Binding of the C-terminal sterile alpha motif (SAM) domain of human p73 to lipid membranes

The alpha splice variant of p73 (p73alpha), a homologue of the tumor suppressor p53, has close to its C terminus a sterile alpha motif (SAM), SAMp73, that is thought to be involved in protein-protein interactions. Here, we report the lipid binding properties of this domain. Binding was assayed against zwitterionic (phosphatidylcholine) and anionic (phosphatidic acid) lipids and was studied by different biophysical techniques, namely, circular dichroism and fluorescence spectroscopies and differential scanning calorimetry. These techniques unambiguously indicate that SAMp73 binds to lipids. The binding involves protein surface attachment and partial membrane penetration, accompanied by changes in SAMp73 structure.     

Structure of the p53 transactivation domain in complex with the nuclear receptor coactivator binding domain of CREB binding protein

The activity and stability of the tumor suppressor p53 are regulated by interactions with key cellular proteins such as MDM2 and CBP/p300. The transactivation domain (TAD) of p53 contains two subdomains (AD1 and AD2) and interacts directly with the N-terminal domain of MDM2 and with several domains of CBP/p300. Here we report the NMR structure of the full-length p53 TAD in complex with the nuclear coactivator binding domain (NCBD) of CBP. Both the p53 TAD and NCBD are intrinsically disordered and fold synergistically upon binding, as evidenced by the observed increase in helicity and increased level of dispersion of the amide proton resonances. The p53 TAD folds to form a pair of helices (denoted Pα1 and Pα2), which extend from Phe19 to Leu25 and from Pro47 to Trp53, respectively. In the complex, the NCBD forms a bundle of three helices (Cα1, residues 2066-2075; Cα2, residues 2081-2092; and Cα3, residues 2095-2105) with a hydrophobic groove into which p53 helices Pα1 and Pα2 dock. The polypeptide chain between the p53 helices remains flexible and makes no detectable intermolecular contacts with the NCBD. Complex formation is driven largely by hydrophobic contacts that form a stable intermolecular hydrophobic core. A salt bridge between D49 of p53 and R2105 of NCBD may contribute to the binding specificity. The structure provides the first insights into simultaneous binding of the AD1 and AD2 motifs to a target protein.     

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.     

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.     

Identification of p53 Sequence Elements That Are Required for MDM2-Mediated Nuclear Export

It has been demonstrated that MDM2 can differentially regulate subcellular distribution of p53 and its close structural homologue p73. In contrast to MDM2-mediated p53 nuclear export, p73 accumulates in the nucleus as aggregates that colocalize with MDM2. Distinct distribution patterns of p53 and p73 suggest the existence of unique structural elements in the two homologues that determine their MDM2-mediated relocalization in the cell. Using a series of p53/p73 chimeric proteins, we demonstrate that three regions of p53 are involved in the regulation of MDM2-mediated nuclear export. The DNA binding domain (DBD) is involved in the maintenance of a proper conformation that is required for functional activity of the nuclear export sequence (NES) of p53. The extreme C terminus of p53 harbors several lysine residues whose ubiquitination by MDM2 appears to be the initial event in p53 nuclear export, as evidenced by the impaired nucleocytoplasmic shuttling of p53 mutants bearing simultaneous substitutions of lysines 370, 372, 373, 381, 382, and 386 to arginines (6KR) or alanines (6KA). Finally, the region between the DBD and the oligomerization domain of p53, specifically lysine 305, also plays a critical role in fully revealing p53NES. We conclude that MDM2-mediated nuclear export of p53 depends on a series of ubiquitination-induced conformational changes in the p53 molecule that lead to the activation of p53NES. In addition, we demonstrate that the p53NES may be activated without necessarily disrupting the p53 tetramer.     

Over-expression of the human MDM2 p53 binding domain by fusion to a p53 transactivation peptide

MDM2 binds to the tumor suppressor protein p53 and regulates the level of p53 in cells. Although it is possible to prepare a small amount of the region of MDM2 that binds to p53, the expression level of this fragment of MDM2 is relatively low, limiting the studies involving this protein. Here, we describe a construct for the optimized bacterial expression and purification of the MDM2 p53 binding domain. We found that the expression level of the soluble MDM2 p53 binding domain in bacteria was increased dramatically by fusing it to its interaction partner, the p53 transactivation peptide. Attachment of the p53 transactivation peptide (residues 17-29) to the N-terminus of MDM2 resulted in a more than 200-fold increase of soluble protein expression of the p53 binding domain in bacteria. To obtain the final MDM2 p53 binding domain (residues 5-109) we inserted a tobacco etch virus protease recognition site between the P53 peptide and the MDM2 p53 binding domain. To weaken the protein/peptide interaction and facilitate the separation of the protein from the complex, we introduced a point mutation of one of the key interaction residues (F19A or W23A) in the p53 peptide. The advantages of our new construct are high yield and easy purification of the MDM2 protein.     

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.     

Systematic Mutational Analysis of Peptide Inhibition of the p53-MDM2/MDMX Interactions

Inhibition of the interaction between the tumor suppressor protein p53 and its negative regulators MDM2 and MDMX is of great interest in cancer biology and drug design. We previously reported a potent duodecimal peptide inhibitor, termed PMI (TSFAEYWNLLSP), of the p53-MDM2 and -MDMX interactions. PMI competes with p53 for MDM2 and MDMX binding at an affinity roughly 2 orders of magnitude higher than that of ^17-28p53 (ETFSDLWKLLPE) of the same length; both peptides adopt nearly identical α-helical conformations in the complexes, where the three highlighted hydrophobic residues Phe, Trp, and Leu dominate PMI or ^17-28p53 binding to MDM2 and MDMX. To elucidate the molecular determinants for PMI activity and specificity, we performed a systematic Ala scanning mutational analysis of PMI and ^17-28p53. The binding affinities for MDM2 and MDMX of a total of 35 peptides including 10 truncation analogs were quantified, affording a complete dissection of energetic contributions of individual residues of PMI and ^17-28p53 to MDM2 and MDMX association. Importantly, the N8A mutation turned PMI into the most potent dual-specific antagonist of MDM2 and MDMX reported to date, registering respective K_d values of 490 pM and 2.4 nM. The co-crystal structure of N8A-PMI-^25-109MDM2 was determined at 1.95 Å, affirming that high-affinity peptide binding to MDM2/MDMX necessitates, in addition to optimized intermolecular interactions, enhanced helix stability or propensity contributed by non-contact residues. The powerful empirical binding data and crystal structures present a unique opportunity for computational studies of peptide inhibition of the p53-MDM2/MDMX interactions.     

MDM2 can promote the ubiquitination, nuclear export, and degradation of p53 in the absence of direct binding

MDM2 can bind the N terminus of p53 and promote its ubiquitination and export from the nucleus to the cytoplasm, where p53 can then be degraded by cytoplasmic proteasomes. Several studies have reported that an intact MDM2 binding domain is necessary for p53 to be targeted for ubiquitination, nuclear export, and degradation by MDM2. In the current study, we examined whether the MDM2 binding domain of p53 could be provided in trans through oligomerization between two p53 molecules. p53 proteins mutated in their MDM2 binding domains were unable to bind MDM2 directly and were resistant to MDM2-mediated ubiquitination, nuclear export, and degradation when expressed with MDM2 alone. However, these same p53 mutants formed a complex with MDM2 and were efficiently ubiquitinated, exported from the nucleus, and degraded when co-expressed with MDM2 and wild-type p53. Moreover, this effect required MDM2 binding by wild-type p53 as well as oligomerization between wild-type p53 and the MDM2 binding-deficient p53 mutants. Taken together, these results support a model whereby MDM2 binding-deficient forms of p53 can bind MDM2 indirectly through oligomerization with wild-type p53 and are subsequently targeted for ubiquitination, nuclear export, and degradation. These findings may have important implications regarding the DNA damage response of p53.     

MDM4 binds ligands via a mechanism in which disordered regions become structured

MDM2 and MDM4 are proteins involved in regulating the tumour suppressor p53. MDM2/4 and p53 interact through their N-terminal domains and disrupting this interaction is a potential anticancer strategy. The MDM2-p53 interaction is structurally and biophysically well characterised, whereas equivalent studies on MDM4 are hampered by aggregation of the protein. Here we present the NMR characterization of MDM4 (14-111) both free and in complexes with peptide and small-molecule ligands. MDM4 is more dynamic in its apo state than is MDM2, with parts of the protein being unstructured. These regions become structured upon binding of a ligand. MDM4 appears to bind its ligand through conformational selection and/or an induced fit mechanism; this might influence rational design of MDM4 inhibitors.

Structured summary

MINT-7896835: p53 (uniprotkb:P04637) and MDM4 (uniprotkb:O15151) bind (MI:0407) by isothermal titration calorimetry (MI:0065)MINT-7896820: p53 (uniprotkb:P04637) and MDM4 (uniprotkb:O15151) bind (MI:0407) by nuclear magnetic resonance (MI:0077)     

Regulation of the E3 ubiquitin ligase activity of MDM2 by an N-terminal pseudo-substrate motif

The tumor suppressor p53 has evolved a MDM2-dependent feedback loop that promotes p53 protein degradation through the ubiquitin–proteasome system. MDM2 is an E3-RING containing ubiquitin ligase that catalyzes p53 ubiquitination by a dual-site mechanism requiring ligand occupation of its N-terminal hydrophobic pocket, which then stabilizes MDM2 binding to the ubiquitination signal in the DNA-binding domain of p53. A unique pseudo-substrate motif or “lid” in MDM2 is adjacent to its N-terminal hydrophobic pocket, and we have evaluated the effects of the flexible lid on the dual-site ubiquitination reaction mechanism catalyzed by MDM2. Deletion of this pseudo-substrate motif promotes MDM2 protein thermoinstability, indicating that the site can function as a positive regulatory element. Phospho-mimetic mutation in the pseudo-substrate motif at codon 17 (MDM2^S17D) stabilizes the binding of MDM2 towards two distinct peptide docking sites within the p53 tetramer and enhances p53 ubiquitination. Molecular modeling orientates the phospho-mimetic pseudo-substrate motif in equilibrium over a charged surface patch on the MDM2 at Arg⁹⁷/Lys⁹⁸, and mutation of these residues to the MDM4 equivalent reverses the activating effect of the phospho-mimetic mutation on MDM2 function. These data highlight the ability of the pseudo-substrate motif to regulate the allosteric interaction between the N-terminal hydrophobic pocket of MDM2 and its central acidic domain, which stimulates the E3 ubiquitin ligase function of MDM2. This model of MDM2 regulation implicates an as yet undefined lid-kinase as a component of pro-oncogenic pathways that stimulate the E3 ubiquitin ligase function of MDM2 in cells.