1H, 13C and 15N backbone assignments for oncogenic RAS Q61 mutants: NRAS Q61R, NRAS Q61K and KRAS Q61R

Activating NRAS Q61R and Q61K mutations are prevalent in cutaneous melanoma and thyroid cancer, with NRAS Q61 mutations representing a major oncogenic driver of melanoma. Despite their importance, oncogenic NRAS mutants remain relatively understudied compared with KRAS and HRAS. Most structural studies of active-state RAS proteins have used X-ray crystallography, as a major limitation of NMR is the absence of backbone resonances from the functionally critical Switch and P-loop regions, which undergo broadening due to conformational exchange on the NMR timescale. Here, we present NMR investigations of GTPγS-bound NRAS Q61R and Q61K to provide a defined active-state system for comparison with WT, G12D, and other hydrolysis-competent RAS proteins. Notably, the highly oncogenic NRAS Q61R and Q61K mutants show observable resonances from both the Switch and P-loop regions, suggesting that these highly melanogenic Q61 mutations alter the conformational dynamics of these functionally critical regions. To determine whether these findings are specific to NRAS, we further obtained backbone NMR resonance assignments for KRAS Q61R and found that this KRAS oncogenic mutant also exhibits observable Switch and P-loop backbone resonances. These findings indicate that the altered dynamics of the Switch regions are associated with the Q61R mutation rather than being isoform-specific. Here, we present the ¹H, ¹³C, and ¹⁵N backbone assignments for active-state NRAS Q61R (93% completeness), NRAS Q61K (86% completeness), and KRAS Q61R (94% completeness). These assignments provide a spectroscopic framework for investigating mutation- and isoform-dependent differences in RAS conformational dynamics and facilitate NMR-based structural and drug-discovery studies of oncogenic RAS proteins.