Evolution and Engineering of Allosteric Regulation in Protein Kinases

Phosphoregulation – in which the addition of a negatively charged phosphate group modulates protein activity – is a common feature of proteins that enables dynamic cellular responses. To understand how new phosphoregulation might be acquired, we mutationally scanned the surface of a prototypical yeast kinase (Kss1) to identify potential regulatory sites. The data reveal a set of spatially distributed “hotspots” that coevolve with the active site and preferentially modulate kinase activity. By engineering simple consensus phosphorylation sites at these hotspots we rewired cell signaling in yeast. Following the same approach in a homolog (Hog1), we introduced new phosphoregulation that modifies localization and signaling dynamics. Beyond synthetic biology, the hotspots are used by the diversity of natural allosteric regulatory mechanisms in the kinase family and exploited in human disease.