A Molecular Mechanistic Deep Dive into 14–3–3Molecular Glue Cooperativity

Abstract Molecular glues (MGs) stabilize protein–protein interactions (PPIs) through interactions at composite binding interfaces, thereby promoting cooperative ternary complex formation. For hub proteins that engage in multiple PPIs with widely varying intrinsic affinities, the interplay between binary PPI affinity and MG cooperativity is therefore a key determinant of selective stabilization. Here, we use the multiclient 14–3–3 scaffold protein as a model system to systematically dissect the relationship between binary 14–3–3/client affinity (KDI) and MG-induced cooperativity (α). Client peptide affinity was systematically tuned by modifying residues N-terminal to the phosphorylated 14–3–3 binding motif while preserving the C-terminal composite interface required for MG recognition. Using a combination of biophysical techniques and protein crystallography, we show that changes in KDI alter the thermodynamic and kinetic parameters of both binary and ternary complex formation, but do not affect MG cooperativity. This principle was observed for the noncovalent MG fusicoccin-A as well as covalent MGs targeting 14–3–3σ/client complexes. Competitive binding experiments and thermodynamic modeling further revealed that, although α is independent of KDI, the interplay between KDI, MG affinity (KDII), and cooperativity determines which PPIs are preferentially stabilized in a multiclient environment. Together, these findings establish cooperativity, intrinsic PPI affinity, and MG affinity as key parameters governing MG activity and selectivity, providing a framework for the rational design of MGs targeting hub protein interactomes.