NMR H, C, and N resonance assignments of the oncogenic Q61R variant of human NRAS in the active, GTP-bound conformation.

NRAS is a frequent mutation in melanoma. Hydrolysis of GTP by NRAS is reported to be much slower than other KRAS and NRAS mutants. Recent structural biology efforts for KRAS and NRAS proteins have been limited to X-ray crystallography and therefore lack insight into the structure and dynamics of these proteins in solution. Here we report the H, N, and C backbone and sidechain resonance assignments of the G-domain of oncogenic NRAS-GTP (MW 19.3 kDa; aa 1-169) using heteronuclear, multidimensional NMR spectroscopy. NRAS-GTP is a conformationally stable protein in solution. The H-N correlation cross-peaks in a 2D H-N HSQC spectrum collected after 48 h at 298 K remained intact and only minimal signs of peak-broadening were noted for select residues. High resolution NMR allowed unambiguous assignments of the H-N correlation cross-peaks for all aa residues, except Y40, in addition to a significantly large number of aliphatic and aromatic sidechain resonances. NRAS-GTP exhibits canonical secondary structural elements in the 5 (five) α-helices, 6 (six) β-strands, and associated loop regions as predicted in TALOS-N and CSI. Order parameter (RCI-S) values predicted by TALOS-N indicate that the NRAS-GTP switch (SW) regions and overall backbone are less flexible than observed in KRAS4b-GTP. The SW region rigidification was validated in heteronuclear NOE measurements. P NMR experiments indicate that the G-domain of NRAS-GTP is in a predominant state 2 (active) conformation.