Enzymatic Regulation of Protein–Protein Interactions in Artificial Cells

Abstract

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.

Competitive protein recruitment in artificial cells

“…1 ). This local concentration represents a 350-fold enhancement of concentration compared to the bulk concentration of 100 nM and is comparable to the 14-3-3 loading that was determined in our previous work 26 . The fluorescein isothiocyanate (FITC)-labeled bivalent c-Raf pS233/pS259 (c-Raf pS) client peptide was added to the separate populations of coacervates at a concentration of 25 nM yielding a 14-3-3/c-Raf pS binding site ratio of 2:1 (Fig.…”

“…Although we have previously used a bioluminescence assay for PPIs in coacervates 26 , confocal microscopy is more suited for determining the localization of a client in a multi-population coacervate sample over time since it additionally provides spatial information and is not enzyme substrate-dependent. Confocal micrographs of the single population coacervates with the different 14-3-3 isoforms showed that, indeed, similar recruitment levels of c-Raf pS were observed after overnight incubation (Fig.…”

“…Exchange or recruitment of client proteins in synthetic cells can also be governed by protein-protein interactions (PPIs) 23 – 27 . For us, in particular interactions of client proteins with the hub protein 14-3-3, a native protein which is important for the regulation of many signaling pathways 28 , 29 , yielded specific uptake based on affinity for 14-3-3 26 . 14-3-3 generally binds to serine/threonine phosphorylated client proteins, although there are also examples of nonphosphorylated binding motifs in client proteins, such as the 14-3-3-binding motif of the bacterial toxin Exoenzyme S 30 , 31 .…”

Rational design and engineering of polypeptide/protein vesicles for advanced biological applications

“…Consequently, the incorporation of proteins into synthetic vesicles has attracted massive attention for advanced biological applications, including drug delivery vehicles 21 and artificial cell platforms. 22,23…”