilitates studies involving binding partners that would be hampered by the low solubility of the wild-type RING domain. Furthermore, it will be valuable for the development of MDM2 RING inhibitors.     

Structural basis of competitive recognition of p53 and MDM2 by HAUSP/USP7: implications for the regulation of the p53-MDM2 pathway

Herpesvirus-associated ubiquitin-specific protease (HAUSP, also known as USP7), a deubiquitylating enzyme of the ubiquitin-specific processing protease family, specifically deubiquitylates both p53 and MDM2, hence playing an important yet enigmatic role in the p53–MDM2 pathway. Here we demonstrate that both p53 and MDM2 specifically recognize the N-terminal tumor necrosis factor–receptor associated factor (TRAF)–like domain of HAUSP in a mutually exclusive manner. HAUSP preferentially forms a stable HAUSP–MDM2 complex even in the presence of excess p53. The HAUSP-binding elements were mapped to a peptide fragment in the carboxy-terminus of p53 and to a short-peptide region preceding the acidic domain of MDM2. The crystal structures of the HAUSP TRAF-like domain in complex with p53 and MDM2 peptides, determined at 2.3-Å and 1.7-Å resolutions, respectively, reveal that the MDM2 peptide recognizes the same surface groove in HAUSP as that recognized by p53 but mediates more extensive interactions. Structural comparison led to the identification of a consensus peptide-recognition sequence by HAUSP. These results, together with the structure of a combined substrate-binding-and-deubiquitylation domain of HAUSP, provide important insights into regulation of the p53–MDM2 pathway by HAUSP.     

Structure of the MDM2/MDMX RING domain heterodimer reveals dimerization is required for their ubiquitylation in trans

MDM2, a ubiquitin E3-ligase of the RING family, has a key role in regulating p53 abundance. During normal non-stress conditions p53 is targeted for degradation by MDM2. MDM2 can also target itself and MDMX for degradation. MDMX is closely related to MDM2 but the RING domain of MDMX does not possess intrinsic E3-ligase activity. Instead, MDMX regulates p53 abundance by modulating the levels and activity of MDM2. Dimerization, mediated by the conserved C-terminal RING domains of both MDM2 and MDMX, is critical to this activity. Here we report the crystal structure of the MDM2/MDMX RING domain heterodimer and map residues required for functional interaction with the E2 (UbcH5b). In both MDM2 and MDMX residues C-terminal to the RING domain have a key role in dimer formation. In addition we show that these residues are part of an extended surface that is essential for ubiquitylation in trans. This study provides a molecular basis for understanding how heterodimer formation leads to stabilization of MDM2, yet degradation of p53, and suggests novel targets for therapeutic intervention.     

Structural Basis of Competitive Recognition of p53 and MDM2 by HAUSP/USP7: Implications for the Regulation of the p53–MDM2 Pathway

Herpesvirus-associated ubiquitin-specific protease (HAUSP, also known as USP7), a deubiquitylating enzyme of the ubiquitin-specific processing protease family, specifically deubiquitylates both p53 and MDM2, hence playing an important yet enigmatic role in the p53–MDM2 pathway. Here we demonstrate that both p53 and MDM2 specifically recognize the N-terminal tumor necrosis factor–receptor associated factor (TRAF)–like domain of HAUSP in a mutually exclusive manner. HAUSP preferentially forms a stable HAUSP–MDM2 complex even in the presence of excess p53. The HAUSP-binding elements were mapped to a peptide fragment in the carboxy-terminus of p53 and to a short-peptide region preceding the acidic domain of MDM2. The crystal structures of the HAUSP TRAF-like domain in complex with p53 and MDM2 peptides, determined at 2.3-Å and 1.7-Å resolutions, respectively, reveal that the MDM2 peptide recognizes the same surface groove in HAUSP as that recognized by p53 but mediates more extensive interactions. Structural comparison led to the identification of a consensus peptide-recognition sequence by HAUSP. These results, together with the structure of a combined substrate-binding-and-deubiquitylation domain of HAUSP, provide important insights into regulation of the p53–MDM2 pathway by HAUSP.     

Regulation of MDM2 E3 ligase activity by phosphorylation after DNA damage

MDM2 is a major regulator of p53 by acting as a ubiquitin E3 ligase. The central acidic domain and C-terminal RING domain of MDM2 are both indispensable for ubiquitination of p53. Our previous study suggested that ATM phosphorylation of MDM2 near the C terminus inhibits RING domain oligomerization, resulting in p53 stabilization after DNA damage. We present here evidence that these modifications allosterically regulate the functions of both acidic domain and RING domain of MDM2. Using chemical cross-linking, we show that the MDM2 RING domain forms oligomers including dimer and higher-order complexes in vivo. RING domain dimerization efficiency is negatively regulated by upstream sequence. ATM-mediated phosphorylation of the upstream sequence further inhibits RING dimerization. Forced oligomerization of MDM2 partially overcomes the inhibitory effect of phosphorylation and stimulates p53 ubiquitination. Furthermore, the ability of MDM2 acidic domain to bind p53 core domain and induce p53 misfolding are also suppressed by the same C-terminal ATM sites after DNA damage. These results suggest that the acidic domain and RING domain of MDM2 are both allosterically coupled to the intervening ATM sites, which enables the same modification to regulate multiple MDM2 functions critical for p53 ubiquitination.     

人EEN蛋白的结构预测和功能分析

采用基于神经网络的算法预测了我们自行克隆的新的白血病相关蛋白ＥＥＮ（ｅｘｔｒａ ｅｌｅｖｅｎｎｉｎｅｔｅｅｎ, ＥＥＮ）全长分子的二级结构,结果表明：ＥＥＮ 蛋白可能有三个结构域,Ｎ 端由三段α螺旋和短β折叠组成,中间为四段α螺旋组成的四螺旋结构,Ｃ端为ＳＨ３结构域,类似于在受体酪氨酸激酶信号传导途径中起重要作用的ＳＥＭ－５／ＧＲＢ２ Ｃ端ＳＨ３结构域;利用同源蛋白结构模拟的方法,模拟了ＥＥＮ ＳＨ３结构域的三维结构,结果表明：ＥＥＮ ＳＨ３结构域与ＳＥＭ－５／ＧＲＢ２ ＳＨ３结构域具有相近的结构,构成脯氨酸结合区的氨基酸非常保守．上述结果提示：ＥＥＮ 蛋白可能为新的信号蛋白,可能涉及新的信号传导途径或新的信号传导旁路,ＳＨ３结构域是其功能区域．     

Dual-site regulation of MDM2 E3-ubiquitin ligase activity

The control of p53 ubiquitination by MDM2 provides a model system to define how an E3-ligase functions on a conformationally flexible substrate. The mechanism of MDM2-mediated ubiquitination of p53 has been analyzed by deconstructing, in vitro, the MDM2-dependent ubiquitination reaction. Surprisingly, ligands binding to the hydrophobic cleft of MDM2 do not inhibit its E3-ligase function. However, peptides from within the DNA binding domain of p53 that bind the acid domain of MDM2 inhibit ubiquitination of p53, localizing a motif that harbors a key ubiquitination signal. The binding of ligands to the N-terminal hydrophobic cleft of MDM2 reactivates, in vitro and in vivo, MDM2-catalyzed ubiquitination of p53F19A, a mutant p53 normally refractory to MDM2-catalyzed ubiquitination. We propose a model in which the interaction between the p53-BOX-I domain and the N terminus of MDM2 promotes conformational changes in MDM2 that stabilize acid-domain interactions with a ubiquitination signal in the DNA binding domain of the p53 tetramer.     

Why is F19Ap53 unable to bind MDM2? Simulations suggest crack propagation modulates binding

Why doesn’t the F19A mutant of p53 bind to MDM2? Binding thermodynamics have suggested that the loss of packing interactions upon mutating Phe into Ala sidechain results in destabilizing the binding free energy between p53 and MDM2. Does this mutation also modulate the initial recognition between p53 and MDM2? We look at atomistic computer simulations of the process of the initial encounter between wild type p53 peptide and its F19A mutant with the N-terminal domain of MDM2. These simulations show that binding is characterized by a complex multistep process. It starts with the capture of F19 of wild type p53 by certain residues in the MDM2 binding pocket. This initial step anchors the peptide onto the surface of MDM2, and with the consequent reduction in the search space of the peptide, the peptide docks into the partially occluded surface of MDM2. This is similar to a crack forming in an otherwise occluded hydrophobic cavity in MDM2, and the peptide, docked through F19, modulates the propagation of this crack, which subsequently results in the stepwise docking of the rest of the peptide through insertions of W23 and L26. The lack of the bulky sidechain of F in the F19A mutant results in the absence of the initial “grasp” complex, and hence the mutant peptide diffuses randomly on the surface of MDM2 without binding. This is the first such demonstration of the possibility that a “kinetic” effect may partly underlie the destabilized thermodynamics of binding of F19A and is a feature that appears to be conserved in evolution. The observations by Wallace et al. (Mol Cell 2006; 23:251–63) that despite the inability of F19A to bind at the N-terminal domain of MDM2, it gets ubiquitinated, can now be partly understood based on a mechanism whereby the occupation of the binding pocket by ligands/peptides induces, via crack propagation and the dynamics of gatekeeper Y100, the ubiquitination signal for interactions between the acidic domain of MDM2 and the DNA binding domain of p53.     

MDM2 protein-mediated ubiquitination of numb protein: identification of a second physiological substrate of MDM2 that employs a dual-site docking mechanism

The E3 ubiquitin ligase, MDM2, uses a dual-site mechanism to ubiquitinate and degrade the tumor suppressor protein p53, involving interactions with the N-terminal hydrophobic pocket and the acidic domain of MDM2. The results presented here demonstrate that MDM2 also uses this same dual-site mechanism to bind to the cell fate determinant NUMB with both the N-terminal hydrophobic pocket and the acidic domain of MDM2 also involved in forming the interaction with NUMB. Furthermore, the acidic domain interactions are crucial for MDM2-mediated ubiquitination of NUMB. Contrary to p53, where two separate domains form the interface with MDM2, only one region within the phosphotyrosine binding domain of NUMB (amino acids 113-148) mediates binding to both these regions of MDM2. By binding to both domains on MDM2, NUMB disrupts the MDM2-p53 complex and MDM2-catalyzed ubiquitination of p53. Therefore, we have identified the mechanism NUMB uses to regulate the steady-state levels of the p53 in cells. By targeting the acidic domain of MDM2 using acid domain-binding ligands we can overcome MDM2-mediated ubiquitination and degradation of NUMB impacting on the stabilization of p53 in cells. Furthermore, delivery of MDM2 acid domain-binding ligands to cancer cells promotes p53-dependent growth arrest and the induction of apoptosis. This highlights the dual-site mechanism of MDM2 on another physiological substrate and identifies the acid domain as well as N terminus as a potential target for small molecules that inhibit MDM2.     

The tumor suppressor p53 and the oncoprotein simian virus 40 T antigen bind to overlapping domains on the MDM2 protein.

The oncogene mdm2 has been found to be amplified in human sarcomas, and the gene product binds to the tumor suppressor p53. In this report, we describe the dissection of the MDM2-binding domain on p53 as well as the p53-binding domain on MDM2. We also demonstrate that the oncoprotein simian virus 40 T antigen binds to the product of cellular oncogene mdm2. We have constructed several N- and C-terminal deletion mutants of p53 and MDM2, expressed them in vitro, and assayed their in vitro association capability. The N-terminal boundary of the p53-binding domain on MDM2 is between amino acids 1 and 58, while the C-terminal boundary is between amino acids 221 and 155. T antigen binds to an overlapping domain on the MDM2 protein. On the other hand, the MDM2-binding domain of p53 is defined by amino acids 1 and 159 at the N terminus. At the C terminus, binding is progressively reduced as amino acids 327 to 145 are deleted. We determined the effect of human MDM2 on the transactivation ability of wild-type human p53 in the Saos-2 osteosarcoma cell line, which does not have any endogenous p53. Human MDM2 inhibited the ability of human p53 to transactivate the promoter with p53-binding sites. Thus, human MDM2 protein, like the murine protein, can inactivate the transactivation ability of human p53. Interestingly, both the transactivation domain and the MDM2-binding domain of p53 are situated near the N terminus. We further show that deletion of the N-terminal 58 amino acids of MDM2, which eliminates p53 binding, also abolishes the capability of inactivating p53-mediated transactivation. This finding suggests a correlation of in vitro p53-MDM2 binding with MDM2's ability in vivo to interfere with p53-mediated transactivation.