Molecular assemblies of the catalytic domain of SOS with KRas and oncogenic mutants

Menée in vitro à l'aide de la spectrométrie de masse et de la cryo-microscopie électronique, cette étude analyse les interactions moléculaires entre le domaine catalytique du facteur d'échange nucléotidique "Son of Sevenless" et les formes mutées oncogènes de la protéine KRAS

Résumé en anglais

Disrupting the interaction between Ras and SOS has emerged as an attractive therapeutic strategy. Here, we characterized the assembly of the catalytic domain of SOS (SOScat) with oncogenic KRas mutants using native mass spectrometry. By resolving distinct molecular species, we show KRas mutants engage SOScat differently and the G13D KRas mutant robustly engages SOScat. A structure of KRasG13D in complex with SOScat shows repositioning of switch I and II regions within KRasG13D. Moreover, potent small-molecule Ras•SOS disruptors do not dissociate KRasG13D•SOScat complexes when KRasG13D–GTP is bound at allosteric site of SOScat. These results underscore the need for more potent Ras•SOS disruptors when considering targeting the disruption of different oncogenic Ras mutants in complex with SOS.Ras is regulated by a specific guanine nucleotide exchange factor Son of Sevenless (SOS), which facilitates the exchange of inactive, GDP-bound Ras with GTP. The catalytic activity of SOS is also allosterically modulated by an active Ras (Ras–GTP). However, it remains poorly understood how oncogenic Ras mutants interact with SOS and modulate its activity. Here, native ion mobility–mass spectrometry is employed to monitor the assembly of the catalytic domain of SOS (SOScat) with KRas and three cancer-associated mutants (G12C, G13D, and Q61H), leading to the discovery of different molecular assemblies and distinct conformers of SOScat engaging KRas. We also find KRasG13D exhibits high affinity for SOScat and is a potent allosteric modulator of its activity. A structure of the KRasG13D•SOScat complex was determined using cryogenic electron microscopy providing insight into the enhanced affinity of the mutant protein. In addition, we find that KRasG13D–GTP can allosterically increase the nucleotide exchange rate of KRas at the active site more than twofold compared to KRas–GTP. Furthermore, small-molecule Ras•SOS disruptors fail to dissociate KRasG13D•SOScat complexes, underscoring the need for more potent disruptors. Taken together, a better understanding of the interaction between oncogenic Ras mutants and SOS will provide avenues for improved therapeutic interventions.All study data are included in the article and/or SI Appendix.