cancers Article Structural Basis for the Functional Changes by EGFR Exon 20 Insertion Mutations Mahlet Z. Tamirat 1, Kari J. Kurppa 2, Klaus Elenius 2,3,4 and Mark S. Johnson 1,* 1 Structural Bioinformatics Laboratory, Biochemistry, Faculty of Science and Engineering, Åbo Akademi University, 20520 Turku, Finland; mahlet.tamirat@abo.fi 2 MediCity Research Laboratories, Institute of Biomedicine, University of Turku, 20520 Turku, Finland; kjkurp@utu.fi (K.J.K.); klaus.elenius@utu.fi (K.E.) 3 Department of Oncology, Turku University Hospital, 20521 Turku, Finland 4 Turku Bioscience Center, University of Turku and Åbo Akademi University, 20520 Turku, Finland * Correspondence: mark.s.johnson@abo.fi Simple Summary: Non-small cell lung cancer (NSCLC) is the most common type of lung cancer that claims the lives of many worldwide. Activating mutations occurring on the epidermal growth factor receptor (EGFR) protein have been associated with the pathogenesis of NSCLC, among which exon 20 insertion mutations play a significant role. The objective of this study is to examine the dynamic structural changes occurring on the EGFR protein as a result of two common EGFR exon 20 insertion mutations, V769insASV and D770insNPG. The study further aims to uncover the mechanisms by which the insertion mutations increase kinase activity. Our results suggest that the insertion mutations stabilize structural elements key to maintaining the active EGFR conformation. Furthermore, the insertions disrupt an interaction essential in stabilizing the inactive conformation, which could drive the kinase from an inactive to an active EGFR state. Citation: Tamirat, M.Z.; Kurppa, K.J.; Elenius, K.; Johnson, M.S. Structural Abstract: Activating somatic mutations of the epidermal growth factor receptor (EGFR) are frequently Basis for the Functional Changes by implicated in non-small cell lung cancer (NSCLC). While L858R and exon 19 deletion mutations are EGFR Exon 20 Insertion Mutations. most prevalent, exon 20 insertions are often observed in NSCLC. Here, we investigated the structural Cancers 2021, 13, 1120. https:// implications of two common EGFR exon 20 insertions in NSCLC, V769insASV and D770insNPG. The doi.org/10.3390/cancers13051120 active and inactive conformations of wild-type, D770insNPG and V769insASV EGFRs were probed Academic Editor: Mumtaz V. Rojiani; with molecular dynamics simulations to identify local and global alterations that the mutations exert Srikumar Chellappan; Amyn on the EGFR kinase domain, highlighting mechanisms for increased enzymatic activity. In the active M. Rojiani conformation, the mutations increase interactions that stabilize the αC helix that is essential for EGFR activity. Moreover, the key Lys745–Glu762 salt bridge was more conserved in the insertion mutations. Received: 14 January 2021 The mutants also preserved the state of the structurally critical aspartate–phenylalanine–glycine Accepted: 27 February 2021 (DFG)-motif and regulatory spine (R-spine), which were altered in wild-type EGFR. The insertions Published: 5 March 2021 altered the structure near the ATP-binding pocket, e.g., the P-loop, which may be a factor for the clinically observed tyrosine kinase inhibitor (TKI) insensitivity by the insertion mutants. The inactive Publisher’s Note: MDPI stays neutral state simulations also showed that the insertions disrupt the Ala767–Arg776 interaction that is key with regard to jurisdictional claims in for maintaining the “αC-out” inactive conformation, which could consequently fuel the transition published maps and institutional affil- from the inactive towards the active EGFR state. iations. Keywords: EGFR tyrosine kinase; exon 20 insertion mutations; non-small cell lung cancer; molecular dynamics simulation; structural biology Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article 1. Introduction distributed under the terms and conditions of the Creative Commons Epidermal growth factor receptor (EGFR) is a membrane-bound signaling protein Attribution (CC BY) license (https:// essential for the development of organisms, owing to its role in cell proliferation, differenti- creativecommons.org/licenses/by/ ation, migration and survival [1]. EGFR belongs to the ERBB family of receptor tyrosine 4.0/). kinases (RTK), which additionally includes ERBB2, ERBB3 and ERBB4 [2,3]. As with other Cancers 2021, 13, 1120. https://doi.org/10.3390/cancers13051120 https://www.mdpi.com/journal/cancers Cancers 2021, 13, x 2 of 23 1. Introduction Epidermal growth factor receptor (EGFR) is a membrane-bound signaling protein Cancers 2021, 13, 1120 essential for the development of organisms, owing to its role in cell proliferation, differ-2 of 21 entiation, migration and survival [1]. EGFR belongs to the ERBB family of receptor tyro- sine kinases (RTK), which additionally includes ERBB2, ERBB3 and ERBB4 [2,3]. As with otherfamily family members, members, the EGFR the EGFR monomer monomer is composed is composed of an of extracellularan extracellular domain, domain, a single- a sin- gle-passpass transmembrane transmembrane domain domain (TM), (TM), an intracellularan intracellular juxtamembrane juxtamembrane (JM) (JM) segment, segment, a cy- a cytoplasmictoplasmic kinase kinase domain domain and and a a C-terminal C-terminal tail tail (Figure (Figure1 A)1A) [ 3[3].]. ActivationActivation of EGFR is inducedinduced by by binding binding of of growth growth factor factor to to the the ectodomain ectodomain [3], [3], which which triggers triggers a a large large confor- confor- mationalmational change fromfrom aa tethered tethered to to an an extended extended ectodomain ectodomain state state as seenas seen by comparingby comparing the themonomeric monomeric [4] and[4] growth-factorand growth-factor bound bound homodimeric homodimeric [5] X-ray [5] structures.X-ray structures. Consequently, Conse- quently,EGFR monomers EGFR monomers on binding on binding a recognized a recogn growthized growth factor, e.g.,factor, EGF, e.g., form EGF, homodimers form homodi- or mersheterodimers or heterodimers with other with ERBB other familyERBB family monomers, monomers, and the and intracellular the intracellular kinase kinase domains do- mainsassociate associate as the asymmetricas the asymmetric dimer required dimer required for activation. for activation. Activated Activated kinase domains kinase bind do- mainsATP and bind catalyze ATP and the catalyze autophosphorylation the autophosphorylation of tyrosine residuesof tyrosine located residues at the located C-terminal at the C-terminaltail that act tail as dockingthat act as sites docking for various sites for other various proteins, other initiating proteins, intracellularinitiating intracellular signaling signalingpathways pathways [6,7]: in the [6,7]: case ofin EGFRthe case homodimers, of EGFR pathwayshomodimers, such aspathways the MAPK/ERK such as andthe MAPK/ERKPI3K-AKT that and are PI3K-AKT important that in cellare proliferation,important in cell differentiation, proliferation, migration differentiation, and inhibition migra- tionof apoptosis and inhibition [8,9]. of apoptosis [8,9]. Figure 1. EpidermalEpidermal growth growth factor receptor (EGFR) and struct structuralural features of the tyrosine kinase domain. ( A) The The different different domains that make up the EGFR protein and structure of the intracellular kinase domain (Protein Data Bank (PDB) ID 2GS2 [10]); [10]); key structural elements an andd active site-bound ATP are highlighted. ATP was positioned based on AMP-PNP bound EGFR structure (PDB ID 2ITX [[11]).11]). ( B) The active (cyan) and inactive (purple)(purple) conformations of the EGFR kinase domain as seen by comparing PDB ID 2GS2 with PDB ID 2GS7 [[10]:10]: the “αC-in” and “αC-out” conformations of the αC helix, the open and extended state of the activation loop (A-loop), the essential K745–E762 ionic interaction broken in the inactive state, and conformational differences within the aspartate–phenylalanine–glycine (DFG) motif. inactive state, and conformational differences within the aspartate–phenylalanine–glycine (DFG) motif. TheThe EGFR kinase domain exists in an equilibrium between an inactive and active conformationconformation (Figure (Figure 1B)1B) and and the the ATP ATP binding binding pocket pocket lies lies between between the the N-terminal N-terminal and and C- terminalC-terminal lobes lobes (Figure (Figure 1A)1A) [10,12,13]. [ 10,12,13 Both]. Both lobes lobes contribute contribute structural structural units, units, such such as the as αtheC helixαC helix and activation and activation loop loop(A-loop), (A-loop), which which are critical are critical for the for regulation the regulation of EGFR of kinase EGFR activity.kinase activity. On activation, On activation, the αC the helixαC of helix the EGFR of the EGFRkinase kinase domain domain assumes assumes the “α theC-in” “αC-in” con- formation,conformation, where where the α theC helixαC helixlocates locates near where near whereATP binds ATP within binds withinthe active the site active (Figure site 1B).(Figure Consequently,1B). Consequently, a conserved a conserved ionic interaction ionic interaction between between Glu762 Glu762 of the of α theC helixαC helixand and Lys745 of the β3 strand forms, stabilizing the active conformation. Furthermore, in the activated state, the A-loop attains an extended orientation, opening up space near the binding pocket. The aspartate–phenylalanine–glycine (DFG) motif that is located in the A-loop is placed so that the catalytic