Allosteric changes imposed by post-translational modifications regulate and differentiate the functions of proteins with intrinsic disorder regions. HDM2 is a hub protein with a large interactome and with different cellular functions. It is best known for its regulation of the p53 tumour suppressor. Under normal cellular conditions, HDM2 ubiquitinates and degrades p53 by the 26S proteasome but after DNA damage, HDM2 switches from a negative to a positive regulator of p53 by binding to p53 mRNA to promote translation of the p53 mRNA. This change in activity is governed by the ataxia telangiectasia mutated kinase via phosphorylation on serine 395 and is mimicked by the S395D phosphomimetic mutant. Here we have used different approaches to show that this event is accompanied by a specific change in the HDM2 structure that affects the HDM2 interactome, such as the N-termini HDM2-p53 protein-protein interaction. These data will give a better understanding of how HDM2 switches from a negative to a positive regulator of p53 and gain new insights into the control of the HDM2 structure and its interactome under different cellular conditions and help identify interphases as potential targets for new drug developments.

Department of Medical Biosciences Umeå University SE 90185 Umeå Sweden

Équipe Labellisée Ligue Contre le Cancer INSERM UMR 1162 Université Paris 7 27 Rue Juliette Dodu 75010 Paris France

Laboratorio de Interacciones Biomoleculares y Cáncer Instituto de Física Universidad Autónoma de San Luis Potosí Zona Universitaria Manuel Nava 6 78290 San Luis Potosí México

Regional Centre for Applied Molecular Oncology Masaryk Memorial Cancer Institute Zluty Kopec 7 656 53 Brno Czech Republic

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Guo J. and Zhou H.X. (2016) Protein allostery and conformational dynamics. Chem. Rev. 116, 6503–6515 10.1021/acs.chemrev.5b00590 PubMed DOI PMC

Rubin S.M. (2013) Deciphering the retinoblastoma protein phosphorylation code. Trends Biochem. Sci. 38, 12–19 10.1016/j.tibs.2012.10.007 PubMed DOI PMC

Akabas M.H. (2015) Cysteine modification: probing channel structure, function and conformational change. Adv. Exp. Med. Biol. 869, 25–54 10.1007/978-1-4939-2845-3_3 PubMed DOI

Kitayner M., Rozenberg H., Kessler N., Rabinovich D., Shaulov L., Haran T.E. et al. (2006) Structural basis of DNA recognition by p53 tetramers. Mol. Cell 22, 741–753 10.1016/j.molcel.2006.05.015 PubMed DOI

Nussinov R. and Tsai C.J. (2015) Allostery without a conformational change? Revisiting the paradigm. Curr. Opin. Struct. Biol. 30, 17–24 10.1016/j.sbi.2014.11.005 PubMed DOI

Burch L.R., Midgley C.A., Currie R.A., Lane D.P. and Hupp T.R. (2000) Mdm2 binding to a conformationally sensitive domain on p53 can be modulated by RNA. FEBS Lett. 472, 93–98 10.1016/S0014-5793(00)01427-7 PubMed DOI

Fahraeus R. and Olivares-Illana V. (2014) MDM2's social network. Oncogene 33, 4365–4376 10.1038/onc.2013.410 PubMed DOI

Jones S.N., Roe A.E., Donehower L.A. and Bradley A. (1995) Rescue of embryonic lethality in Mdm2-deficient mice by absence of p53. Nature 378, 206–208 10.1038/378206a0 PubMed DOI

Kubbutat M.H., Jones S.N. and Vousden K.H. (1997) Regulation of p53 stability by Mdm2. Nature 387, 299–303 10.1038/387299a0 PubMed DOI

Honda R., Tanaka H. and Yasuda H. (1997) Oncoprotein MDM2 is a ubiquitin ligase E3 for tumor suppressor p53. FEBS Lett. 420, 25–27 10.1016/S0014-5793(97)01480-4 PubMed DOI

Oliner J.D., Pietenpol J.A., Thiagalingam S., Gyuris J., Kinzler K.W. and Vogelstein B. (1993) Oncoprotein MDM2 conceals the activation domain of tumour suppressor p53. Nature 362, 857–860 10.1038/362857a0 PubMed DOI

Tao W. and Levine A.J. (1999) Nucleocytoplasmic shuttling of oncoprotein Hdm2 is required for Hdm2-mediated degradation of p53. Proc. Natl Acad. Sci. U.S.A. 96, 3077–3080 10.1073/pnas.96.6.3077 PubMed DOI PMC

Dumaz N. and Meek D.W. (1999) Serine15 phosphorylation stimulates p53 transactivation but does not directly influence interaction with HDM2. EMBO J. 18, 7002–7010 10.1093/emboj/18.24.7002 PubMed DOI PMC

Chehab N.H., Malikzay A., Stavridi E.S. and Halazonetis T.D. (1999) Phosphorylation of Ser-20 mediates stabilization of human p53 in response to DNA damage. Proc. Natl Acad. Sci. U.S.A. 96, 13777–13782 10.1073/pnas.96.24.13777 PubMed DOI PMC

Chao C., Hergenhahn M., Kaeser M.D., Wu Z., Saito S., Iggo R. et al. (2003) Cell type- and promoter-specific roles of Ser18 phosphorylation in regulating p53 responses. J. Biol. Chem. 278, 41028–41033 10.1074/jbc.M306938200 PubMed DOI

Sluss H.K., Armata H., Gallant J. and Jones S.N. (2004) Phosphorylation of serine 18 regulates distinct p53 functions in mice. Mol. Cell Biol. 24, 976–984 10.1128/MCB.24.3.976-984.2004 PubMed DOI PMC

Wu Z., Earle J., Saito S., Anderson C.W., Appella E. and Xu Y. (2002) Mutation of mouse p53 Ser23 and the response to DNA damage. Mol. Cell Biol. 22, 2441–2449 10.1128/MCB.22.8.2441-2449.2002 PubMed DOI PMC

Khosravi R., Maya R., Gottlieb T., Oren M., Shiloh Y. and Shkedy D. (1999) Rapid ATM-dependent phosphorylation of MDM2 precedes p53 accumulation in response to DNA damage. Proc. Natl Acad. Sci. U.S.A. 96, 14973–14977 10.1073/pnas.96.26.14973 PubMed DOI PMC

Maya R., Balass M., Kim S.T., Shkedy D., Leal J.F., Shifman O. et al. (2001) ATM-dependent phosphorylation of Mdm2 on serine 395: role in p53 activation by DNA damage. Genes Dev. 15, 1067–1077 10.1101/gad.886901 PubMed DOI PMC

Gannon H.S., Woda B.A. and Jones S.N. (2012) ATM phosphorylation of Mdm2 Ser394 regulates the amplitude and duration of the DNA damage response in mice. Cancer Cell 21, 668–679 10.1016/j.ccr.2012.04.011 PubMed DOI PMC

Naski N., Gajjar M., Bourougaa K., Malbert-Colas L., Fahraeus R. and Candeias M.M. (2009) The p53 mRNA-Mdm2 interaction. Cell Cycle 8, 31–34 10.4161/cc.8.1.7326 PubMed DOI

Gajjar M., Candeias M.M., Malbert-Colas L., Mazars A., Fujita J., Olivares-Illana V. et al. (2012) The p53 mRNA-Mdm2 interaction controls Mdm2 nuclear trafficking and is required for p53 activation following DNA damage. Cancer Cell 21, 25–35 10.1016/j.ccr.2011.11.016 PubMed DOI

Medina-Medina I., Garcia-Beltran P., de la Mora-de la Mora I., Oria-Hernandez J., Millot G., Fahraeus R. et al. (2016) Allosteric interactions by p53 mRNA governs HDM2 E3 ubiquitin ligase specificity under different conditions. Mol. Cell Biol. 36, 2195–2205 10.1128/MCB.00113-16 PubMed DOI PMC

Sakaguchi K., Sakamoto H., Lewis M.S., Anderson C.W., Erickson J.W., Appella E. et al. (1997) Phosphorylation of serine 392 stabilizes the tetramer formation of tumor suppressor protein p53. Biochemistry 36, 10117–10124 10.1021/bi970759w PubMed DOI

Worrall E.G., Worrall L., Blackburn E., Walkinshaw M. and Hupp T.R. (2010) The effects of phosphomimetic lid mutation on the thermostability of the N-terminal domain of MDM2. J. Mol. Biol. 398, 414–428 10.1016/j.jmb.2010.03.023 PubMed DOI

Fraser J.A., Worrall E.G., Lin Y., Landre V., Pettersson S., Blackburn E. et al. (2015) Phosphomimetic mutation of the N-terminal lid of MDM2 enhances the polyubiquitination of p53 through stimulation of E2-ubiquitin thioester hydrolysis. J. Mol. Biol. 427, 1728–1747 10.1016/j.jmb.2014.12.011 PubMed DOI

Hernández-Vidales K., Guevara E., Olivares-Illana V. and González F.J. (2019) Characterization of wild-type and mutant p53 protein by Raman spectroscopy and multivariate methods. J. Raman Spectrosc. 50, 1388–1394 10.1002/jrs.5655 DOI

Wallace M., Worrall E., Pettersson S., Hupp T.R. and Ball K.L. (2006) Dual-site regulation of MDM2 E3-ubiquitin ligase activity. Mol. Cell 23, 251–263 10.1016/j.molcel.2006.05.029 PubMed DOI

Kavan D. and Man P. (2011) MSTools—web based application for visualization and presentation of HXMS data. Int. J. Mass Spectrom. 302, 53–58 10.1016/j.ijms.2010.07.030 DOI

Coufalova D., Vojtesek B. and Hernychova L. [Utilization of hydrogen/deuterium exchange in biopharmaceutical industry]. Klin. Onkol. 29 (Suppl. 4), 59–63 10.14735/amko20164S59 PubMed DOI

Klotz I.M. and Frank B.H. (1965) Deuterium–hydrogen exchange in amide N–H groups. J. Am. Chem. Soc. 87, 2721–2728 10.1021/ja01090a033 PubMed DOI

Reyes-Vivas H., Martinez-Martinez E., Mendoza-Hernandez G., Lopez-Velazquez G., Perez-Montfort R., Tuena de Gomez-Puyou M. et al. (2002) Susceptibility to proteolysis of triosephosphate isomerase from two pathogenic parasites: characterization of an enzyme with an intact and a nicked monomer. Proteins 48, 580–590 10.1002/prot.10179 PubMed DOI

Xu D. and Zhang Y. (2012) Ab initio protein structure assembly using continuous structure fragments and optimized knowledge-based force field. Proteins 80, 1715–1735 PubMed PMC

Xu D. and Zhang Y. (2013) Toward optimal fragment generations for ab initio protein structure assembly. Proteins 81, 229–239 10.1002/prot.24179 PubMed DOI PMC

Peng Z., Mizianty M.J. and Kurgan L. (2014) Genome-scale prediction of proteins with long intrinsically disordered regions. Proteins 82, 145–158 10.1002/prot.24348 PubMed DOI

Ishida T. and Kinoshita K. (2007) PrDOS: prediction of disordered protein regions from amino acid sequence. Nucleic Acids Res. 35, W460–W464 10.1093/nar/gkm363 PubMed DOI PMC

Momand J., Zambetti G.P., Olson D.C., George D. and Levine A.J. (1992) The mdm-2 oncogene product forms a complex with the p53 protein and inhibits p53-mediated transactivation. Cell 69, 1237–1245 10.1016/0092-8674(92)90644-R PubMed DOI

Medina-Medina I., Martinez-Sanchez M., Hernandez-Monge J., Fahraeus R., Muller P. and Olivares-Illana V. (2018) P53 promotes its own polyubiquitination by enhancing the HDM2 and HDMX interaction. Protein Sci. 27, 976–986 10.1002/pro.3405 PubMed DOI PMC

Wei X., Wu S., Song T., Chen L., Gao M., Borcherds W. et al. (2016) Secondary interaction between MDMX and p53 core domain inhibits p53 DNA binding. Proc. Natl Acad. Sci. U.S.A. 113, E2558–E2563 10.1073/pnas.1603838113 PubMed DOI PMC

Shimizu H., Burch L.R., Smith A.J., Dornan D., Wallace M., Ball K.L. et al. (2002) The conformationally flexible S9-S10 linker region in the core domain of p53 contains a novel MDM2 binding site whose mutation increases ubiquitination of p53 in vivo. J. Biol. Chem. 277, 28446–28458 10.1074/jbc.M202296200 PubMed DOI

Yu G.W., Rudiger S., Veprintsev D., Freund S., Fernandez-Fernandez M.R. and Fersht A.R. (2006) The central region of HDM2 provides a second binding site for p53. Proc. Natl Acad. Sci. U.S.A. 103, 1227–1232 10.1073/pnas.0510343103 PubMed DOI PMC

Kawai H., Wiederschain D. and Yuan Z.M. (2003) Critical contribution of the MDM2 acidic domain to p53 ubiquitination. Mol. Cell Biol. 23, 4939–4947 10.1128/MCB.23.14.4939-4947.2003 PubMed DOI PMC

Toledo F. and Wahl G.M. (2007) MDM2 and MDM4: p53 regulators as targets in anticancer therapy. Int. J. Biochem. Cell Biol. 39, 1476–1482 10.1016/j.biocel.2007.03.022 PubMed DOI PMC

Toledo F. and Wahl G.M. (2006) Regulating the p53 pathway: in vitro hypotheses, in vivo veritas. Nat. Rev. Cancer 6, 909–923 10.1038/nrc2012 PubMed DOI

Oliner J.D., Saiki A.Y. and Caenepeel S. (2016) The role of MDM2 amplification and overexpression in tumorigenesis. Cold Spring Harb. Perspect. Med. 6 10.1101/cshperspect.a026336 PubMed DOI PMC

Coindre J.M., Pedeutour F. and Aurias A. (2010) Well-differentiated and dedifferentiated liposarcomas. Virchows Arch. 456, 167–179 10.1007/s00428-009-0815-x PubMed DOI

Momand J., Jung D., Wilczynski S. and Niland J. (1998) The MDM2 gene amplification database. Nucleic Acids Res. 26, 3453–3459 10.1093/nar/26.15.3453 PubMed DOI PMC

DeLano W.L. (2002) PyMOL, DeLano Scientific LLC, San Carlos, CA

DeLano W.L. (2009) The PyMOL Molecular Graphics System, DeLano Scientific LLC, San Carlos, CA

Cryptic in vitro ubiquitin ligase activity of HDMX towards p53 is probably regulated by an induced fit mechanism

Resistance mechanisms to inhibitors of p53-MDM2 interactions in cancer therapy: can we overcome them?