RalA and RalB, members of the Ras superfamily, play a pivotal role in tumour progression in Ras-driven cancers, hence there is a growing need to understand these proteins in terms of their structure and function to facilitate future pharmaceutical intervention in this critical oncogenic pathway.

So far, biophysical characterisation of Ral proteins has typically been carried out with truncated constructs that exclude the C-terminal ~20 residues. However, this C-terminal hypervariable region is not only the main source of variation between RalA and RalB, but also serves as a membrane anchor via a polybasic region and prenylation and has been suggested to play key roles in modulation of the function of these proteins. A more robust understanding of this hypervariable region and its function is required before attempting to unpick how it may translate into the biological differences exhibited between RalA and RalB.

This project uses solution NMR spectroscopy and nanodiscs as membrane mimetic tools to investigate the interaction of RalA with biological membranes at the molecular level. Clear differences were found in how RalA interacts with the membrane mimic based on the nucleotide status of the protein and the lipid composition of the nanodiscs. Furthermore, phosphomimic mutations were used to investigate the effects of phosphorylation of RalA at Ser194 by Aurora kinase on membrane interactions. Phosphorylation of this residue appears to have consequences for membrane interaction that extend beyond the HVR to the G-domain of the protein, demonstrating that this short intrinsically disordered region of the GTPase could hold the key to unlocking the complex regulation of these proteins in vivo.

There has been growing interest in the interaction between the HVR of certain small GTPases and Calmodulin but as of yet, no structures are available of Calmodulin in complex with these singly lipidated and polybasic motifs. This study has used solution NMR, in tandem with other biophysical techniques such as SPR and ITC, to elucidate the structure of the RalA HVR in complex with Calmodulin and the molecular basis for recognition of this GTPase in a membrane environment. The interaction between Calmodulin and RalA levers the prenyl anchor out of the lipid bilayer and results in dissociation of RalA from the membrane. The functional consequence of this interaction is still unclear but raises questions about where this molecular taxi is taking RalA, and why. Preliminary work into the interaction between RalB and Calmodulin has revealed fundamental differences in the binding of these two GTPases to this ubiquitous calcium-sensor with this posing interesting questions about whether the differences in binding to Calmodulin could underpin the functional diversity displayed by RalA and RalB in vivo.