Switchable Control of Scaffold Protein Activity via Engineered Phosphoregulated Autoinhibition

Scaffold proteins operate as organizing hubs to enable high-fidelity signaling, fulfilling crucial roles in the regulation of cellular processes. Bottom-up construction of controllable scaffolding platforms is attractive for the implementation of regulatory processes in synthetic biology. Here, we present a modular and switchable synthetic scaffolding system, integrating scaffold-mediated signaling with switchable kinase/phosphatase input control. Phosphorylation-responsive inhibitory peptide motifs were fused to 14-3-3 proteins to generate dimeric protein scaffolds with appended regulatory peptide motifs. The availability of the scaffold for intermolecular partner protein binding could be lowered up to 35-fold upon phosphorylation of the autoinhibition motifs, as demonstrated using three different kinases. In addition, a hetero-bivalent autoinhibitory platform design allowed for dual-kinase input regulation of scaffold activity. Reversibility of the regulatory platform was illustrated through phosphatase-controlled abrogation of autoinhibition, resulting in full recovery of 14-3-3 scaffold activity.

Automated summary

The study presents a modular and switchable synthetic scaffolding system that integrates scaffold-mediated signaling with switchable kinase/phosphatase input control. By fusing phosphorylation-responsive inhibitory peptide motifs with 14-3-3 proteins, dimeric protein scaffolds with appended regulatory peptide motifs were generated. The availability of the scaffold for intermolecular partner protein binding could be regulated by phosphorylation of the autoinhibition motifs, allowing precise control of signaling.