Abstract

In this 3-year project grant we will combine and integrate insights from an array of advanced structural techniques, including cryo-EM, H-D exchange- and ion mobility-MS, and methyl-resolved and paramagnetic NMR, to effect a step change in our understanding of how FGFRs (hence RTKs more broadly) interact with the Cdc37-HSP90 chaperone system. Over a number of years we have established in our labs the means to generate heterotrimeric/tetrameric complexes that represent defined points on the kinase-co-chaperone-chaperone interaction cycle through in vitro reconstitution of individually expressed and purified components carrying functional mutations, e.g. E47A and D93N ATP-binding/hydrolysis HSP90 mutants, I538F DFG-latch mutant FGFR3, thus overcoming what can often be a major limiting factor in structurally-based projects. Our methyl-resolved NMR platform will enable us to define the rules for discrimination by Cdc37 between kinase variants on the strong-weak client continuum, and how these features are selected for presentation to HSP90. Furthermore, we will be able to probe the effect of HSP90 inhibitors such as PU-H71 on the full complex structure and dynamics, and answer the question of whether there is obligate order and homogeneity in the assembly of the kinase-co-chaperone-chaperone complexes.