The GTPase κB-Ras is an essential subunit of the RalGAP tumor suppressor complex.

κB-Ras1 and κB-Ras2 are small GTPases with non-canonical features that act as tumor suppressors downstream of Ras. Via interaction with the RalGAP (GTPase activating protein) complex, they limit activity of Ral GTPases and restrict anchorage-independent proliferation. We here present the crystal structure of κB-Ras1 in complex with the N-terminal domain of RGα2. The structure suggests a mechanism of intrinsic GTP hydrolysis of κB-Ras1 that relies on a scaffolding function of the GTPase rather than on catalytic residues, which we confirm by mutational analysis. The interaction with RGα2 is nucleotide-independent and does not involve κB-Ras1 switch regions, which establishes κB-Ras proteins as a constitutive third subunit of RalGAP complexes. Functional studies demonstrate that κB-Ras proteins are not required for RalGAP catalytic activity in vitro, but for functionality in vivo. We propose that κB-Ras may thus act as regulator of RalGAP localization and thereby control the Ras/Ral signaling pathway.