NMR H-1, C-13, N-15 backbone resonance assignments of the T35S and oncogenic T35S/Q61L mutants of human KRAS4b in the active, GppNHp-bound conformation

RAS proteins cycling between the active-form (GTP-bound) and inactive-form (GDP-bound) play a key role in cell signaling pathways that control cell survival, proliferation, and differentiation. Mutations at codon 12, 13, and 61 in RAS are known to attenuate its GTPase activity favoring the RAS active state and constitutively active downstream signaling. This hyperactivation accounts for various malignancies including pancreatic, lung, and colorectal cancers. Active KRAS is found to exist in equilibrium between two rapidly interconverting conformational states (State1-State2) in solution. Due to this dynamic feature of the protein, the H-1-N-15 correlation cross-peak signals of several amino acid (AA) residues of KRAS belonging to the flexible loop regions are absent from its 2D H-1-N-15 HSQC spectrum within and near physiological solution pH. A threonine to serine mutation at position 35 (T35S) shifts the interconverting equilibrium to State1 conformation and enables the emergence of such residues in the 2D H-1-N-15 HSQC spectrum due to gained conformational rigidity. We report here the H-1(N), N-15, and C-13 backbone resonance assignments for the 19.2 kDa (AA 1-169) protein constructs of KRAS-GppNHp harboring T35S mutation (KRAS(T35S/C118S)-GppNHp) and of its oncogenic counterpart harboring the Q61L mutation (KRAS(T35S/Q61L/C118S)-GppNHp) using heteronuclear, multidimensional NMR spectroscopy at 298 K. High resolution NMR data allowed the unambiguous assignments of H-1-N-15 correlation cross-peaks for all the residues except for Met1. Furthermore, 2D H-1-N-15 HSQC overlay of two proteins assisted in determination of Q61L mutation-induced chemical shift perturbations for select residues in the regions of P-loop, Switch-II, and helix alpha 3.

Automated summary

RAS proteins play a crucial role in cell signaling pathways, with mutations affecting its activity and leading to cancer. The dynamic nature of active KRAS protein results in absence of certain amino acid residues' H-1-N-15 correlation cross-peak signals in the 2D spectrum, but mutations can alter conformation dynamics.