Structural characterisation of MDM2 RING domain: E2-ubiquitin binding and activation by phosphorylation

Magnussen, Helge Magnus (2020) Structural characterisation of MDM2 RING domain: E2-ubiquitin binding and activation by phosphorylation. PhD thesis, University of Glasgow.

Abstract

The RING E3 ligase MDM2 is a primary negative regulator of the tumour suppressor protein p53. It blocks transcriptional activity and ubiquitinates p53, resulting in proteasomal degradation. MDM2’s ligase activity depends on the dimerisation of its C-terminal RING domain with either itself or its homologue MDMX. The crystal structure of the MDM2-MDMX heterodimer RING domain in complex with E2-ubiquitin has recently been crystallised. In this complex, only the MDM2 RING domain binds an E2-ubiquitin complex whereas the MDMX RING domain does not. However, MDMX’s C-terminal tail supports ubiquitin binding. This complex assembly results in one MDM2-MDMX RING heterodimer bound to one E2-ubiquitin complex. Due to extensive aggregation of the MDM2 homodimer, no structural information of the homodimeric MDM2-E2-ubiquitin complex has been obtained to date. During the course of my studies, I developed a purification protocol to generate non-aggregated homodimeric MDM2 RING domain. Sufficient amounts of homogeneous protein could be isolated for crystallisation purposes and crystal structures of the MDM2 homodimer alone and in complex with E2-ubiquitin were obtained. The crystal structures show that the homodimer can simultaneously bind two molecules of E2ubiquitin. The E2-ubiquitin binding surface resembles the heterodimer but shows significant differences in the arrangement of helices adjacent to the RING domain. Upon DNA damage, p53 needs to be stabilised in order to trigger cell cycle arrest or apoptosis. This requires p53 to be uncoupled from MDM2-mediated downregulation and is achieved by a number of phosphorylation events on both proteins, which reduce the binding affinity between p53 and MDM2. However, mouse models suggest that additional mechanisms exist as p53 is stabilised even when the corresponding phosphorylation sites are mutated. In addition, p53 is reportedly stabilised by phosphorylation of MDM2 near the RING domain, a region that is sequentially far away from the p53-binding domain. So far, the molecular basis of this mechanism has been elusive. Here, I show that phosphorylation near the RING domain enhances MDM2’s catalytic activity. With my MDM2 purification protocol, homodimeric phospho-MDM2 was generated and the crystal structure in complex with E2-ubiquitin was obtained. The molecular basis and homodimer-specificity of this novel phosphoregulation will be discussed. The results presented here help to understand the molecular function of MDM2, particularly under DNA damage conditions, and might be beneficial in diagnostics. The purification protocol of homogeneous MDM2 RING domain will be helpful for further structure-based studies such as the design of an MDM2 RING domain inhibitor.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Subjects: Q Science > QR Microbiology
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Cancer Sciences > Beatson Institute of Cancer Research
• Supervisor's Name: Huang, Professor Danny T.
• Date of Award: 2020
• Depositing User: Mr Helge M Magnussen
• Unique ID: glathesis:2020-79033
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 12 Aug 2020 13:27
• Last Modified: 21 Feb 2023 12:57
• Thesis DOI: 10.5525/gla.thesis.79033
• URI: https://theses.gla.ac.uk/id/eprint/79033