C-RAF Sequence-Encoded Cooperativity Hotspots for 14-3-3/C-RAF Molecular Glues.

14-3-3 Proteins are hub proteins with an extensive interactome of phosphorylated client proteins, making them attractive targets for molecular glues (MGs) that stabilize specific 14-3-3/client protein complexes. However, selectively stabilizing individual 14-3-3 protein-protein interactions (PPIs) remains a key challenge for effectively drugging the 14-3-3 interactome. Here, we systematically investigated the sequence-encoded determinants underlying selective stabilization of the 14-3-3/C-RAF complex by covalent MGs. Using stepwise C-terminal peptide sequence extension, alanine scanning, and MG activity analysis on a large 14-3-3 peptide interactome panel, we demonstrate that residues beyond the canonical +1 and +2 positions contribute to cooperative ternary complex formation. The C-RAF +4Val and +6Met residues promote stabilization by an MG through sequence-dependent conformational arrangements that position the peptide backbone and enable the +6Met side chain to wrap around the MG, thereby reducing ternary complex dissociation. These findings provide general principles for the rational design of selective cooperative MGs targeting hub protein PPIs.