Allosteric regulation, the process by which a protein’s activity can be modulated by binding of an effector molecule distal to the active site, is vital for cellular signaling. However, its evolution is largely unexplored territory. I will describe our experimental exploration of the evolution over 1.5 billion years of two allosteric regulation mechanisms widely found in the modern protein kinase superfamily, phosphorylation of the activation loop and binding of a regulatory partner protein. Using Ancestral Sequence Reconstruction (ASR) we unravel the origins of allosteric activation including surprising mechanistic features. Moreover, ASR enabled identification of the underlying allosteric network that spans the kinase from the N-terminal to the C-terminal lobes. In the second part of the talk I describe how we exploit this new knowledge for the development of allosteric inhibitors and activators. This latter approach delivered novel kinase inhibitors and activators with extreme specificity and high affinity thereby opening the road to new cancer treatment. Third, I will address the evolution of enzyme catalysis in response to one of the most fundamental evolutionary drivers, temperature. Using ASR, we answer the question of how enzymes coped with an inherent drop in catalytic speed caused as the earth cooled down over 3.5 billion years. Tracing the evolution of enzyme activity and stability from the hot-start towards modern hyperthermophilic, mesophilic and psychrophilic organisms illustrates active pressure versus passive drift in evolution on a molecular level.

















Dorothee Kern (Brandeis University)