Structural analysis of cancer- associated mutations in the Notch Ankyrin domain

Abstract

This thesis explores how changes in the Ankyrin domain of the Notch signalling pathway, specifically mutations linked to cancer, impact normal Notch activity and lead to irregular cellular signalling. The study centres around three key mutations—A2059V, A2060V (Su42c), and V2061L—known to disrupt normal Notch activity, ultimately contributing to aberrant cellular signalling. The study employs a mix of methods, including stability experiments, nuclear magnetic resonance (NMR) spectroscopy, and advanced computational modelling, to unravel the complex relationship between the structure and function of Notch in the three cancer-associated mutations. The investigation begins by establishing an efficient protein expression protocol, optimising conditions for the Drosophila Notch Ankyrin domain to ensure robust sample quality. Subsequently, stability experiments provide crucial insights into the comparative stability profiles of the mutants, revealing A2059V's notable instability, A2060V's surprising stability akin to WT despite impaired Notch signalling, and V2061L's enhanced stability correlating with reduced Notch activity. NMR spectroscopy shows the structural nuances of the WT and mutant Ankyrin domains, shedding light on their folding patterns and dynamic interactions with the Dx protein. The study then uses advanced computational modelling, including AlphaFold and GROMACS, to predict and analyse the 3D structures and dynamic behaviour of the WT Ankyrin domain and its mutants. Computational findings validate experimental observations, highlighting key structural motifs and conformational changes associated with the mutations. These observations also indicate that crystal contacts may be influencing local conformation in the WT Ankyrin domain. Molecular dynamics simulations show some dynamic transitions in the Notch Ankyrin domain, offering deeper insights into its flexibility and potential implications on cellular functionality. Through a comprehensive analysis of experimental and computational results, this thesis advances our understanding of how structural alterations induced by specific mutations in the Notch Ankyrin domain contribute to the dysregulation of Notch signalling in cells, leading to possibly fatal outcomes. The nuanced relationships between structural stability, flexibility, and functional outcomes provide a foundation for future investigations aiming to decipher the intricate mechanisms underlying Notch-related pathologies.

Date of Award	28 May 2024
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Martin Baron (Main Supervisor) & Jonathan Waltho (Co Supervisor)