Disordered Motifs and Domains in Cell Control - October 11-15, 2014

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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