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Structural analysis of MDM2 RING separates degradation from regulation of p53 transcription activity

Nature Structural & Molecular Biology volume 24, pages 578–587 (2017)Cite this article

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Abstract

MDM2–MDMX complexes bind the p53 tumor-suppressor protein, inhibiting p53's transcriptional activity and targeting p53 for proteasomal degradation. Inhibitors that disrupt binding between p53 and MDM2 efficiently activate a p53 response, but their use in the treatment of cancers that retain wild-type p53 may be limited by on-target toxicities due to p53 activation in normal tissue. Guided by a novel crystal structure of the MDM2–MDMX–E2(UbcH5B)–ubiquitin complex, we designed MDM2 mutants that prevent E2–ubiquitin binding without altering the RING-domain structure. These mutants lack MDM2's E3 activity but retain the ability to limit p53′s transcriptional activity and allow cell proliferation. Cells expressing these mutants respond more quickly to cellular stress than cells expressing wild-type MDM2, but basal p53 control is maintained. Targeting the MDM2 E3-ligase activity could therefore widen the therapeutic window of p53 activation in tumors.

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Figure 1: Structure of MDM2R–MDMXR–UbcH5B–Ub.
Figure 2: Importance of MDMX's C-terminal tail.
Figure 3: Mechanism of E2∼Ub recruitment and activation by MDM2RH.
Figure 4: Redesigning ligase-dead MDM2 mutants.
Figure 5: Reactivation of MDMX's ligase activity.
Figure 6: MDM2 I440 and R479 mutants can limit p53 activity.
Figure 7: Cells expressing MDM2 I440 and R479 mutants rapidly respond to stress.

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Acknowledgements

We would like to thank L. Buetow for her comments on the manuscript and DLS for access to beamlines I24 beamlines (mx8659) that contributed to the results presented here. This work was supported by Cancer Research UK (C596/A17196) and D.T.H. received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (grant agreement no. 647849).

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  1. Karen H Vousden

    Present address: The Crick Institute, London, UK

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  1. Cancer Research–UK Beatson Institute, Garscube Estate, Glasgow, UK

    Koji Nomura, Marta Klejnot, Dominika Kowalczyk, Andreas K Hock, Gary J Sibbet, Karen H Vousden & Danny T Huang

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Contributions

K.N., A.K.H. and K.H.V. designed cell-based experiments. K.N. performed all cell-based experiments and analyzed the data. M.K. and D.T.H. performed crystallization and structural determination. M.K., D.K. and D.T.H. performed protein purification and in vitro biochemical assays. G.J.S. performed and analyzed SPR experiments. K.N., A.K.H., K.H.V. and D.T.H. wrote the manuscript. All authors read and approved the final manuscript.

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Correspondence to Karen H Vousden or Danny T Huang.

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Integrated supplementary information

Supplementary Figure 1 UbcH5B~Ub discharge catalyzed by MDM2 and MDM2–MDMX

(a) A representative nonreduced SDS-PAGE showing the effects of UbcH5B mutations in discharging UbcH5B variant~Ub to L-lysine in 1 min catalyzed by MDM2R-MDMXR. (b) A bar graph showing the fraction of UbcH5B variant~Ub left in a. (c) A representative nonreduced SDS-PAGE showing the effects of Ub mutations in discharging UbcH5B~Ub variant to L-lysine in 1 min catalyzed by MDM2R-MDMXR. (d) A bar graph showing the fraction of UbcH5B variant~Ub left in c. (e) A representative nonreduced SDS-PAGE showing the effects of MDM2 mutations in discharging UbcH5B~Ub to L-lysine in 1 min catalyzed by MDM2R-MDMXR variants. (f) A bar graph showing the fraction of UbcH5B~Ub left in e. (g) A representative nonreduced SDS-PAGE showing the discharge of UbcH5B~Ub to L-lysine in 1 min catalyzed by MDM2R-MDMXR variants. (h) A bar graph showing the fraction of UbcH5B~Ub left in g. (i) A representative nonreduced SDS-PAGE showing the discharge of UbcH5B~Ub to L-lysine in 1 min catalyzed by MDM2R-MDMXR variants. (j) A bar graph showing the fraction of UbcH5B~Ub left in i. (k) A representative nonreduced SDS-PAGE showing the discharge of UbcH5B~Ub to L-lysine in 1 min catalyzed by GST-MDM2 variants. (l) A bar graph showing the fraction of UbcH5B~Ub left in k. Three independent reactions were performed (n=3) and error bars indicate standard deviation.

Supplementary Figure 2 Redesigning ligase-dead MDM2 mutants

(a) Nonreduced SDS-PAGE showing the discharge of UbcH5B~Ub to L-lysine over time catalyzed by MDM2 I440 and R479 variants. (b) SDS-PAGE showing the pull-down experiments of GST-MDM2 I440E, I440K and R479P with His-MDM2 I440E, I440K and R479P, respectively. GST-MDM2 variant and the corresponding His-MDM2 variant were co-expressed in E. coli and purified by Ni-NTA affinity followed by glutathione sepharose affinity chromatography. (c) SDS-PAGE showing the pull-down experiments of GST-MDM2 variants with His-MDMX. GST-MDM2 variant and His-MDMX were co-expressed in E. coli and purified by Ni-NTA affinity followed by glutathione sepharose affinity chromatography. (d) Nonreduced SDS-PAGE showing the discharge of UbcH5B~Ub to L-lysine over time catalyzed by MDM2-MDMX variants. Asterisks indicate contaminants.

Supplementary Figure 3 SPR analyses of MDM2 and MDMX variants binding affinities for UbcH5B–Ub

(a) Representative sensorgrams (left) and binding curves (right) for GST-MDM2 398-C variants with UbcH5B–Ub in the presence of UbΔGG are shown. Only sensorgram is shown for GST-MDM2 398-C variants that displayed no measurable UbcH5B–Ub binding in the presence of UbΔGG. (b) Representative sensorgrams (left) and binding curves (right) for GST-MDMXR variants with UbcH5B–Ub are shown. Wild type MDMX displayed no UbcH5B–Ub binding up to 100 μM UbcH5B–Ub whereas both K478R and N448C, K478R mutants exhibited UbcH5B–Ub binding. However, Kd could not be estimated due to the weak binding affinity. All experiments in a and b were performed in duplicates.

Supplementary Figure 4 Validation of tetracycline-inducible p53 knockdown and MDM2 knockout cells.

U2OS cells were infected with pLKO tet-on shp53. (a) Western blot showing that doxycycline treatment causes p53 knock-down in 2 days and this effect can be washed off in 5 days. (b) MDM2 was then disrupted by CRISPR. Immunoblotting showing CRISPR disruption (target p53 binding domain) resulted in MDM2 knock-out. (c) Genomic PCR followed by sequencing showing that CRISPR disruption (target p53 binding domain) caused MDM2 knock-out. (d) MDM2 knock-out cells were treated with indicated drugs for 4 hours and phosphorylated p53 (serine 15 and serine 20) are analyzed by western blot.

Supplementary Figure 5 MDM2 mutants can limit induction of p53 target genes.

qPCR showing mRNA expression of p53 target genes (GADD45β is not a p53 target gene) is attenuated by MDM2 I440K, I440E but not by C464A. n = 3 independent experiments each. P values (one-way ANOVA with Tukey's post hoc test): v.s. EV No doxy (vertical), v.s. C464A No doxy (horizontal). “n.s.” indicates not significant vs. EV No doxy. F statistics and degrees of freedom of ANOVAs are reported in Supplementary Table 3.

Supplementary Figure 6 MDM2 I440 and R479 mutants can rapidly respond to the stress.

(a) Immunoblots showing that following wild type p53 re-expression by removal of doxycycline (Doxy), p21 is more rapidly induced in cells expressing MDM2 I440 and R479 in response to actinomycin D (10 nM) treatment compared to cells expressing wild type MDM2 (see Fig. 7c). Similar responses in cells treated with 4 μM nutlin (b) and 1 μM doxorubicin (c).

Supplementary Figure 7 Purified proteins used in the assays and size-exclusion chromatography profiles.

(a) SDS-PAGE showing MDM2R-MDMXR variants. (b) SDS-PAGE showing GST-MDM2R-His-MDMXR and GST-MDMXR-His-MDM2R variants. (c) SDS-PAGE showing MDMXR variants, MDM2R-MDMXR and MDMXRH. (d) SDS-PAGE showing GST-MDMXR variants. (e) SDS-PAGE showing GST-MDM2 398-C variants. (f) Superdex 75 10/300 gel filtration chromatography profile of MDM2R-MDMXR and MDMXR variants. Full length MDMX has been reported to dimerize with Kd of 1 μM (Bista, M. et al., Proceedings of the National Academy of Sciences of the United States of America 110, 17814-17819, 2013). To assess the dimerization state of MDMXR variant, protein samples (10 μM, 100 μl) were loaded onto superdex 75 10/300. The dotted line indicates the elution volume of MDM2R-MDMXR heterodimer. MDMXR variants containing N448C substitution eluted similar or slightly earlier than this line indicating dimer formation, whereas MDMX variants lacking N448C substitution eluted later than this line, presumably exist as monomer. (g) HiLoad 26/600 Superdex 200 chromatography profile of MDM2RH purification. His-GST-MDM2R was incubated with TEV protease to release His-GST tag, followed by Ni-NTA pass-back. The cleaved sample was loaded directly onto HiLoad 26/600 Superdex 200. Trace amount of MDM2R was found throughout the elution profile (45-95 ml) with the majority of MDM2R eluted between 95-110 ml consistent with an earlier report showing that MDM2R exists as dimer and aggregate (Poyurovsky, M.V. et al Embo Journal 26, 90-101, 2007). MDM2RH peak (indicated by arrow) eluted after His-GST peak (indicated by arrow; 50 kDa) was assumed as dimer and was pooled and concentrated for activity assay in Fig. 3d.

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Supplementary Data Set 1

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Nomura, K., Klejnot, M., Kowalczyk, D. et al. Structural analysis of MDM2 RING separates degradation from regulation of p53 transcription activity. Nat Struct Mol Biol 24, 578–587 (2017). https://doi.org/10.1038/nsmb.3414

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