Interactive Functional Dynamics of Human K-Ras, Its Oncogenic Mutants and their Binding Partners

About This Grant

PROJECT SUMMARY The overall goal of this project is the comprehensive structural and dynamic characterization of the highly flexible human K-Ras oncoprotein and its interactions with protein-binding partners and small-molecule ligands in both its normal and dysregulated states by the combined use of state-of-the-art experimental and computational methods. K-Ras is well-known to be exceptionally susceptible to carcinogenic mutations in certain key amino-acid positions, which through a cascade of protein signaling processes dysregulate cell proliferation. K-Ras is directly associated with about 25% of all human cancers. As a GTPase, K-Ras is a molecular switch with wild-type K-Ras being in its on-state when bound to GTP and turning to the off-state through catalytic hydrolysis of GTP to GDP. In oncogenic mutants, such as G12C, G12D, and G12V, K-Ras is perpetually locked into active signaling of the Ras-Raf-MEK-ERK pathway leading up to malignancy. This project builds on recent breakthroughs in the applicant’s lab overcoming two key obstacles that have severely impaired past efforts to understand K-Ras and its interactions with its protein partners and potential drug ligands: they made the functionally critical Switch I and Switch II regions of K-Ras fully visible and assigned by NMR and overcame the need to work with non-hydrolyzable GTP-analogs instead of native GTP as an integral K-Ras ligand. Leveraging these advances, it is proposed to comprehensively investigate the structural-dynamics ensembles of K-Ras in the presence and absence of its functionally critical protein binding partners and small-molecule ligands by NMR and computational modeling. This entails the full characterization of the modes of interactions of K-Ras wild-type vs. G12 mutants and GTP- vs. GDP-bound with the small-molecule drug candidate MRTX1133, protein GTPase activating protein (GAP), and the RBD and CRD domains of the downstream signaling protein-kinase B-Raf. The wealth of quantitative NMR data at atomic resolution will give novel information essential for our understanding of the driving forces underlying K-Ras and its function in health and disease. These data will provide powerful synergies with computational approaches, such as AlphaFold and extended molecular dynamics computer simulations for obtaining a realistic, experimentally validated in silico description of K-Ras behavior in the presence of its binding partners for wild-type and the mutants. Validated conformational ensembles will be subsequently mined for allosteric effects and used for virtual ligand screening including cryptic pockets uncovered during this process. Due to its highly dynamic nature, the fully quantitative atomic-level structural-dynamic model of K-Ras and its binding partners is likely to be directly beneficial enabling the discovery of key molecular determinants of K- Ras cancer biology and guiding the design of new therapeutic strategies to silence mutationally activated K-Ras.