| dc.description.abstract | Mutations in RAS are associated with many different cancers, and RAS has been a therapeutic target for more than three decades. Studying the biochemical properties of RAS mutants and most importantly the interaction with activator and downstream effectors lay a foundation to better understand how RAS functions in signaling pathway. In chapter 2, high-resolution native mass spectrometry (MS) was used to determine the kinetics and transition state thermodynamics of intrinsic hydrolysis for KRAS and oncogenic mutants. MS data reveal heterogeneity where both 2’-deoxy and 2’-hydroxy forms of GDP (Guanosine diphosphate) and GTP (Guanosine triphosphate) are bound to the recombinant enzyme. In addition, MS results show that the transition state thermodynamics for the intrinsic GTPase activity of KRAS is both enthalpically and entropically unfavorable. The oncogenic mutants, G12C, Q61H and G13D unexpectedly exhibit a higher 2’-deoxy GTP intrinsic hydrolysis rate compared to that for GTP.
In chapter 3, the interaction of RAS mutants with a specific guanine nucleotide exchange factor, Son of Sevenless (SOS), is studied. Native ion mobility-mass spectrometry was 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. Also, 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 two-fold compared to KRAS-GTP. Furthermore, small molecule RAS•SOS disruptors fail to dissociate KRASG13D•SOScat complexes underscoring the need for more potent disruptors.
Lastly, chapter 4 is focused on the biochemical characterization of BRAF, a downstream effector of RAS. Our results indicate that BRAF populates different stoichiometries with MEK1 and 14-3-3 dimers. In the presence of ATP, BRAF undergoes dimerization suggesting that ATP promotes the activation of BRAF. In addition, copper stimulates the dimerization of BRAF in MEK1-independent manner. | en |
