The MDM2 protein regulates the tumour suppressor protein p53, acting as its chaperone,
regulating its translation and targeting p53 for degradation by the 26s proteasome via its E3
ligase activity. The E3 ligase activity of MDM2 is dependent on its C-terminal RING
domain. E3 ligases containing a RING domain are traditionally thought to catalyse the
transfer of ubiquitin from their conjugating enzyme (E2) partner to the target protein, in the
final step of the ubiquitination cascade. Various E2 enzymes have been shown to interact
with their partner E3 ligases, yet evidence for the interaction between MDM2 and its partner
E2, UbcH5α has not yet been shown. It has been reported that the reason for this lack of
evidence is that the interaction between the two is highly unstable.
Here I show that MDM2 forms a stable isolatable interaction with UbcH5α, the C-terminal
tail of MDM2 is not necessary for this interaction. Although RING E3 ligases were not
previously thought to interact with ubiquitin, preliminary evidence is emerging that suggests
that this interaction is possible indeed I show that MDM2 and ubiquitin form a stable
complex. I demonstrate that UbcH5α and ubiquitin both interact with the RING of MDM2,
specifically the 20 most C-terminal amino acids of MDM2. My results show that both these
proteins can bind this region of the RING simultaneously. I also highlight specific residues
including tyrosine 489 and arginine 479 important for UbcH5α and ubiquitin binding
respectively and the negative affect that these mutations have on the E3 ligase activity of
MDM2 towards p53. Furthermore I show by limited proteolysis and hydrogen deuterium
exchange that UbcH5α can be allosterically activated by MDM2. A novel peptide phage
display technique linked to next generation sequencing was developed to further confirm an
allosteric change and demonstrates that UbcH5α has different binding specificity for peptides
when in a free or ligand bound conformation.
MDM2 is a popular target for cancer therapeutics due to its dysregulation throughout many
cancer types, including 30% of soft tissue sarcomas. Dissecting the mechanism of MDM2
function is an important step in identifying specific drugable interfaces on MDM2 and its
interacting partners so that effective therapeutics can be designed
