1. Switchable Control of Scaffold Protein Activity via Engineered Phosphoregulated Autoinhibition.
- Author
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Hazegh Nikroo A, Lemmens LJM, Wezeman T, Ottmann C, Merkx M, and Brunsveld L
- Subjects
- Phosphoric Monoester Hydrolases metabolism, Phosphorylation, Protein Binding, 14-3-3 Proteins chemistry, Peptides metabolism
- Abstract
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.
- Published
- 2022
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