Structural and biochemical insights into heterotetramer formation of oncogenic K-Ras4B^G12Vand Rgl2, a RalA/B activator

AbstractAbout a quarter of total human cancers carry mutations in Ras isoforms. Accumulating evidence suggests that small GTPases, RalA and RalB, and their activators, Ral guanine nucleotide exchange factors (RalGEFs), play an essential role in oncogenic Ras-induced signalling. We studied the interaction between human K-Ras4B and the Ras binding domain (RBD) of Rgl2 (Rgl2RBD), one of the RBD-containing RalGEFs. We show that the G12V oncogenic K-Ras4B mutation increases the affinity with Rgl2RBD. The crystal structure of the K-Ras4BG12V: Rgl2RBDcomplex shows a 2:2 heterotetramer where the Switch I and Switch II regions of each K-RasG12Vinteract with both Rgl2RBDmolecules. This structural arrangement is highly similar to the H-RasE31K:RALGDS crystal structure and is distinct from the well-characterised Ras:RAFRBDcomplexes. Importantly, the G12V mutation was found at the dimer interface of K-Ras4BG12Vwith its partner. Solution state NMR and mass photometry analyses support the heterotetramer formation. Our study reveals a distinct mode of Ras:effector complex formation by RalGEFs, and offers a possible mechanistic explanation for how the oncogenic K-Ras4BG12Vhyperactivates the RalA/B pathway.