Protein interaction kinetics delimit the performance of phosphorylation-driven protein switches

Abstract

AbstractPost-translational modifications (PTMs) such as phosphorylation and dephosphorylation can rapidly alter protein surface chemistry and structural conformation which can, in turn, switch protein-protein interactions (PPIs) within signaling networks. Recently,de novodesigned phosphorylation-responsive protein switches have been created that harness kinase- and phosphatase-mediated phosphorylation that modulate PPIs. PTM-driven protein switches could be useful for investigating PTM dynamics in living cells, developing biocompatible nanodevices, and engineering signaling pathways to program cell behavior. However, little is known about the physical and kinetic constraints of PTM-driven protein switches, which limits their practical application. In this study, we present a theoretical framework to evaluate two-component PTM-driven protein switches based on four performance metrics: effective concentration, dynamic range, response time, and reversibility. Our computational models reveal an intricate relationship between the binding kinetics, phosphorylation kinetics, and switch concentration that governs the sensitivity and reversibility of PTM-driven protein switches. Building upon the insights of our theoretical investigation, we built and evaluated two novel phosphorylation-driven protein switches consisting of phosphorylation-sensitive coiled coils as sensor domains fused to fluorescent proteins as actuator domains. By modulating the phosphorylation state of the switches with a specific protein kinase and phosphatase, we demonstrate fast, reversible transitions between easily differentiated “on” and “off” states. The response of the switches linearly correlated to the concentration of the kinase, demonstrating its potential as a biosensor for kinase measurements in real time. As intended, both switches responded to specific kinase activity with an increase in fluorescence signal and our model could be used to distinguish between two mechanisms of switch activation: dimerization or a structural rearrangement. In summary, the protein switch kinetics model presented here should be useful to guide the design of PTM-driven switches and tune their performance towards concrete applications.