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Structural basis of how stress-induced MDMX phosphorylation activates p53

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Abstract

The tumor-suppressor protein p53 is tightly controlled in normal cells by its two negative regulators—the E3 ubiquitin ligase MDM2 and its homolog MDMX. Under stressed conditions such as DNA damage, p53 escapes MDM2- and MDMX-mediated functional inhibition and degradation, acting to prevent damaged cells from proliferating through induction of cell cycle arrest, DNA repair, senescence or apoptosis. Ample evidence suggests that stress signals induce phosphorylation of MDM2 and MDMX, leading to p53 activation. However, the structural basis of stress-induced p53 activation remains poorly understood because of the paucity of technical means to produce site-specifically phosphorylated MDM2 and MDMX proteins for biochemical and biophysical studies. Herein, we report total chemical synthesis, via native chemical ligation, and functional characterization of (24–108)MDMX and its Tyr99-phosphorylated analog with respect to their ability to interact with a panel of p53-derived peptide ligands and PMI, a p53-mimicking but more potent peptide antagonist of MDMX, using FP and surface plasmon resonance techniques. Phosphorylation of MDMX at Tyr99 weakens peptide binding by approximately two orders of magnitude. Comparative X-ray crystallographic analyses of MDMX and of pTyr99 MDMX in complex with PMI as well as modeling studies reveal that the phosphate group of pTyr99 imposes extensive steric clashes with the C-terminus of PMI or p53 peptide and induces a significant lateral shift of the peptide ligand, contributing to the dramatic decrease in the binding affinity of MDMX for p53. Because DNA damage activates c-Abl tyrosine kinase that phosphorylates MDMX at Tyr99, our findings afford a rare glimpse at the structural level of how stress-induced MDMX phosphorylation dislodges p53 from the inhibitory complex and activates it in response to DNA damage.

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Acknowledgements

This work was partially supported by the US National Institutes of Health grant CA167296 and the Overseas Scholars Collaborative Research grant 81128015 from the National Natural Science Foundation of China (WL), and by the National Basic Research Program of China (973 Program, 2013CB932500) (W-YL). XC was the recipient of a scholarship from the China Scholarship Council. Portions of this research were carried out at the University of Maryland X-ray Crystallography Shared Service and the Stanford Synchrotron Radiation Lightsource, a Directorate of SLAC National Accelerator Laboratory and an Office of Science User Facility operated for the U.S. Department of Energy Office of Science by Stanford University. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research and by the National Institutes of Health, National Center for Research Resources, Biomedical Technology Program (P41RR001209), and the National Institute of General Medical Sciences.

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  1. X Chen and N Gohain: These authors contributing equally to this work.

Authors and Affiliations

  1. Institute of Human Virology and Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD, USA

    X Chen, N Gohain, C Zhan, M Pazgier & W Lu

  2. Key Laboratory of Smart Drug Delivery of MOE and PLA, Fudan University School of Pharmacy, Shanghai, China

    X Chen & W-Y Lu

  3. Center for Translational Medicine, Xi’an Jiaotong University School of Life Science and Technology, Shaanxi, China

    W Lu

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  1. X Chen
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Correspondence to M Pazgier or W Lu.

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Chen, X., Gohain, N., Zhan, C. et al. Structural basis of how stress-induced MDMX phosphorylation activates p53. Oncogene 35, 1919–1925 (2016). https://doi.org/10.1038/onc.2015.255

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