Enzymatic Regulation of Protein-Protein Interactions in Artificial Cells

Membraneless organelles are important for spatial organization of proteins and regulation of intracellular processes. Proteins can be recruited to these condensates by specific protein–protein or protein–nucleic acid interactions, which are often regulated by post-translational modifications. However, the mechanisms behind these dynamic, affinity-based protein recruitment events are not well understood. Here, a coacervate system that incorporates the 14-3-3 scaffold protein to study enzymatically regulated recruitment of 14-3-3-binding proteins is presented, which mostly bind in a phosphorylation-dependent manner. Synthetic coacervates are efficiently loaded with 14-3-3, and phosphorylated binding partners, such as the c-Raf pS233/pS259 peptide (c-Raf), show 14-3-3-dependent sequestration with up to 161-fold increase in local concentration. The c-Raf domain is fused to green fluorescent protein (GFP-c-Raf) to demonstrate recruitment of proteins. In situ phosphorylation of GFP-c-Raf by a kinase leads to enzymatically regulated uptake. The introduction of a phosphatase into coacervates preloaded with the phosphorylated 14-3-3-GFP-c-Raf complex results in a significant cargo efflux mediated by dephosphorylation. Finally, the general applicability of this platform to study protein–protein interactions is demonstrated by the phosphorylation-dependent and 14-3-3-mediated active reconstitution of a split-luciferase inside artificial cells. This work presents an approach to study dynamically regulated protein recruitment in condensates, using native interaction domains.</p

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

Modular bioengineered kinase sensorsviascaffold protein-mediated split-luciferase complementation

Phosphorylation is a key regulation event in cellular signaling. Sensing the underlying kinase activity is of crucial importance for its fundamental understanding and for drug development. For this, modular kinase activity sensing concepts are urgently needed. We engineered modular serine kinase sensors based on complementation of split NanoBiT luciferase on protein assembly platforms generated from the scaffold protein 14-3-3. The bioengineered platforms are modular and easy adaptable as exemplary shown using novel sensors for the kinases PKA, PKB, and CHK1. Two designs were conceptualized, both relying on binding of defined mono- or bivalent kinase recognition motifs to the 14-3-3 platform upon phosphorylation, resulting in reconstitution of active split-luciferase. Especially the design based on double phosphorylation and bivalent 14-3-3 binding exhibits high efficiency for signal amplification (>1000-fold) and sensitivity to specific kinases, including in cellular lysates