For over 30 years, Ras GTPases have been intensively studied in cancer research. Membrane bound Ras proteins (H-Ras, N-Ras, K-Ras 4A, and K-Ras 4B) function as a molecular switch that cycles between GDP “off” state and GTP “on” state. They regulate a diverse array of cellular pathways including proliferation, differentiation, and survival. Oncogenic Ras proteins are found in about 30% of all recorded cancer cases. K-Ras is the highest mutated Ras recorded than all other isoform at 85%. With also a 95% occurrence in pancreatic cancers, discovery for anti-Ras therapeutic strategies have been a highly sought out interest in cancer research. However, despite the research history and high presence in tumors, there is no effective cancer treatment for oncogenic Ras cases. failure to find any treatment is why Ras has been deemed the “undruggable” protein.
Contributing to the little success is that Ras is considered to have a relatively smooth and static surface from X-ray crystal structures. Over 140 crystal structures of Ras are deposited in PDB to date. These crystal structure shows minimal global change in residue position, indicating no plausible allosteric binding site. However, two important loop regions, named Switch I and Switch II, are highly dynamic and may adopt multiple confirmations. These loops regions can adopt an ensemble of excited-state confirmations that X-ray crystallography could not detect. Therefore, if there is an allosteric pocket for drug targets, the pocket would exist in an excited-state conformation.
Nuclear Magnetic Resonance (NMR) has the ability to detect protein dynamics in sparsely populated excited states. We can utilize the potential of NMR to study a wide range of dynamics at atomic resolution in order to monitor the perturbations in the switch loops, then compare these perturbations between wild-type and mutant K-Ras.
Here, I discuss preliminary NMR analysis of both GDP and GTP-bound WT K-Ras and the shortcomings on why traditional NMR methods are not viable to study the switch loops. I will also discuss alternative perspectives using novel NMR techniques to further understand how switch I and II behavior modulate K-Ras intrinsic dynamics and how mutations perturb such dynamics.