Extended Data Figure 6: The APS-2-79 binding site within KSR2 and possible basis for KSR over RAF selectivity.
From: Small molecule stabilization of the KSR inactive state antagonizes oncogenic Ras signalling
a, APS-2-79 and ATP are overlaid in the KSR2 and MEK1 active sites, respectively. ATP was shown here to emphasize the MEK active site, but ATP was not included in the final model. Positive (blue) and negative (red) Fo − Fc electron density maps, calculated before modelling of APS-2-79, are contoured at 3.5σ. Strong-positive-difference density within KSR2 supported modelling of APS-2-79 bound to KSR2 within the KSR2–MEK1 complex. b, Electron density map (blue mesh) for APS-2-79 (sticks) contoured at 4.5σ. Map represents positive difference density within the KSR2 active site before modelling of APS-2-79. c, Superposition of KSR2 (ATP- and APS-2-79-bound) with BRAF monomer (PDB code: 4W05) and BRAF dimer (PDB code: 3C4C) co-crystal structures reveals the possible bases for selectivity of APS-2-79 for KSR over RAF proteins. Residues within the APS-2-79 binding pocket that diverge between KSR and RAF proteins, but which are highly conserved within both sub-families are indicated with arrows. Thr802 in KSR2, which is universally a Gly residue in all active RAF homologues, and also Phe516 and Phe793 in KSR2, which adopt distinct orientations from the equivalent Phe residues in RAF kinases, directly contact the biphenyl ether motif in APS-2-79. The T802G substitution, as well as the positional differences of the above-mentioned aromatic residues, would be predicted to reduce binding of active RAFs with APS-2-79. Another interaction that is probably favoured in KSR includes the contact mediated by the epsilon nitrogen of Arg692 with the –O- linker of the biphenyl motif; the placement of Arg692 is stabilized by Asp803 of the DFG motif. In RAF, the Arg-to-Lys substitution (Lys483 in subdomain II of BRAF), lacks the equivalent nitrogens to bond with both the –O- linker in APS-2-79 and the aspartate of the DFG motif. d, Positive (blue) and negative (red) Fo − Fc electron density map contoured at ± 2.5σ, before modelling of residues I809 to Q814 in KSR2, is shown. e, Sequence alignment of KSR and RAF proteins. Arrows highlight APS-2-79 contact residues.
