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

Mahlet Z Tamirat,Mark S Johnson,Klaus Elenius,Marika Koivu,Freddie Salsbury

doi:10.1371/journal.pone.0222814

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.

Highlights

Epidermal growth factor receptor (EGFR) kinase is a tyrosine kinase involved in multiple cellular processes, such as cell proliferation, differentiation, migration and survival [1]
Kinases are dynamic proteins that exist in an equilibrium between active and inactive states, and access to the active catalytic state is highly regulated and of restricted duration in order to Activating EGFR deletion mutation prevent signaling effects that lead to abnormal biological effects
The percentage occupancy for hydrogen bonds formed by the side-chain polar atoms of E762 and K745 support this view (Fig 7C): the Glu762 –Lys745 hydrogen bond is observed in more frames of the mutant trajectory than for wild-type EGFR. These findings suggest that the stability of the αC helix imparted by ΔELREA mutation enhances the stability of the E762. . .K745 salt bridge, which is key to EGFR kinase activity

Summary

Introduction

Epidermal growth factor receptor (EGFR) kinase is a tyrosine kinase involved in multiple cellular processes, such as cell proliferation, differentiation, migration and survival [1]. The ErbBs are fundamental to the development and growth of organisms, anomalies with their regulation and/or signaling activity often associates them with various cancers, making them key therapeutic targets [2, 3]. Already in the 1980’s it was observed that avian erythroblastosis retrovirus encoded chordate-species EGFR kinase domains that, untethered to a growth factor sensing ectodomain, was associated with development of cancers in the same chordate due to retrovirus infection [4, 5]. Mutations observed in ErbB4 in non-small cell lung cancer patients experimentally lead to changes in ErbB2-ErbB4 heterodimer signaling promoting cell proliferation but not differentiation [9], and to increased phosphorylation likely because of stabilization of the ErbB4 active dimer state [10]

Methods

Results

Conclusion