Small Molecule‐Induced and Cooperative Enzyme Assembly on a 14‐3‐3 Scaffold

Scaffold proteins regulate cell signaling by promoting the proximity of putative interaction partners. While frequently applied in a cellular setting, their fundamental understanding, amongst other in terms of quantitative parameters, has been lagging behind. Here we present a scaffold protein platform based on the native 14-3-3 dimeric protein and under the control of a small-molecule, which allows to be studied in an in vitro setting and mathematically described. Robust small-molecule regulation of caspase-9 activity via induced dimerization on the 14-3-3 scaffold was demonstrated. The individual parameters of this system were precisely determined and used to develop a mathematical model of the scaffolding concept. This model was used to elucidate the strong cooperativity of the 14-3-3 scaffold-mediated enzyme activation. This work provides entry into the long needed quantitative insights in scaffold protein functioning and paves the way for the optimal use of reengineered 14-3-3 proteins as chemical inducible scaffolds in synthetic systems.