MDMX contains an autoinhibitory sequence element

Michal Bista, Miriana Petrovich, and Alan R. Fersht1

Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 0QH, United Kingdom

Contributed by Alan R. Fersht, September 14, 2013 (sent for review August 28, 2013) and MDMX are homologous that bind to p53 and Results regulate its activity. Both contain three folded domains and ∼70% MDMX Reversibly Dimerizes. MDMX was expressed in Escherichia intrinsically disordered regions. Previous detailed structural and coli and purified to homogeneity using standard methods (12, biophysical studies have concentrated on the isolated folded 21). We found by size-exclusion chromatography (SEC) that fi domains. The N-terminal domains of both exhibit high af nity the eluted at volumes typical for ∼400-kDa globular for the disordered N-terminal of p53 (p53TAD) and inhibit its trans- proteins, much earlier than expected from a monomeric 55- activation function. Here, we have studied full-length MDMX and kDa molecule (Fig. 1A). The elution volume was concentration ∼ fi found a 100-fold weaker af nity for p53TAD than does its iso- dependent, suggesting that the protein was undergoing a re- lated N-terminal domain. We found from NMR spectroscopy and versible oligomerization. Multiangle light scattering (MALS) binding studies that MDMX (but not MDM2) contains a conserved, analysis yielded molecular weight of the eluted protein fraction disordered self-inhibitory element that competes intramolecularly to be ∼100–110 kDa, in agreement with a dimer (110 kDa)– for binding with p53TAD. This motif, which we call the WWW monomer (55 kDa) equilibrium. We used analytical ultracen- element, is centered around residues Trp200 and Trp201. Deletion trifugation to find the dimerization K to be 1.12 ± 0.18 μM or mutation of the element increased binding affinity of MDMX to d that of the isolated N-terminal domain level. The self-inhibition of (Fig. S2). C-terminally truncated mutants of MDMX were ex- MDMX implies a regulatory, allosteric mechanism of its activity. clusively monomeric, showing that the RING domain was es- MDMX rests in a latent state in which its binding activity with sential for dimerization. p53TAD is masked by autoinhibition. Activation of MDMX would MDMX Binds to p53. We mixed MDMX with tetrameric p53 and require binding to a regulatory protein. The inhibitory function of the WWW element may explain the oncogenic effects of an alter- subjected the complexes to SEC-MALS analysis. Their sizes were dependent on the stoichiometry, reaching maximal values native splicing variant of MDMX that does not contain the WWW ∼ element and is found in some aggressive cancers. of 400 kDa for equimolar mixtures, which corresponded to octamers built of four MDMX and four p53 subunits (Fig. 1A). fl MDM4 | HDMX | MDMX-S | IDP | TROSY Interestingly, even though interaction of exible, multivalent receptor–ligand pairs (like MDMX and p53) could potentially lead to formation of high–molecular-weight polymers (22), we he homologous proteins MDMX and MDM2 are major found a size cap of 400 kDa that was preserved even at high negative regulators of the level of p53 and its activity in T ratios of MDMX to p53 and vice versa. normal cells. They are frequently up-regulated in many types of – cancers, becoming oncogenes (1 3). Both proteins bind to the MDMX Stimulates Ubiquitination. SEC-MALS analysis confirmed transactivation domain of p53 (p53TAD) and inhibit its in- that the recombinant MDMX contained functional NTD (re- teraction with transcriptional cofactors. In addition, MDM2 is an sponsible for p53 binding) and C-terminal RING domain capa- E3 ligase with a catalytically active RING domain and ble of homodimerization. To confirm the biochemical activity of can target p53 for proteosomal degradation (4). MDMX has an recombinant proteins, we performed an in vitro ubiquitination inactive RING domain but can efficiently inhibit the p53 activity, assay. MDMX did not exhibit intrinsic E3 ligase activity, and either independently [e.g., in the vast majority of primary mel- MDM2 was only weakly active. Mixing the two recombinant anomas (5)] or in association with MDM2 by boosting its E3 proteins yielded a highly active complex capable of performing ligase activity (6). Both proteins are vital for homeostasis, and knockout of either of them in mice models results in p53-dependent Significance lethality, with survival times longer for MDMX deficiency (3). MDM2 and MDMX are intrinsically disordered proteins, each The protein MDMX is an important negative regulator of the with three folded domains (Fig. S1A). The structures of the tumor suppressor p53 in normal cells and can become a pow- isolated domains have been elucidated at atomic resolution (Fig. erful oncogene. Previous studies on isolated protein fragments S1 B–D): the N-terminal, globular SWIB-like domain (NTD) established that the N-terminal domain of MDMX binds tightly binds to the p53 transactivation domain p53TAD (7, 8) [with to the N-terminal domain of p53 and inhibits it. We now find W23p53 contributing the most interaction energy (9, 10)]; the that full-length MDMX contains a regulatory element (the central part of the protein contains a RanBP2 type zinc finger “WWW element”) that binds to its own N-terminal domain and (ref. 11 and PDB ID code 2CR8) and the C terminus is a RING- prevents MDMX from binding to p53. This autoinhibition type domain, which tends to associate and form introduces a new level of regulation of MDMX, whereby ac- homodimers and heterodimers and higher-order oligomers (12, cessory proteins may sequester the WWW sequence and 13). There are specific MDM2 inhibitors that bind to the NTD reactivate MDMX, and may explain why a variant of MDMX [e.g., nutlin-3 (14)], which usually have lower potency against that lacks the WWW sequence, which is found in some cancer cells, might be oncogenic. MDMX (8). The most oncogenic form of these proteins is the – – MDM2 MDMX heterodimer (3, 15 17). Author contributions: M.B., M.P., and A.R.F. designed research; M.B. and M.P. performed There are few detailed biophysical and structural studies on research; M.B. and M.P. analyzed data; and M.B. and A.R.F. wrote the paper. the full-length proteins and how the individual domains interact The authors declare no conflict of interest. – fi (18 21). Here, we analyze MDMX and nd that there is a reg- 1To whom correspondence should be addressed. E-mail: [email protected]. ulatory autoinhibitory sequence within MDMX that regulates its This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. binding characteristics with p53, with biological implications. 1073/pnas.1317398110/-/DCSupplemental.

17814–17819 | PNAS | October 29, 2013 | vol. 110 | no. 44 www.pnas.org/cgi/doi/10.1073/pnas.1317398110 Downloaded by guest on September 30, 2021 +MDMX MDM2 MDMX MDM2

A B Molecular weight [kDa] μ μ 160 kDa μ μμ 110 kDa

80 kDa A280 (relative) 60 kDa

50 kDa

Elution time [min]

Fig. 1. MDMX is dimer capable of stimulating E3 activity of MDM2. (A) SEC-MALS analysis of MDMX and p53; A280 chromatograms were scaled to give the same peak heights. Masses calculated from the static light scattering are shown as thick lines. Composition of samples subjected to the analysis described in the legend. (B) Enzymatic activity of recombinant proteins; in vitro ubiquitination reactions were resolved by denaturing electrophoresis and immunoblotted with anti-ubiquitin antibodies, which recognize polyubiquitinated forms more strongly than monoubiquitinated (see Fig. S3 for detection using other antibodies).

polyubiquitination (Fig. 1B). The results of the in vitro assay NTD Interacts Intramolecularly with a Conserved “WWW Element.” performed with recombinant proteins were in good agreement The spectral changes in the NTD and flexible linkers sug- with the previously reported activities of endogenous proteins in gested that the p53TAD displaces an intramolecular interaction cellular context (6); we found that MDMX stimulated both of the NTD with a segment of MDMX, releasing Trp residues. autoubiquitination of MDM2–MDMX complex and p53 ubiq- Basing on observing that three tryptophans were involved and uitination (Fig. S3) (23). primary sequence analysis, we hypothesized that the interaction might involve a tryptophan-rich segment within residues 190– NMR Spectra of MDMX. We labeled the full-length MDMX with 210 (190FEEWDVAGLPWWFLGNLRSNY210, which we call 15N and recorded a transverse relaxation-optimized spectroscopy the WWW element). Notably, the element is specifictoMDMX, (TROSY) spectrum. The spectrum shown in Fig. 2A was domi- as the corresponding region of MDM2 contains an unrelated nated by signals from highly flexible, intrinsically disordered sequence (Fig. 3A). 15 linkers. Such appearance, with majority of resonances occupying To verify binding of the segment experimentally, we N-labeled the central part of the spectrum is typical of intrinsically dis- the WWW-containing peptide (181–209) and titrated it with – ordered proteins. In addition to these signals from disordered the isolated NTD. The NTD WWW interaction was strong linkers, we could also identify a range of broader and more dis- enough to occur between isolated domains that were not perse resonances originating from the folded NTD and RanBP2 covalently linked. Numerous resonances of the WWW were domains. The absence of RING signals from the spectrum was broadened on binding. We assigned the heteronuclear single- anticipated because in multidomain proteins with intrinsically quantum coherence (HSQC) spectrum of the WWW construct andmappedtheinteractionsitetoaminoacids194–206 (Fig. disordered linkers, central domains are frequently more con- B C strained rotationally than structures located distally (24, 25). 3 and ). Interestingly, the WWW element remained in the fi intermediate timescale of chemical exchange in excess of There were unexpected signi cant differences between the – spectra of the NTD in full-length protein and the isolated MDMX NTD. This suggested that, even in the bound state, domains, implying that MDMX–NTD interacts with another the WWW was not stabilized in a single conformation. The unbound, isolated WWW seemed to lack stable structure, as part of the protein (Fig. 2A). Addition of the p53TAD peptide to its backbone chemical shifts were close to the random coil MDMX gave an NMR spectrum that corresponded to that from values, 1H-15N heteronuclear nuclear Overhauser effect the isolated p53TAD–NTD complex (Fig. 2B). Surprisingly, (NOE) enhancements were low and nuclear Overhauser effect besides anticipated chemical shift perturbations in the canonical spectroscopy (NOESY) spectrum revealed little interresidual p53-binding pocket within the NTD, several sharp resonances contacts (Fig. S4). Notably, we observed that p53TAD and to characteristic for unstructured linkers appeared on addition of a lesser extent also nutlin-3 were capable of dissociating the p53TAD to MDMX. Particularly noticeable were three down- preformed NTD–WWW complex (Fig. S5 A–E). fi eld shifted signals that appeared at frequencies typical for HN Addition of 14N-WWW to 15N-NTD (Fig. 4A) caused nu- groups of Trp side chains. merous NTD cross-peaks to become broadened or perturbed. 15 –14 The TROSY spectrum of a N-MDMX N-p53 complex The chemical shift perturbations in the NTD were analogous to C fl BIOCHEMISTRY (Fig. 2 ) was also dominated by signals of exible linkers, al- those induced by p53TAD; using the NTD backbone assignment, though few residual signals of RanBP2 domain (but not NTD) we confirmed that the WWW and p53 TAD binding sites co- were also identifiable. Notably, the detectable resonances aligned incided (Fig. 4C). The resulting spectrum of the NTD–WWW well with the MDMX–p53TAD complex, and similar patterns complex reproduced that of full-length MDMX (Fig. 4B). were observed in the random coil and Trp regions of the spec- The TROSY spectrum of a monomeric MDMX variant (1–340) trum. The absence of NTD signals is most likely explained by matched both the spectrum of the NTD–WWW complex and exchange between binding of TAD (p53 19–26) and a further that of full-length MDMX, suggesting that the NTD domain transactivation fragment (p53 49–54) (26) and possibly also ro- in MDMX interacts with the WWW element in its own chain CHEMISTRY tational restriction of the domain in the complex. (Fig. S5F).

Bista et al. PNAS | October 29, 2013 | vol. 110 | no. 44 | 17815 Downloaded by guest on September 30, 2021 K A p53 binding was inhibited. The d of NTD and WWW was 1,300 nM. 105 The Kd of MDMX with full-length p53 (1,110 nM) was similar to that with p53TAD (2,870 nM), implying that association of the 110 NTDs is the main energetic component of binding.

115 Deletion or Mutation of the WWW Element Enhances Binding. We –

(ppm) further corroborated the WWW NTD interaction in the full- N

15 120 length protein, by introducing mutations in WWW aiming to

- – – 1 disrupt the WWW NTD interaction. As a key residue for p53 ω 125 NTD interaction is tryptophan (9, 10) and the NMR spectrum of the NTD–WWW complex resembles that of the NTD–p53TAD complex, we focused on mutating tryptophans that were implied 130 by NMR to be directly involved in the interaction (W200 and W201). Although mutation of a single Trp to Ala did not in- 10 9 8 7 1 crease the p53 affinity markedly (Table 1), double mutation to ω2 - H (ppm) Asp had more pronounced effect and complete removal of the B WWW element resulted in MDMX binding p53 almost as tightly 105 as the isolated NTD (90 nM).

110 A

) 190 200 210 115 Human (Homo sapiens) LGF EEWDVAGLPWWF LGNLR SNYTP R SN ppm Mouse (Mus musculus) LDF EEWDVAGLPWWF LGNLRNNC I P KSN

N ( Chicken (Gallus gallus) LVF EEWDVAGLPWWF LGNLR SNYKSR SN

15 120 Green anole (Anolis carolinensis) SVLEEWDVPGLPWWF LGNLRNNYKSR SN - 1 Clawed frog (Xenopus laevis) F I F EEWDEAGLPWWF LGNLRTNYNLQ S I ω Zebrafish (Danio renio) VT LEEWDLSGLPWWF LGNLR SNYTRR SN 125 Elephant shark (C. millii) SVSDHWDAAGLPWWF I KS LQ SNYGSRKS

MDM2 Human (Homo sapiens) SISLSFDESLALCVIREICCERSSSSSE 130 B 105 10 9 8 7 G197 - 1H (ppm) G204 ω2 G189 C 110

105 ) T183 115 S208 ppm N205 S184 F190 W201

N ( E192 W200 V195 110 R207 15 D187 W193 120 E182 - L206 L203 1 F202 E191 L198 ) ω D181 L188 115 R185 D194 125 ppm A196 L186

( N209 N

15 120

- 130 1 ω 10 9 8 7 125 1 ω2 - H (ppm) C 130 105

10 9 8 7 110 1 ω2 - H (ppm) 115 Fig. 2. Overlays of 15N-TROSY spectra of MDMX. (A) 15N-MDMX (red) and isolated 15N-NTD (16–116; gray). (B) 15N-MDMX–14N-p53TAD (17–32) com- N (ppm) 15 14 plex (blue) and N-NTD (16-116)– N-p53TAD (17–32) complex (yellow). (C) 15 120 15 14 - N-MDMX– N-p53 complex (green). Insets show fragments overlaid with 1 spectrum (A). ω 125

WWW Element Inhibits p53 Binding. We used isothermal titration 130 – calorimetry to measure the thermodynamics of MDMX p53 10 9 8 7 1 interactions and the contribution of the WWW element (Table 1). ω2 - H (ppm) p53TAD was tightly bound by isolated NTD (Kd = 30 nM). In K = Fig. 3. WWW element interacts with NTD. (A) Multiple sequence alignment contrast, MDMX bound p53TAD 100-fold less tightly ( d 2900 of WWW sequences from different species; corresponding sequence of nM). Titrations with truncated MDMX variants (Table 1, with MDM2 lacks similarity. (B) 15N-HSQC, assigned spectrum of isolated WWW p53TAD) revealed that as long as the WWW element was present, (181–209). (C) 15N-WWW (181–209) bound to 14N-NTD (1–111).

17816 | www.pnas.org/cgi/doi/10.1073/pnas.1317398110 Bista et al. Downloaded by guest on September 30, 2021 of MDMX-S relative to MDMX may thus have a significant effect on p53 activity. Discussion We noted that the NMR spectrum of the N-terminal domain of MDMX resembled that of the isolated domain when bound to a peptide rather than the unligated domain. Furthermore, we noted that three tryptophan side chains in a disordered region of the protein had perturbed signals. We surmised that an internal sequence of the protein was binding to its N-terminal domain. We located the relevant sequence element by deletion and point mutation analysis, and showed that the isolated sequence ele- ment bound tightly to MDMX–NTD. Furthermore, we found that MDMX lacking the sequence element bound 32-fold more tightly to p53. Consequently, there is an important sequence element in the central region of MDMX (but not MDM2), the existence of which implies a mechanism for its regulation. Residues 190–210 in that intrinsically disordered region constitute an inhibitory module, which we term the WWW element. It competes with the p53TAD for binding to the NTD of MDMX, thus lowering the affinity of MDMX for p53. Efficient binding of MDMX to p53 requires a mechanism to relieve the allosteric self-inhibition. The WWW element does not contain known posttranslational modifications sites, implying that activation of MDMX for p53 binding must be achieved by another mechanism. There is some evidence for two processes: alternative splicing; or by accessory proteins that can bind the WWW element (Fig. 5). Alternative splicing is a very common regulatory mechanism in proteins containing inhibitory modules (28), and indeed, a splic- ing variant MDMX-S, containing only the NTD is overexpressed in some cancers and strongly associated with a negative outcome (29, 30). The affinity of MDMX-S toward p53 was two orders of magnitude higher than for the standard variant, which may be a possible explanation of the high oncogenic potential of the short protein.

Table 1. ITC results

−1 −1 Protein KD,nM ΔH, kcal/mol ΔS,cal·mol ·K

With p53TAD* MDMX 2,870 ± 558 −8.1 ± 0.8 −2.3 MDMX 1–340 2,700 ± 531 −8.3 ± 0.6 −2.8 † MDMX 1–303 8,200 ± 826 −10.3 ± 0.3 −11.9 MDMX 1–238† 5,560 ± 491 −16.3 ± 0.9 −31.5 MDMX 1–111 30.3 ± 3.9 −13.9 ± 0.1 −13.2 ‡ MDMX ΔWWW 92.6 ± 11.7 −17.6 ± 0.2 −27.7 MDMX W200D/201D 385 ± 43.5 −15.3 ± 0.3 −22.7 With p53 MDMX + p53§ 1,110 ± 101 −8.3 ± 0.2 −1.2 Fig. 4. WWW binding resembles p53TAD in binding. (A) Titration of With WWW element { 15N-NTD (16–116) with 14N-WWW (181–209). Reference spectrum in red MDMX 1–111 1,290 ± 641 −3.4 ± 0.4 15.3 jj and NTD–WWW complex in blue. (B) Overlay of full-length MDMX Mutants with p4 spectrum (yellow) with spectra of NTD–WWW complex (blue) and iso- MDMX 508 ± 36.8 −14.4 ± 0.1 −20.3 lated zinc finger domain (pink). (C) WWW binding site mapping. Areas MDMX W200A 209 ± 27.6 −15.8 ± 0.2 −23.4 Δδ 2+ Δδ 2 1/2 > around amides with chemical shift perturbations ( 1H 0.04 15N ) 0.2 MDMX W201A 485 ± 50 −14.3 ± 0.2 −19.9 ppm colored red. Splicing variant**

MDMX-S + p53TAD 40.3 ± 6.3 −15.2 ± 0.2 −18.0 BIOCHEMISTRY

MDMX-S Splicing Variant. MDMX is regulated at the post- *p53TAD, residues 17–32. transcriptional level by alternative splicing. One of the main splicing †Protein has C-term His-tag, which minimizes degradation. forms, MDMX-S, is a product of exon 6 skipping, producing ‡Residues 193–210 deleted. a 127-aa protein sequence containing the NTD and 17 addi- §Independent association of p53 and MDMX monomers assumed; values calculated for monomers concentrations. tional amino acids (27). Recombinant MDMX-S binds p53 { Sequence 181–211 of MDMX. with high affinity similar to the isolated NTD and by two jj fi

P4, high-af nity p53-derived peptide [sequence (5,6-FAM)-LTFEHYWAQLTS] CHEMISTRY orders of magnitude stronger than full-length MDMX. Owing (37). to their large difference in p53 binding affinities, even low levels **MDMX alternative splicing variant (27).

Bista et al. PNAS | October 29, 2013 | vol. 110 | no. 44 | 17817 Downloaded by guest on September 30, 2021 μ – RING solution. The syringe was loaded with 40 L of p53TAD peptide (14 33), 5,6- WWW ZnF FAM-p4 peptide (37) (sequence LTFEHYWAQLTS), MDMX WWW peptide (181–211), or full-length, stabilized quadruple mutant of p53. Titrant and NTD titrand solutions were prepared in a matching buffer (20 mM Tris, pH 7.2, activation 150 mM NaCl, 0.5 mM TCEP), and titrant concentration was kept 10 times higher than the titrand. All solutions were degassed for 10 min before measurement. Titrations were performed at 20 °C with stirring speed 800 rpm, injection volumes of 2 μL, and a spacing of 120 s. The data were fit MDMX-S using a one-site binding model available in the Origin ITC data analysis splicing accessory regulator software (version 7.0).

NMR Spectrometry. NMR spectra were recorded at 293 K using Bruker NMR spectrometers (Avance II+ 700 MHz, Avance III 600 MHz, DRX 600 MHz, or Fig. 5. Regulation of MDMX activity by autoinhibition. Full-length MDMX Avance 500 MHz) equipped with cryogenically cooled probes. The standard rests in a latent state in which its binding activity with p53TAD is masked. NMR buffer contained 20 mM Tris, pH 7.2, 150 mM NaCl, 5% D2O, and Activation of MDMX can achieved either by alternative splicing, yielding buffer-exchange before measurement was achieved either by SEC or using a truncated MDMX variant (MDMX-S), or by binding of the inhibitory Zeba Spin 7K MWCO, 2-mL desalting kit. The spectra were recorded using module by an accessory factor. TROSY (38) or fast HSQC (39) pulse sequences, typically as 160 × 1,024 complex points matrices. Typical sample concentrations were in the range of 20–50 μM for full-length protein constructs and 100 μM for isolated domains The sequence of the WWW has been strongly preserved constructs. Typical measurement time for TROSY spectrum of full-length during evolution (Fig. 3A) (31). High abundance of hydrophobic MDMX was 20 h. Topspin 3.1 was used for data processing; before Fourier fi amino acids and its intrinsically unstructured character, sug- transformation, the free induction decays were zero- lled and apodized gested that the motif is likely to be a promiscuous binding site for using sine-bell squared (QSINE) function. Processed spectra were plotted and visualized using Sparky (University of California, San Francisco). Tryp- other partners besides the NTD of MDMX. Indeed, Chen and tophan indole resonances were identified from a 1H-15N-HSQC spectrum coworkers (32, 33) have recently found that the WWW element 1 α obtained omitting the H refocusing pulse during t1 evolution and mea- provides a docking site for casein kinase 1 , which in turn acti- suring the 1H-15N coupling constants, which were higher for indole side vates MDMX for p53 binding and phosphorylates it on S289. chains (1J = ∼99 Hz). DNA damage relieves the CK1α–MDMX interaction, resulting Resonance assignments were obtained manually using 15N-13C–labeled in release of p53 from the complex. Accordingly, it is likely that samples, using a standard set of triple resonance (HNCA, CBCACONH, other p53TAD binding partners and other proteins might acti- HNCACB, HNCO, HNCACO) (40) and 15N-NOESY-HSQC experiments (41). vate MDMX by binding to the WWW segment. 1H-15N NOE enhancements were calculated from intensities of cross-peaks in presaturated and nonsaturated spectra acquired at 600-MHz 1H Larmor Materials and Methods frequency, in an interleaved mode, according to ref. 42. The 5-s interscan Protein Production. Full-length MDMX, MDM2, as well as truncated constructs relaxation delay was set to avoid water saturation. [MDMX 1–111, MDMX-S, MDMX 16–116, MDMX 1–238 C-terminal 6×His, MDMX 1–303 C-terminal His, MDMX 1–340, MDMX W200,201D, MDMX In Vitro Ubiquitination Assay. Ubiquitination assays were carried out by ΔWWW (lacking residues 193–210), MDMX-WWW 181–209, MDMX zinc a modification of protocol from ref. 43. Reaction buffer contained 40 mM finger 292–340] were cloned into pETM plasmids (34) (obtained from the Tris, pH 7.4, 10 mM MgCl2, 0.6 mM DTT, and 10 mM ATP. Two hundred Protein Expression and Purification Core Facility at the European Molecular microliters of reaction mixes were prepared by mixing the following: 80 ng Biology Laboratory Heidelberg) and purified to homogeneity using a com- of E1 (UbE1), 4 μg of E2 (UbE2D2), 20 μg of ubiquitin, 6 μg of p53, and (i) bination of affinity and size-exclusion chromatographies, using a protocol 1.4 μg of MDMX, (ii)1.6μg of MDM2, and (iii)1.6μg of MDM2 plus 1.4 μgof that essentially has been described in refs. 12 and 21. The final purification MDMX. All of the proteins were recombinant and produced in-house (ex- step for all constructs except for MDMX 181–211 was SEC in a buffer con- cept E1 ligase which was purchased from Boston Biochemicals). Reactions taining 20 mM Tris, pH 7.2, 150 mM NaCl, and 0.5 mM tris(2-carboxyethyl) were preincubated at 37 °C and started simultaneously by addition of ATP. phosphine (TCEP). MDMX WWW 181–209 was additionally purified by Aliquots were mixed with 4× LDS sample buffer (Invitrogen) after 15 min. The RP-HPLC using Discovery BIO Wide Pore C5-5 column (Supelco Analytical) samples were resolved by denaturing electrophoresis on 4–12% precast Bis- and 9–90% (vol/vol) gradient of AcCN in water, 0.1% TFA, and sub- Tris gels (Invitrogen) and subjected to immunoblotting analysis monoclonal 15 15 13 sequently lyophilized. Isotope labeling with Nor N, C was achieved anti-ubiquitin MCA1398g (AbD Serotec, Bio-Rad), anti-MDM2 SMP14 (Milli- 15 by growing the bacteria in M9 medium supplemented with N-NH4Cl or pore), anti-MDMX 8C6 (Millipore), and anti-p53 DO-7 (Dako) antibodies. 15 13 N-NH4Cl and C-glucose, respectively. fi p53 stabilized quadruple mutant was produced and puri ed as described Analytical Ultracentrifugation. Sedimentation equilibrium experiments on (25, 35). MDMX were performed using Beckman XL-I analytical ultracentrifuge equipped with an absorbance and interference detection system with an fi Analytical SEC. Analytical gel ltrations were performed using a GE Health- An50Ti rotor and six-sector 12-mm pathlength cells (Beckman Coulter, USA). fl care Superose 6 10/300GL column with a ow of 0.5 mL/min. One hundred Data were collected at 10 °C following absorbance at 280 nm at 18,000, microliters of protein sample at different concentrations were injected. The 20,000, and 24,000 rpm. The protein concentration was 1.5 μM, and the running buffer contained 20 mM Tris, pH 7.2, 150 mM NaCl, and 0.5 mM TCEP. buffer contained 20 mM Tris, pH 7.4, 500 mM NaCl, and 4 mM TCEP. Scans The chromatography system was coupled to a DAWN HELIOS MALS in- were collected at 8-h intervals until the equilibrium was reached by re- strument (Wyatt Technology) and an Optilab rEX refractometer (Wyatt petitive scans. Data were analyzed using the UltraSpin software (http:// Technology). The measurements of the intensity of the Rayleigh scattering as www.mrc-lmb.cam.ac.uk/dbv/ultraspin2/). Data were fittedtoadouble- a function of the angle and differential refractive index were performed exponential model indicating equilibrium of two components with simultaneously on-line and used to determine the weight average molar mass masses corresponding to that of monomer and dimer, respectively. of eluted oligomers and protein complexes (36). Data analysis was performed using the ASTRA5 (Wyatt Technology) software. ACKNOWLEDGMENTS. We acknowledge Drs. Stefan M. Freund, Christopher M. Johnson, and Stephen H. McLaughlin for advice and helpful comments Isothermal Titration Calorimetry. The isothermal titration calorimetry (ITC) and for access to MRC-LMB biophysics and NMR facility instrumentation. experiments were performed using a Microcal ITC200 instrument (Microcal). This work was funded by Medical Research Council Programme Grant The sample cell of the calorimeter was loaded with 207 μL of MDMX G0901534.

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