Volume 23, Issue 7, Pages 837-848 (July 2016) Molecular Basis for Redox Activation of Epidermal Growth Factor Receptor Kinase  Thu H. Truong, Peter Man-Un Ung, Prakash B. Palde, Candice E. Paulsen, Avner Schlessinger, Kate S. Carroll  Cell Chemical Biology  Volume 23, Issue 7, Pages 837-848 (July 2016) DOI: 10.1016/j.chembiol.2016.05.017 Copyright © 2016 Elsevier Ltd Terms and Conditions

Cell Chemical Biology 2016 23, 837-848DOI: (10. 1016/j. chembiol. 2016 Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 1 EGFR Catalytic Kinase Domain and Covalent Inhibitors (A) Crystal structure of the EGFR kinase domain (PDB: 3VJO) bound to AMP-PNP showing all six conserved cysteine residues. The catalytic loop is highlighted in lime green, T790/L858 residues in magenta, and E746/A750 residues in lilac. (B) Top: structure of two electrophilic covalent EGFR inhibitors, afatinib and PD168393. The acrylamide functional groups in each inhibitor are highlighted in the gray circle. Bottom: crystal structure of the EGFR kinase domain (PDB: 4G5J) in a covalent thioether bond with afatinib at C797. Cell Chemical Biology 2016 23, 837-848DOI: (10.1016/j.chembiol.2016.05.017) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 2 H2O2 Stimulation of EGFR Autophosphorylation and Biochemical Properties of C797 (A) Full-length native EGFR was treated with EGF (3.3 ng/μl) or H2O2 (0–100 μM). Western blot shows autophosphorylated (p) and total EGFR. (B) Densitometric quantification of (A). Data are normalized to untreated control and are representative of three independent readings (mean ± SEM). *p < 0.05, **p < 0.01, ***p < 0.001 compared with untreated control. (C) Bromobimane fluorescence labeling of recombinant EGFR over a pH range (2–6.5) yields a single pKa value for C797 of 5.5 ± 0.1. Data are representative of two independent readings. (D) Plotting the rate of sulfenic acid formation over a range of H2O2 concentrations (10, 20, 40, 80 μM) yields a second-order rate constant for the H2O2 reactivity of C797 of ≥110 M−1 s−1. Data represent the average of two independent readings (mean ± SEM). Cell Chemical Biology 2016 23, 837-848DOI: (10.1016/j.chembiol.2016.05.017) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 3 Effect of C797 Mutation on Recombinant and Cellular EGFR Kinase Activity (A) Catalytic efficiency (kcat/KM) with respect to ATP. Relative efficiency was calculated by determining kcat/KM for each protein with varying ATP and saturating peptide substrate (RAHEEIYHFFFAKKK) and dividing by the kcat/KM of wild-type (WT) enzyme. (B) Catalytic efficiency (kcat/KM) with respect to peptide substrate. Relative efficiency was calculated by determining kcat/KM for each protein with varying peptide substrate (poly[Glu4Tyr1]) and saturating ATP and dividing by the kcat/KM of wild-type (WT) enzyme. (C and E) EGF- (C) and H2O2-mediated (E) activation of C797 mutants. Wild-type (WT), C797S, and C797A EGFR were transfected into HeLa cells and stimulated with EGF or H2O2 for 5 min. Western blots show phosphorylated (p) and total EGFR. Mutants exhibit decreased autophosphorylation levels compared with wild-type. (D and F) Densitometric quantification of (C) and (E), respectively. At higher H2O2 concentrations (i.e., 500 μM), C797S phosphorylation levels (##) increase, which can be attributed to PTP inhibition. Data are normalized to untreated wild-type (WT) control and are representative of three independent readings (mean ± SEM). *p < 0.05, ***p < 0.001 compared with untreated wild-type control. Cell Chemical Biology 2016 23, 837-848DOI: (10.1016/j.chembiol.2016.05.017) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 4 MD Simulation of Reduced and Sulfenylated EGFR (A and B) Dashed yellow lines indicate interactions predicted to occur between the C797 thiolate (A) or sulfenic acid (B), ATP substrate and/or the backbone carbonyl of R841. Figure shows movie frame depicting the shortest distance (1.6 Å) for sulfenic acid hydrogen bonding with the R841 carbonyl. (C and E) EGF- (C) and H2O2-mediated (E) activation of R841 mutants. Wild-type (WT), R841K, and R841A EGFR were transfected into HeLa cells and stimulated with EGF or H2O2 for 5 min. Western blots show phosphorylated (p) and total EGFR. (D and F) Densitometric quantification of (C) and (E), respectively. Data are normalized to untreated wild-type (WT) control and are representative of three independent readings (mean ± SEM). *p < 0.05, **p < 0.01, ***p < 0.001 compared with untreated wild-type control. Cell Chemical Biology 2016 23, 837-848DOI: (10.1016/j.chembiol.2016.05.017) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 5 Sulfenylation Profiles of Oncogenic EGFR Mutations (A–C) Western blots show sulfenylated and total EGFR. Lung cancer cells expressing wild-type (WT) A549 (A), or EGFR mutants NCI-H1975 (L858R/T790M) (B) and HCC827 (ΔE746-A750) (C) were stimulated with EGF or vehicle for 2 min and sulfenic acids were detected by streptomycin-horseradish peroxidase western blot. (D) Densitometric quantification of (A–C). Data are normalized to untreated control for each respective cell line and are representative of three independent readings (mean ± SEM). *p < 0.05, **p < 0.01 compared with the untreated control for each cell line. Cell Chemical Biology 2016 23, 837-848DOI: (10.1016/j.chembiol.2016.05.017) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 6 The Effect of Chronic Oxidative Stress on Irreversible EGFR Inhibitors and Model for Redox Regulation of EGFR in Cells (A and B) A431 cells were incubated with 2 U/ml glucose oxidase for 3 hr to simulate chronic H2O2 stress, treated with (A) afatinib or (B) PD168393 for 1 hr, and subsequently treated with 100 ng/ml EGF for 5 min. Plots represent densitometric quantification of western blotting for phosphorylated (p) and total EGFR. Data are normalized to untreated control for each respective condition and are representative of two independent readings (mean ± SEM). (C) Model for H2O2 modification of EGFR C797 in cells. Cell Chemical Biology 2016 23, 837-848DOI: (10.1016/j.chembiol.2016.05.017) Copyright © 2016 Elsevier Ltd Terms and Conditions