NMR 1H, 13C, and 15N resonance assignments of the oncogenic Q61R variant of human NRAS in the active, GTP-bound conformation.

Abstract

NRAS^Q61R is a frequent mutation in melanoma. Hydrolysis of GTP by NRAS^Q61R 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 ¹H^N, ¹⁵N, and ¹³C backbone and sidechain resonance assignments of the G-domain of oncogenic NRAS^Q61R-GTP (MW 19.3 kDa; aa 1-169) using heteronuclear, multidimensional NMR spectroscopy. NRAS^Q61R-GTP is a conformationally stable protein in solution. The ¹H-¹⁵N correlation cross-peaks in a 2D ¹H-¹⁵N 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 ¹H-¹⁵N correlation cross-peaks for all aa residues, except Y40, in addition to a significantly large number of aliphatic and aromatic sidechain resonances. NRAS^Q61R-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-S²) values predicted by TALOS-N indicate that the NRAS^Q61R-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. ³¹P NMR experiments indicate that the G-domain of NRAS^Q61R-GTP is in a predominant state 2 (active) conformation.