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

Abstract

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.

Altered conformational landscape and dimerization dependency underpins the activation of EGFR by α C– β 4 loop insertion mutations

“…The pEGFP-N1-EGFR plasmid from our previous studies was used for mutagenesis (25,26). The PCR primers for site-directed mutagenesis are designed using the NEBaseChanger v1.2.6 web server from New England Biolabs.…”

“…MD studies on other recurring mutations such as R776H in the αC-β4 loop have identified novel autoinhibitory interactions associated with kinase activation (25) and protonation-dependent changes in mutant EGFR functions (30). In addition, MD-based free-energy methods (26,31), molecular mechanics calculations (32), and molecular modeling studies (33) have provided new insights into mutation-induced drug resistance mechanisms and the conformational transitions connecting active and inactive states (34)(35)(36).…”

“…The dimerized or multimerized EGFR complex is catalytically active due to the stabilization of key catalytic motifs that prime the kinase domain for autophosphorylation and downstream signaling (9,21). Many activating oncogenic mutations, such as L858R, E746 A750del, G719S, G724S, R776H, and M766T, strictly rely on the asymmetric dimer for their activity (22)(23)(24)(25)(26). Intriguingly, some other complex…”

Molecular dynamics simulation-guided drug sensitivity prediction for lung cancer with rare EGFR mutations

“…Recently, computational structural modeling and molecular dynamics (MD) simulations have helped us clarify the activation mechanism of EGFR at the atomic level (25–27). In addition, predictions of sensitivity of EGFR mutants to EGFR tyrosine kinase inhibitors were performed for several EGFR mutations using binding free energy calculated with MD simulation (28, 29) and fitness scores calculated by molecular docking simulation (30).…”

Integrative annotation and knowledge discovery of kinase post-translational modifications and cancer-associated mutations through federated protein ontologies and resources

“…The procedure for EGFR mutational analysis is described in our previous studies 40 , 41 . Briefly, desired point mutations (S768D, S768E, S768I) were introduced into a pEGFP-N1-EGFR plasmid using QuikChange Site-Directed Mutagenesis Kits (Agilent Genomics, CA, USA).…”