Visualizing the assembly of MAPK cell signalling complexes using NMR spectroscopy – MAPKassembly

Mitogen-activated protein kinases (MAPKs) are components of eukaryotic signal transduction networks that enable cells to respond appropriately to extracellular stimuli. The MAPK pathways display remarkably high levels of protein intrinsic disorder, however, current knowledge about signal transmission is essentially limited to crystal structures of folded, catalytic domains of kinases and phosphatases. Intrinsically disordered proteins (IDPs) play crucial roles in ensuring signalling specificity in the MAPK pathways by assembling multiple kinases into dynamic signalling complexes. This assembly occurs either through intrinsically disordered regulatory domains of the kinases themselves, or via an “external” disordered scaffold protein that binds three kinases of a given signalling module. In this project, we will provide an entirely new perspective on the MAPK interactome by studying long disordered regions of kinases and scaffold proteins (up to 500 amino acids in length) and visualize their step-wise assembly into large multi-enzyme complexes that mediate and regulate MAPK signalling specificity. We will rely on nuclear magnetic resonance (NMR) spectroscopy for tackling these dynamic systems, and we will develop novel NMR methods for obtaining atomic resolution models of IDP complexes that so far have remained beyond the reach of classical structural biology techniques due to their inherent flexibility. Elucidating the mechanism of action of these disordered domains is particularly pertinent as deregulation of the MAPK pathways has been strongly linked to a number of human cancers. We will make available high resolution structural models of IDP complexes controlling specificity thereby paving the way for structure-based development of novel drugs targeting specific steps of these cancer-related pathways. Finally, the developed NMR methodologies will be applicable to a number of biological systems as we continue to explore the “dark proteome” at atomic resolution.