MDM2, murine double minute 2, is a RING type-E3 ligase protein and also an oncogene.
MDM2 plays a critical role in determining the steady levels and activity of p53 in cells using
two mechanisms. The N-terminal domain of MDM2 binds to the transactivation domain of
p53 and inhibits its transcriptional activity. The RING domain of MDM2 plays a role in the
ubiquitination (and degradation) of p53. Several proteins are responsible for the
ubiquitination mechanism including the ubiquitin-activating enzyme (E1), ubiquitin-conjugating
enzyme (E2) and ubiquitin ligase (E3). Since the E2-E3 interaction is essential
for ubiquitination, the protein-protein recognition site is a potential drug target.
Two different MDM2 RING constructs were expressed and purified: MDM2RING (residues
386-491) and MDM2RING△C (residues 386-478). Both constructs were characterised
using dynamic light scattering, size exclusion chromatography, mass spectrometry, NMR
and electron microscopy. E3 ligase activity in vitro was also studied. Taken together these
results showed that the MDM2RING construct formed a concentration-dependent oligomeric
structure. In contrast, the MDM2RING△C construct formed a dimer at all concentrations.
Both MDM2RING and MDM2RING △ C retain E3 ligase activity. However, the
MDM2RING△C construct is less active. Full length E2 enzyme UbcH5a was also purified.
Various biophysical techniques were used to study its interaction with MDM2 as well as
with potential small molecule inhibitors as in principle, small molecules which disrupt the
interaction between MDM2 and UbcH5a, could prevent/promote ubiquitination of p53.
The dimerisation of MDM2 is important for its E3 activity and the C8-binding site
potentially provides a second druggable site. In this work, peptide 9, which has the same
sequence as the C-terminus of MDMX (an MDM2 homologue) was found to inhibit MDM2
E3 activity. Various biological techniques including NMR, fluorescence anisotropy, and
electrospray mass spectrometry were used to investigate the interaction between two
inhibitory peptides and MDM2. A major part of project involved virtual screening (VS) to
search for small molecules which can affect MDM2-dependent ubiquitination. Three
potential targets were considered: (1) the C8-binding site of MDM2; (2) the UbcH5a-binding
site of MDM2; and (3) the MDM2-binding site of UbcH5a. Several small molecules were
identified using our virtual screening database-mining and docking programs that were
shown to affect MDM2-dependent ubiquitination of p53.
In terms of understanding the complex biochemical mechanism of MDM2 this work
provides two interesting and functionally relevant observations: (i) the MDM2 RING△C
construct is a dimer as this would not be expected form the existing studies, and has less E3
ligase activity than MDM2RING; (ii) small molecules that bind MDM2 on the E2 binding
site enhanced E3 ligase activity. One model to explain these observations is that binding of
small molecule activators family to the RING induces a change in the conformation of the Cterminal
tail residues which may enhance E2 binding
