A Structural and Bio chemical Insight into RAS Mediated Activation of S mall G Protein GEF

Ras belongs to a family of small G-proteins and acts as a signalling hub, initiating multiple downstream pathways. These include MAPK, PI3K, PLC, and small GTPases, RalA/B in humans. Accumulating evidence suggests that RalA and RalB, and their activators, Ral guanine nucleotide exchange factors (RalGEFs), play an essential role in oncogenic Ras-induced signalling. This is also the case in fission yeast, where a constitutively-active RAS impacts little on the MAPK pathway but induces prolonged activation of a small G-protein Cdc42 through its GEF, Scd1 (Kelsall et al., 2019). Therefore, it is important to obtain more structural insights into the interaction between RAS and small G-protein GEFs, the less extensively studied Ras effectors. In this project, I first examined the interaction between fission yeast Ras, Ras1, and Scd, hoping to reveal a conserved feature of the interaction between Ras and small G-GEF in this highly tractable model organism. I next examined the interaction between human KRas and Rgl2, the most ubiquitously expressed RalGEF across different cell types and tissues (Catozzi et al., 2021). NMR analysis showed the interaction between oncogenic Ras1.G17V and Scd1, and oncogenic KRas.G12V and Rgl2-RBD, confirming that the Ras-mediated small G-protein GEF activation is a highly conserved cellular process. Strikingly, biolayer interferometry (BLI) assay revealed oncogenic V12 mutation increased KRas affinity to Ras binding domain (RBD) of Rgl2. Crystal structure of a 2:2 heterotetramer KRas.G12V:Rgl2-RBD complex revealed that the V12 oncogenic mutation resides at the KRas.G12V- KRas.G12V interface of the complex, providing a structural explanation for the complex stabilisation by the oncogenic G12V mutation. Finally, the heterotetramer formation was confirmed by the mass-photometry measurement. Collectively, the study reports a distinct Ras-effector interface, which can be exploited as a novel therapeutic target to disrupt the oncogenic activation of the RalA/B pathway.