Computational and Experimental Characterization of Patient Derived Mutations Reveal an Unusual Mode of Regulatory Spine Assembly and Drug Sensitivity in EGFR Kinase

The
catalytic activation of protein kinases requires precise positioning
of key conserved catalytic and regulatory motifs in the kinase core.
The Regulatory Spine (RS) is one such structural motif that is dynamically
assembled upon kinase activation. The RS is also a mutational hotspot
in cancers; however, the mechanisms by which cancer mutations impact
RS assembly and kinase activity are not fully understood. In this
study, through mutational analysis of patient derived mutations in
the RS of EGFR kinase, we identify an activating mutation, M766T,
at the RS3 position. RS3 is located in the regulatory αC-helix,
and a series of mutations at the RS3 position suggest a strong correlation
between the amino acid type present at the RS3 position and ligand
(EGF) independent EGFR activation. Small polar amino acids increase
ligand independent activity, while large aromatic amino acids decrease
kinase activity. M766T relies on the canonical asymmetric dimer for
full activation. Molecular modeling and molecular dynamics simulations
of WT and mutant EGFR suggest a model in which M766T activates the
kinase domain by disrupting conserved autoinhibitory interactions
between M766 and hydrophobic residues in the activation segment. In
addition, a water mediated hydrogen bond network between T766, the
conserved K745-E762 salt bridge, and the backbone amide of the DFG
motif is identified as a key determinant of M766T-mediated activation.
M766T is resistant to FDA approved EGFR inhibitors such as gefitinib
and erlotinib, and computational estimation of ligand binding free
energy identifies key residues associated with drug sensitivity. In
sum, our studies suggest an unusual mode of RS assembly and oncogenic
EGFR activation, and provide new clues for the design of allosteric
protein kinase inhibitors.