Kinases function as signallers within the cell; using phosphorylation of the proteins with which they interact as molecular switches to perform functions in cells. Outside of their core catalytic domain, kinases can contain intrinsically disordered regions (IDRs) as part of their structure or interact with intrinsically disordered proteins (IDPs). The disorder, or lack of stable structure, of IDRs and IDPs can result in a protein or protein region being without any structure, or dynamically processing between stable structures, giving rise to an ensemble of conformations. The disorder and dynamic properties of such proteins makes the structural biologists’ task of elucidating their molecular mechanism harder. But this is pressing work in the context of kinases, since their dis- and mis- regulation is a common trait of cancer, and drugging them is a topic of considerable interest. In this thesis, I investigate the value of chemical crosslinking mass spectrometry (XL-MS) in studying dysregulated kinases that feature IDRs or dynamic conformations. We used the mitotic serine/threonine kinase Aurora-A (AurA) and its activating intrinsically disordered partner protein TPX2 as a testbed for kinases as a target type, to explore the practicalities and limits of applying XL-MS to such a dynamic system.
XL-MS is an in-solution technique that uses a bi-functional reagent to generate distance restraints between the reactive side chains of residues that can be mapped to previously generated structures or models. We used the mass cleavable amine and hydroxyl reactive crosslinking disuccinimidyl dibutyric urea (DSBU), in conjunction with the software MeroX, to demonstrate that it was possible to generate useful structural data for dynamic kinases and their complexes, with this requiring good crosslink coverage. We found that the use of crosslink and monolink scoring in conjunction with MD simulations of kinase structures/conformers and literature data were required to adequately describe this system.
Ultimately, this thesis demonstrates the value of this methodology, as one that is both cost-effective and time efficient to study kinase structure and function, so long as it is used in combination with structural techniques that disentangle the conformational ensemble.
