Extended Data Fig. 6: PP1C interaction sites.
From: Structural basis for SHOC2 modulation of RAS signalling
a, Overview of technical principle of peptide dephosphorylation based affinity assays. At a fixed concentration of peptide, binding of SHOC2 and RAS will induce a higher PP1C catalytic activity. Data points are from two time points (n = 2) b, Representative plot of complex formation with different SHOC2 mutants. [PP1C] = 25 nM, [MRAS(GCP)] = 1 µM. Data points are from two time points (n = 2) (left). Table summarizing KDapp values for different SHOC2 mutants from three independent experiments (n = 3) (right). Mutation of SHOC2 charged patches reduces affinity beyond the limit of detection of the assay. c, Left, SLiM binding site (blue) on PP1C (purple), with structure of several interacting SLiMs (white), “Opposing” site (yellow) with structures of several interacting proteins (white). d, Detailed view of the SHOC2 N terminal “VAF” motif (green, left) binding to PP1C with unsharpened cryoEM map (grey surface). Alignment of an existing high resolution x-ray crystal structure containing an RVxF motif bound in this region (yellow, right, from PDB: 5INB) overlays well with the cryoEM map (grey surface). Alignments performed on PP1C. e, SHOC2:PP1C:MRAS cryoEM structure with SLiM (blue) and “opposing” (yellow) binding sites highlighted. The majority of SHOC2 interactions with PP1C are away from the SLiM binding and opposing sites. The SHOC2 “VAF” motif binds to the SLiM binding site. MRAS binds solely to the “opposing” site. f, SDS22 (white) is another LRR protein which interacts with PP1C, though utilizing different binding surfaces to SHOC2, bridging the SLiM and opposing sites.
