Structural characterization of EGFR exon 19 deletion mutation using molecular dynamics simulation

Abstract

Epidermal growth factor receptor (EGFR) is a tyrosine kinase receptor important in diverse biological processes including cell proliferation and survival. Upregulation of EGFR activity due to over-expression or mutation is widely implicated in cancer. Activating somatic mutations of the EGFR kinase are postulated to affect the conformation and/or stability of the protein, shifting the EGFR inactive-active state equilibrium towards the activated state. Here, we examined a common EGFR deletion mutation, Δ746ELREA750, which is frequently observed in non-small cell lung cancer patients. By using molecular dynamics simulation, we investigated the structural effects of the mutation that lead to the experimentally reported increases in kinase activity. Simulations of the active form wild-type and ΔELREA EGFRs revealed the deletion stabilizes the αC helix of the kinase domain, which is located adjacent to the deletion site, by rigidifying the flexible β3-αC loop that accommodates the ELREA sequence. Consequently, the αC helix is stabilized in the “αC-in” active conformation that would prolong the time of the activated state. Moreover, in the mutant kinase, a salt bridge between E762 and K745, which is key for EGFR activity, was also stabilized during the simulation. Additionally, the interaction between EGFR and ATP was favored by ΔELREA EGFR over wild-type EGFR, as reflected by the number of hydrogen bonds formed and the free energy of binding. Simulation of inactive EGFR suggested the deletion would promote a shift from the inactive conformation towards active EGFR, which is supported by the inward movement of the αC helix. The MDS results also align with the effects of tyrosine kinase inhibitors on ΔELREA and wild-type EGFR lung cancer cell lines, where more pronounced inhibition was observed against ΔELREA than for wild-type EGFR by inhibitors recognizing the active kinase conformation.

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“…Periodic boundary conditions were implemented, and the particle-mesh Ewald algorithm was applied 79 for electrostatic interactions with a distance cutoff of 9 Å. For full details of the simulation protocol see Tamirat et al, 2019 80 . Conformations were saved every 10 ps and the resulting MD trajectories were analysed further by calculating the root-mean-square deviations (RMSD; over backbone atoms) and root-mean-square fluctuations (RMSF; over Cα atoms), as well as monitoring hydrogen bond interactions using CPPTRAJ 81 and VMD 82 .…”

Structural Basis for the Functional Changes by EGFR Exon 20 Insertion Mutations

“…As a result, the β3-αC loop attains different conformations, accounting for the higher RMSF of this region in D770insNPG EGFR in contrast to V769insASV EGFR. Indeed, the β3-αC loop is the location for multiple activating deletion mutations, where the shortening of this loop is postulated to impart structural stability to the nearby αC helix [ 27 ].…”

“…The deletions occur at the β3-αC loop and are suggested to affect the structural stability of the adjacent αC helix by limiting its range of motions [ 26 ]. Our previous study has indeed demonstrated that the most common EGFR exon 19 deletion, Δ746ELREA750, appears to stabilize the activated kinase domain state and drives a conformational shift from the inactive state towards the active EGFR state by affecting the structural and positional stability of the αC helix [ 27 ]. NSCLC patients with the L858R and exon19 deletion mutations respond to first- and second-generation tyrosine kinase inhibitors (TKIs), such as gefitinib, erlotinib, and afatinib; however, resistance is inevitably conferred mainly due to acquisition of a second mutation, the T790M “gate-keeper” mutation [ 28 ].…”

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“…This results from a decrease in the β3-αC loop flexibility, which stabilizes the αC-helix in the active αC-in conformation. Furthermore, the ∆ 746 ELREA 750 deletion causes an inwards conformational shift of the αC-helix, which disrupts the hydrophobic cluster in between the αC-helix, thereby promoting the activation of the TK domain [86].…”

“…Similarly, to L858R point-mutants, Del19 EGFR mutants display a decreased affinity for ATP binding (K M ) and a lower K i for first-generation TKIs such as erlotinib [66,86]. However, exon 19 deletions and consequent residue insertions display large heterogeneity and thus exhibit differential drug sensitivity [85,91].…”