De novo design of a reversible phosphorylation-dependent switch for membrane targeting

Modules that switch protein-protein interactions on and off are essential to develop synthetic biology; for example, to construct orthogonal signaling pathways, to control artificial protein structures dynamically, and for protein localization in cells or protocells. In nature, the E. coli MinCDE system couples nucleotide-dependent switching of MinD dimerization to membrane targeting to trigger spatiotemporal pattern formation. Here we present a de novo peptide-based molecular switch that toggles reversibly between monomer and dimer in response to phosphorylation and dephosphorylation. In combination with other modules, we construct fusion proteins that couple switching to lipid-membrane targeting by: (i) tethering a 'cargo' molecule reversibly to a permanent membrane 'anchor'; and (ii) creating a 'membrane-avidity switch' that mimics the MinD system but operates by reversible phosphorylation. These minimal, de novo molecular switches have potential applications for introducing dynamic processes into designed and engineered proteins to augment functions in living cells and add functionality to protocells. The ability to dynamically control protein-protein interactions and localization of proteins is critical in synthetic biological systems. Here the authors develop a peptide-based molecular switch that regulates dimer formation and lipid membrane targeting via reversible phosphorylation.The authors thank the Biochemistry Core Facility of the Max Planck Institute of Biochemistry for LC-MS and CD spectroscopy services, Stefan Pettera and Stephan Uebel for assistance with peptide synthesis and analytical HPLC, and Katharina Nakel for assistance with cloning