Understanding the role of intrinsic disorder in MAPK cell signalling by NMR spectroscopy

The mitogen-activated protein kinase (MAPK) cell signalling pathways are essential components of eukaryotic signal transduction networks that enable cells to respond appropriately to extracellular stimuli. These signalling pathways generally feature a signalling module composed of a small GTPase and three protein kinases that are sequentially activated by phosphorylation. Intrinsically disordered scaffold proteins play crucial roles in ensuring signalling specificity by mediating the assembly of specific signalling modules. Our research group is interested in revealing the mechanism of action of these disordered scaffolds at atomic resolution using NMR spectroscopy. This includes revealing the structural and dynamic basis of the recruitment of GTPases and kinases onto the scaffolds and studying how phosphorylation of the scaffold proteins modulates complex assembly.

The presentation will focus on scaffold proteins within the stress-activated JNK signalling pathway and, in particular, on the recruitment of the small GTPase Rac1 to the disordered scaffold protein POSH. Using a combination of NMR and X-ray crystallography, we show that POSH adopts a novel fold in complex with Rac1 by exploiting two separate molecular recognition elements (MRE1 and MRE2) that fold upon binding to Rac1. Using NMR exchange experiments, we map the kinetic details of the folding trajectory of POSH revealing that the interaction initially proceeds through binding and instantaneous folding of MRE1 followed by a reversible folding event of MRE2 on the second time scale on the surface of Rac1. Our work provides insight into the complexity of binding mechanisms employed by intrinsically disordered proteins and offers novel structural insight into effector recruitment by Rac1.

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