Soutenance de thèse : Structure, dynamics, and assembly of scaffolding complexes in MAPK cell signalling

Par Lenette Kjaer (IBS/Groupe Flexibilité et Dynamique des Protéines par RMN)

Intrinsically disordered scaffold proteins are essential components of many eukaryotic signalling pathways. Acting as molecular hubs they bind and organise multiple components of a signalling module and thereby help preserve pathway integrity and efficiency. In the pro-apoptotic c-Jun N-terminal kinase (JNK) pathway signalling is initiated by binding of the small GTPase Rac1 to the scaf-fold protein POSH (Plenty Of SH3s). In this work we identify a novel recognition mode for Rac1 binding to a non-canonical CRIB motif in an intrinsically disordered region of the scaffold protein. Using ¹⁵N nuclear relaxation rates to delineate the precise binding site, we demonstrate that the interaction involves two molecular recognition elements (MRE1 and MRE2) covering an impressive 50 amino acids of POSH. We obtain the crystal structure of the POSH-Rac1 complex at 1.2 Å resolution showing complete folding of both MREs of POSH upon binding to Rac1. Using an extensive set of chemical exchange saturation transfer (CEST) experiments, we map the kinetic details of the folding trajectory of POSH upon binding to Rac1. We show that the interaction initially proceeds through binding and instantaneous folding of MRE1 followed by a reversible folding event of MRE2 on the seconds timescale on the surface of Rac1. Extending our approach to the oncogenic splice variant Rac1b, which displays 15-fold lower affinity for POSH, we attribute this affinity difference to a reduction in the association rate between the GTPase and POSH supporting the hypothesis that Rac1b is unable to stabilise its binding-competent closed-switch conformation. Overall, our work contributes to our understanding of molecular recognition and signalling regulation by intrinsically disordered proteins in eukaryotic cell signalling. Additionally, it provides insights into how Rac1b becomes defective in downstream signalling pathways and contributes to tumorigenesis in multi-ple human cancers.