Fig. 2: KRAS Q61H is insensitive to GEF and GAP activities. | Nature Communications

Fig. 2: KRAS Q61H is insensitive to GEF and GAP activities.

From: The Q61H mutation decouples KRAS from upstream regulation and renders cancer cells resistant to SHP2 inhibitors

Fig. 2

a Intrinsic nucleotide-exchange curves of wild-type (WT) and mutant KRAS obtained from real-time NMR experiments. A tenfold molar excess of GTPγS was added to GDP-loaded KRAS at t = 0 and the GDP-bound fraction remaining was monitored over time by NMR. The data were fit to one-phase exponential decay functions. The nucleotide-exchange curves do not go to zero, because the nucleotide exchange equilibrates at a certain ratio, which is a function of the relative affinity of GDP:GTPγS. b Histogram of intrinsic exchange rates of each mutant determined in a, presented as fold changes for the mutants relative to wild-type KRAS. The error bars indicate the SD of the fold changes. c GEF-assisted nucleotide-exchange curves in the presence of SOScat (1 : 600 molar ratio SOS : KRAS), measured using real-time NMR. Data were fit as in a. d Histogram representing the mean intrinsic and SOScat (1 : 600) assisted exchange rates for wild-type KRAS and mutants obtained from three independent fitting curves for each mutation. Three pair of peak intensities for each group were used to generate the fitting curves. The error bars represent SEM of the three exchange rates. e A snapshot ribbon diagram of SOS1 (red) in complex with WT-KRAS (blue) (top panel). Time evolution of hydrogen bonds formed between the Thr935 of SOS and the sidechain of KRAS Q61 (bottom panel). f A snapshot ribbon diagram of SOS1 (red) in complex with KRAS Q61H (orange) (top panel). Time evolution of hydrogen bonds formed between the Thr935 of SOS and the sidechain of KRAS His61. g Intrinsic and GAP-assisted GTP hydrolysis curves for the wild type and Q61H mutant. The GAP domain of RASA1 was added at a ratio of 1 : 3000 to KRAS-GTP (a, c, e). h A ribbon diagram of RasGAP (blue) and KRAS Q61H (orange) catalytic site. Dots represent the fraction of GDP-loaded KRAS based on the intensities of GDP- and GTP-specific peaks (IGDP/IGDP + IGTPγS). a, b, g Error bars represent SEM of the three independent pairs of peak intensities used for the calculation.

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