Fig. 3: Rasopathy mutations and RAF binding predicted to affect complex formation.

a, Surface representation of the SHOC2–PP1Cα–MRAS holoenzyme highlighting the active site and accessible acidic (red), C-terminal (black) and hydrophobic (yellow) substrate-binding grooves of PP1Cα. The RVXF and other regulatory surfaces of PP1Cα are not occupied in the SHOC2(80–582)–MRAS(Q71R, 1–178)–PP1Ca(7–300) ternary complex. b, GOF rasopathy mutations localize to PPI interfaces. The indicated GOF and LOF rasopathy mutations are shown as sticks highlighted in yellow and orange, respectively; SHOC2 and MRAS are shown in grey with SWI and SWII indicated. SHOC2 Ile173 (Met173 in WT SHOC2) fills a hydrophobic space produced by SWI, SWII and SHOC2 LRR domains. SHOC2 Gln269/His270 resides at the interface of all three subunits, whereas SHOC2 Asp175 coordinates a water to MRAS Tyr81. PP1Cα Pro50 sits at the PPI interface but cannot make substantial interactions owing to the limitations of the side chain. c, Schematic of the M2H system (left). The Nluc reporter signal resulting from concurrent expression and interaction between GAL4(DBD)–MRAS and SHOC2–VP16(TAD) of WT and mutant variants (right). Data are mean ± s.e.m. n = 3 independent biological replicates. The diagram was created with BioRender.com. d, Alignment of the MRAS–SHOC2–PP1C ternary holophosphatase structure with a structure of KRAS bound to the CRAF RBD-CRD domain (PDB: 6XHA). Note that the alignment (using RAS as the key object) results in multiple clashes between RBD-CRD and PP1C–SHOC2. e, Untagged full-length SHOC2 disrupts the interaction of MRAS(Q71R)–GppNHp and RBD as assessed in a TR-FRET assay (IC₅₀ 913nM), indicating that SHOC2 and RBD binding to RAS is incompatible. Technical replicates are shown as individual data points; one of three independent experiments is shown. Competition IC₅₀ values for additional tested combinations are provided in Extended Data Table 3.