Studies of the Allosteric Activation of the Epidermal Growth Factor Receptor

Ligand binding to the extracellular domain of the epidermal growth factor receptor (EGFR) results in receptor dimerization and allosteric activation of the kinase domain through formation of a specific asymmetric dimer. This asymmetric dimer is required for EGFR to undergo autophosphorylation and propagate the signal. Structurally, formation of the asymmetric dimer stabilizes the active conformation of the kinase domain, wherein the structural rearrangements create a hydrophobic pocket on the amino-terminal lobe (N-lobe) of one kinase at the dimer interface. However, the affinity for formation of the dimer by the kinase domain alone in solution is very weak. As a result the kinase domain is predominantly monomeric and bears very low catalytic activity. Studies of mechanisms that promote formation of the asymmetric dimer or otherwise stabilize the active conformation may provide additional insights into the allosteric activation of the kinase domain./nWe hypothesized that a small molecule could allosterically activate the kinase domain by binding at the N-lobe hydrophobic pocket. Furthermore, a compound that binds at the N-lobe may sterically block formation of the asymmetric dimer and inhibit dimerization-mediated autophosphorylation. A small molecule library was screened for activation of the EGFR kinase domain, and five compounds were discovered to increase kinase activity. Characterization of the compounds shows that each compound has unique properties of binding to EGFR, dependence on reductant, reversibility, and activity with EGFR mutants, which suggests distinct mechanisms for each compound in activation of EGFR. The molecular mechanisms by which the compounds activate EGFR are unknown due to the difficulty of obtaining co-crystal structures. Nonetheless, it appears that at least one compound may block formation of the asymmetric dimer of kinase domains. This work demonstrates that the EGFR kinase domain may be activated by small molecules, and the small molecules that were discovered may help elucidate the mechanisms that control allosteric activation of the EGFR kinase domain./nThe EGFR kinase domain phosphorylates most peptide substrates with a relatively low catalytic efficiency, likely due to the poor affinity for kinase domain dimerization in solution. Peptide C is a synthetic peptide substrate of EGFR developed by others that is phosphorylated with a significantly higher catalytic efficiency, and we sought to understand the basis for this. Peptide C was found to increase EGFR kinase activity by promoting formation of the EGFR kinase domain asymmetric dimer. Activation of the kinase domain by Peptide C also enhances phosphorylation of other substrates. Aggregation of the EGFR kinase domain by Peptide C likely underlies activation, and Peptide C precipitates several other proteins. Peptide C was found to form fibrils independent of the presence of EGFR, and these fibrils may facilitate aggregation and activation of the kinase domain. These results establish that a peptide substrate of EGFR may increase catalytic activity by promoting kinase domain dimerization and allosteric activation by an aggregation-mediated mechanism.

Advisor: Kuriyan, John