Biophysical Characterization of RAS-SOS Complexes by Native Mass Spectrometry

RAS is regulated by specific guanine nucleotide exchange factors, such as Son of Sevenless (SOS), that activates RAS by facilitating the exchange of inactive, GDP-bound RAS with GTP. The catalytic activity of SOS is known to be allosterically modulated by an active, GTP-bound RAS. However, it remains poorly understood how oncogenic RAS mutants interact with SOS and modulate its activity. In this chapter, we describe the application of native mass spectrometry (MS) to monitor the assembly of the catalytic domain of SOS (SOS^cat) with RAS and cancer-associated mutants. Results from this approach have led to the discovery of different molecular assemblies and distinct conformers of SOS^cat engaging KRAS. It was also found that KRAS^G13D exhibits high affinity for SOS^cat and is a potent allosteric modulator of its SOS^cat activity. KRAS^G13D-GTP can allosterically increase the nucleotide exchange rate of KRAS at the active site by more than twofold compared to the wild-type protein. Furthermore, small-molecule RAS•SOS disruptors fail to dissociate KRAS^G13D•SOS^cat complexes, underscoring the need for more potent disruptors targeting oncogenic RAS mutants. Taken together, native MS will be instrumental in better understanding the interaction between oncogenic RAS mutants and SOS, which is of crucial importance for development of improved therapeutics.