Extended Data Fig. 8: RAS binding interactions.
From: Structural basis for SHOC2 modulation of RAS signalling
a, SEC trace showing SHOC2:PP1C:KRAS cannot interact with BRAF RBD (green) or CRAF RBD (orange) (top) with SDS-PAGE analysis of SEC fractions (bottom). A FLAG tag on the CRAF RBD increases its mass slightly compared to the BRAF RBD. Results representative of two independent experiments b, BRAF pS365 peptide dephosphorylation by PP1C at varying SHOC2 and KRAS concentrations shows a dependence of the Kdapp of one binding partner on concentration of the other. Data points are from two time points (n = 2). c, Dephosphorylation of BRAF pS365 peptide (0.2 mM) by PP1C:SHOC2 in the presence of varying RAS concentrations. Data points are from two time points (n = 2). MRAS exhibits a lower KDapp than KRAS, though also shows decreasing activity at higher concentrations, possibly because higher concentrations of the SHOC2:PP1C:MRAS trimer promote formation of a SHOC2:PP1C:MRAS hexamer which may be catalytically inactive. [PP1C] = 10 nM, [SHOC2] = 100 nM. d, Sequence alignment between M, K, H and NRAS N termini, Switch I and II (top) and C termini (bottom) e, Top: TR-FRET measuring association between PP1C and SHOC2 in the presence of different GCP bound MRAS/KRAS chimeras, showing that 8 residues on MRAS which contact PP1C (see yellow highlighted residues in (b)) are critical for high binding affinity. Neither the MRAS N nor C termini improve KRAS binding affinity. The KRAS:MRAS chimera showed a modest but statistically significant binding affinity improvement compared to KRAS WT (103 µM vs 128 µM, p = 0.02), but did not fully recapitulate the strong affinity of MRAS WT, showing that other factors such as MRAS Switch I and II dynamics are also important in driving complex formation. Data points are from measurements taken at two time points (n = 2) (top). Bottom: Table summarizing KDapp values from three independent experiments (n = 3). [Tb-PP1C] = 2 nM, [Red-SHOC2] = 200 nM. f, chronos score correlations plotted separately for HRAS, KRAS and NRAS vs SHOC2. Main Fig. 3c represents the combination of these plots, with the lowest of the three H/K/NRAS chronos scores for each cell line selected for display in Fig 3c.
