Nmr Experiments On Wild-Type And Mutant Fibroblast Growth Factor Receptor Kinases Reveal Conformational Dynamics Associated With Enzyme Activation

The kinase domain of receptor tyrosine kinases (RTKs) is responsible for phosphorylating intracellular substrates upon hormone binding to the extracellular ectodomain. The resulting signal cascade is essential for several biological processes in human biology, including development and metabolism. Mutations in the kinase domain of RTKs can enhance the intrinsic activity of the enzyme and over-stimulate the resulting signal cascade leading to a wide variety of cancers and growth defects. Although a plethora of kinase crystal structures have been resolved, the dynamics associated with pathogenic mutations remain poorly understood. Here we analyzed the dynamics of pathogenic mutations within the fibroblast growth factor receptor kinase domain (FGFRK) as probed using Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments. Previous work showed that mutations at a conserved lysine residue in the activation loop bypassed the need for activation loop phosphorylation by shifting the population of the kinase from an autoinhibited state toward an active conformation. Interestingly, the degree of active state directly correlated with the kinase activity both in vitro and in human disease. The most prominent gain-of-function mutant was a lysine to glutamic acid substitution, which was found to stabilize the active conformation of the activation loop without the need for tyrosine phosphorylation (i.e., normal activation). The observed dispersions acquired on these pathogenic kinases using the CPMG experiments reveal conformational dynamics of the kinase in switching between active and inactive conformations and thus enabled us along with kinase crystal structures to identify an allosteric pathway spanning from the activation loop to the kinase hinge. These data agree well with the structural differences observed between kinases harboring different activating mutations, thus explaining how intrinsically activating mutations propagate their effects through the kinase.