Fig. 2. KRAS^Q61 proteins show distinct biochemical phenotypes.

(A) Quantification of nucleotide exchange rates of recombinant KRAS^Q61 mutant proteins (amino acids 2-169) in the absence (left) and in the presence of equimolar concentration of the catalytic domain of RASGRP1. Data are mean ± S.E.M. from three or more independent experiments. ** P ≤ 0.01 and *** P ≤ 0.001 by one-way ANOVA. (B) Quantification of pulldown assay for KRAS-GTP levels in RIE-1 cells using CRAF-RBD shown as mean ± S.E.M. of three independent experiments. * P ≤ 0.05 **, P ≤ 0.01, and *** P ≤ 0.001 by one-way ANOVA. Error bars, (C) Ribbon diagram showing X-ray structural overlays of KRAS^Q61E (teal, 7LZ5) with KRAS^WT (silver, 4DSO). Proteins were crystallized bound to non-hydrolyzable GMPPCP. The Q61 sidechain is indicated in red and the E61 sidechain is indicated in blue. (D) KRAS^Q61E NMR chemical shifts resemble KRAS^WT. ¹H-¹⁵N HSQC NMR overlay of KRAS^WT (red) and KRAS^Q61E (blue) in the GMPPCP-bound (left) and GDP-bound (right) states. Data are representative of two biological replicates. (E) Relative binding affinities of KRAS^Q61 proteins to select RAS binding (RBD) and association (RA) domains. Values were normalized to KRAS^WT for each indicated effector. Data are averages from three or more independent experiments.