Architecture of the MKK6-p38α complex defines the basis of MAPK specificity and activation

The MAP kinase p38α is a central component of signalling in inflammation and the immune response and is, therefore, an important drug target. Little is known about the molecular mechanism of its activation by double-phosphorylation from MAP2Ks, due to the challenge of trapping a transient and dynamic hetero-kinase complex. Here, we applied a multidisciplinary approach to generate the first structure of p38α in complex with its MAP2K MKK6 and understand the activation mechanism. Integrating cryo-EM with MD simulations, HDX-MS and in cellulo experiments, we demonstrate a dynamic, multi-step, phosphorylation mechanism, reveal new catalytically relevant interactions, and show that MAP2K disordered N-termini determine pathway specificity. Our work captures, for the first time, a fundamental step of cell signalling: a kinase phosphorylating its downstream target kinase. One-Sentence Summary Integrative Cryo-EM and MD analysis of an active hetero-kinase complex reveals details of cellular signal transmission