NMR studies of the role of intrinsic disorder in mitogen- activated protein kinase cell signalling pathways – NMRSignal

Over the last decade, classical structural biology has experienced a shift towards a more dynamic paradigm with the realization that a protein can be fully functional even in the absence of a stable, folded structure. It is estimated that up to 40% of the proteins encoded by the human genome are intrinsically disordered or contain disordered regions of significant length (> 50 amino acids). Bioinformatics studies have placed proteins implicated in cell signalling among those with the highest levels of intrinsic disorder in the human genome. In this project, we will focus on the role of intrinsic disorder in the mitogen-activated protein kinase (MAPK) cell signalling pathways, which are essential components of eukaryotic signal transduction networks that enable cells to respond appropriately to extracellular stimuli.
In recent years, it has become increasingly clear that intrinsically disordered proteins (IDPs) play key roles in maintaining MAPK signalling specificity by assisting the context-specific assembly of members of the MAPK pathways into highly dynamic signalling complexes. Within these complexes, phosphorylation of the catalytic domains can efficiently occur without cross-reactivity with neighbouring signalling pathways. These signalling complexes have until now escaped characterization by structural biologists due to their highly dynamic nature. In this project, we will elucidate the molecular basis for signalling specificity within the MAPK pathways by developing experimental and analytical tools to characterize the assembly, structure, dynamics, stoichiometry and affinity of signalling complexes, with particular emphasis on the protein-protein interactions that contribute to signalling specificity within the c-Jun N-terminal kinase (JNK) pathway. This includes the atomic resolution characterization of the intrinsically disordered domains of kinases and scaffold proteins, interaction studies with cognate and non-cognate kinases, studies of post-translational modifications within the disordered domains and their role in protein-protein interactions, as well as assembly of entire multi-kinase complexes.
To achieve this goal, we will use nuclear magnetic resonance (NMR) spectroscopy that allows characterization of IDPs and their interaction mechanisms at atomic resolution, together with our recently developed computational approaches providing representative structural ensembles of IDPs on the basis of extensive sets of experimental NMR data. In addition, we will rely on complementary experimental techniques such as X-ray crystallography for obtaining crystal structures of IDP complexes that mediate signalling specificity as well as small angle X-ray scattering (SAXS) for mapping the conformational space available to these highly dynamic complexes.