Abstract
The human MDM2 protein regulates the tumor suppressor protein p53 by restricting its transcriptional activity and by promoting p53 degradation. MDM2 is ubiquitously expressed, with its overexpression implicated in many forms of cancer. The inhibitory effects of MDM2 on p53 have been shown to involve its N-terminal p53-binding domain and its C-terminal RING domain. The presence of an intact central acidic domain of MDM2 has also been shown to regulate p53 ubiquitination, with this domain shown to directly interact with the p53 DNA-binding domain to regulate the DNA binding activity of p53. To date, little structural information has been obtained for the MDM2 acidic domain. Thus, to gain insight into the structure and function relationship of this region, we have applied solution-state NMR spectroscopy to characterize the segment of MDM2 spanning residues 215–300. These boundaries for the acidic domain were determined on the basis of consensus observed in multiple sequence alignment. Here, we report the 1H, 15N and 13C backbone and 13Cβ chemical shift assignments and steady-state {1H}-15N heteronuclear NOE enhancement factors as a function of residue for the acidic domain of MDM2. We show that this domain exhibits the hallmarks of being a disordered protein, on the basis both of assigned chemical shifts and residue-level backbone dynamics, with localized variation in secondary structure propensity inferred from chemical shift analysis.
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Data Availability
The assignments for the backbone resonances and the {1H}-15N heteronuclear NOE enhancement factors have been deposited at the Biological Magnetic Resonance Data Bank under the Accession Number 51334. The datasets and materials generated during and/or analyzed during the current study are available from the corresponding authors on reasonable request.
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Acknowledgements
J.K.R. is grateful to Dr. Michael W. Gray for generous donation of the FPLC system. NMR experiments were acquired at QANUC and we thank Dr. Tara Sprules, NMR facility manager at QANUC, for her expert NMR data acquisition and technical support. We are also grateful for access to the NMRbox resource for data processing and analysis.
Funding
This work was supported by Discovery Grants from the Natural Sciences and Engineering Research Council of Canada (NSERC, RGPIN/05907 − 2017 to J.K.R. and RGPIN/06083 − 2020 to X.-Q.L.) Key equipment was provided by NSERC Research Tools and Instruments Grants (to J.K.R. and X.-Q.L.) and Dalhousie Medical Research Foundation Capital Equipment Grants (to J.K.R. and X.-Q.L.) The Québec/Eastern Canada High Field NMR Facility (QANUC) is supported by the Canada Foundation for Innovation and the McGill University Faculty of Science and Department of Chemistry. NMRbox: National Center for Biomolecular NMR Data Processing and Analysis NMRbox is a Biomedical Technology Research Resource (BTRR), which is supported by NIH grant P41GM111135 (NIGMS).
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All authors contributed to the study conception and design. QS: material preparation was performed. QS: data processing and analysis were performed primarily working with JKR. X-QL and JKR: Funding for this work was obtained. QS: The manuscript, including figure preparation, was drafted and all authors edited the manuscript. All authors read and approved the final manuscript.
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Song, Q., Liu, XQ. & Rainey, J.K. 1H, 15N and 13C backbone resonance assignments of the acidic domain of the human MDM2 protein. Biomol NMR Assign 17, 9–16 (2023). https://doi.org/10.1007/s12104-022-10112-4
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DOI: https://doi.org/10.1007/s12104-022-10112-4