Disordered Motifs and Domains in Cell Control
Poster Session I
10-POS
Board 10
The Use of Ion Mobility Mass Spectrometry to Probe Modulation of Function of p53 by a
Small Molecule Inhibitor
Eleanor Dickinson
1
, Joanna Zawacka-Pankau
2
, Galina Selivanova
2
, Perdita Barran
1
.
1
University of Manchester, Manchester, United Kingdom,
2
Karolinska Institutet, Stockholm,
Sweden.
The transcription factor, p53 is heavily implicated in tumour suppression pathways, blocking
tumour development by triggering cellular senescence or apoptosis. This partially disordered
protein is implicated in over 50% of tumours, its function often rendered inert by overexpression
of the ubiquitin E3 ligase MDM2, also intrinsically disordered. p53 binds to MDM2 directly via
their respective N-terminal domains. Release of p53 from MDM2 would reactivate the tumour
suppressor, providing an attractive cancer therapy drug target. The drug candidate RITA has
been shown to restore wild-type p53 function in tumour cells by preventing the p53/MDM2
interaction. In contrast to the previously reported inhibitor Nutlin which binds MDM2, RITA
binds to the p53 N-terminus. It is hypothesised that RITA binds outside of the p53/MDM2
binding cleft, allosterically exerting its effect via a conformational change in p53.
This study aims to probe the mechanism of RITA inhibition of p53 using a multi-technique
approach. We employ the gas phase techniques Mass Spectrometry (MS) and Ion Mobility-Mass
Spectrometry (IM-MS) to study the conformational space occupied by both wild-type and mutant
N-terminal p53 during RITA inhibition. Hydrogen deuterium exchange coupled to MS (HDX-
MS) is used to study the interaction in the solution phase. IM-MS measurements reveal that
RITA binds to p53 N-terminus transiently and with low affinity. Despite this it reconfigures the
wild-type protein to a unique compact structure. We hypothesise that this conformational change
in p53 prevents MDM2 from binding. HDX-MS data reveals the highly flexible nature of p53 N-
terminus and highlights the unique capability of IM-MS to capture transient structural
intermediates in dynamic protein systems.
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