Transitions to catalytically inactive conformations in EGFR kinase

Abstract

The epidermal growth factor receptor (EGFR) is a key protein in cellular signaling, and its kinase domain (EGFR kinase) is an intensely pursued target of small-molecule drugs. Although both catalytically active and inactive conformations of EGFR kinase have been resolved crystallographically, experimental characterization of the transitions between these conformations remains difficult. Using unbiased, all-atom molecular dynamics simulations, we observed EGFR kinase spontaneously transition from the active to the so-called "Src-like inactive" conformation by way of two sets of intermediate conformations: One corresponds to a previously identified locally disordered state and the other to previously undescribed "extended" conformations, marked by the opening of the ATP-binding site between the two lobes of the kinase domain. We also simulated the protonation-dependent transition of EGFR kinase to another ["Asp-Phe-Gly-out" ("DFG-out")] inactive conformation and observed similar intermediate conformations. A key element observed in the simulated transitions is local unfolding, or "cracking," which supports a prediction of energy landscape theory. We used hydrogen-deuterium (H/D) exchange measurements to corroborate our simulations and found that the simulated intermediate conformations correlate better with the H/D exchange data than existing active or inactive EGFR kinase crystal structures. The intermediate conformations revealed by our simulations of the transition process differ significantly from the existing crystal structures and may provide unique possibilities for structure-based drug discovery.

Fig. 1.

Comparison of key structural elements of simulation and crystal structures of EGFR kinase. (A) Superposition of the crystal structures of the active and the Src-like inactive conformations (PDB ID codes 2ITP and 2GS7). (B) Superposition of the conformation reached in our simulation with the Src-like inactive crystal structure.

Fig. 2.

Transition to the Src-like inactive state. (A) Cartoon representation of the transition pathway. The extended conformation features an open interlobe arrangement and cracking at the hinge region. (B) Extended conformation (blue) compared with the initial active conformation (red). The αC helix and the activation loop are highlighted. The distance between the C_α atoms of Val762 and Thr879, which is used as an indicator of the protein’s degree of extension, is marked. (C) Close-up of the hinge region, with (blue) and without (red) cracking. (D) Transition to the Src-like inactive conformation measured by the switch of salt bridges, RMSD, radius of gyration, and the number of helical residues in the hinge region (residues 795–831). The locally disordered state is highlighted by a yellow background and the extended conformation by a light green background. (Bottom) Dissolution of helical structure in the hinge region indicates that these intermediate conformations were accompanied by cracking. (E) Average and residue-specific root-mean-square fluctuation (RMSF) as functions of time in the simulation. The RMSF was calculated with a running time window of 1 µs with respect to the average conformation in that time window. Only the C_α atoms of each residue were used in the calculation. As highlighted by the green background and orange rectangles, the extended conformations preceding the transition to the Src-like inactive conformation were marked by high RMSF, indicative of high conformational entropy, particularly at the regions of the hinge and the activation loop. (F) Transitions projected onto a 2D space of RMSD with respect to the active and Src-like inactive crystal structures, respectively. RMSD is calculated using the Cα atoms of the αC and the two-turn helices (residues 756–769 and 857–863). Each dot represents a snapshot of the trajectory; the interval between the snapshots shown is 25 ns. The light blue region includes conformations within 8.2 Å RMSD of both active and Src-like inactive crystal structures. As shown, the transition (red dots, pink arrows) passed through two sets of intermediate conformations, which were identified by visual inspection. The simulation starting from the inactive conformation is also shown (purple dots).

Fig. 3.

Corroboration from H/D exchange experiments. (A) Total SASA of EGFR kinase and its amide groups as functions of the simulation time. Also shown is the correlation of the SASA of the amide groups with the measured H/D exchange rates (pH 8.0). The amide SASA profile was calculated for each snapshot in the simulation and averaged over a time window of 1 µs before comparison with the H/D exchange data. Note that the peak of the correlation (highlighted) occurs for the locally disordered state (Fig. 2D). (B) Correlation of the amide SASA and the measured H/D exchange rates at the highlighted simulation time. Each dot represents a peptide fragment of EGFR kinase (C) for which the H/D exchange rate is measured. Note that a residue may belong to multiple overlapping fragments due to the nonspecific nature of the proteolysis step in the H/D experiment, resulting in more dots on the correlation graph than the total number of fragments shown in C. The amide SASA of a fragment is averaged over its constituent residues. (C) An H/D exchange profile of EGFR kinase measured at pH 8.0.

Fig. 4.

Transition to the DFG-out inactive conformation. (A) Snapshots of the DFG flip, at 0, 13.42, 17.35, and 52.13 µs of the MD simulation, respectively. The snapshot at 0 µs is identical to the crystal structure of the active conformation (PDB ID code 2ITP). Note the motion of the αC helix involved in the DFG flip, measured by radius of gyration, number of helical residues in the hinge region, and RMSD with respect to crystal structures. (B) Snapshot taken at 23.32 µs, in which the Src-like inactive conformation serves as an intermediate of the DFG flip. (C) DFG flip measured by radius of gyration, RMSDs, and other metrics. The running average with windows of 100 ns (green) is also shown in the second panel from the top. The extended conformation (with light blue background) is accompanied by cracking. Three conformational events of the activation loop are marked: 1, visiting the Src-like inactive conformation as an intermediate; 2, visiting the substrate-competitive conformation, which is separated from the initial active conformation by as much as 20 Å RMSD; and 3, return to the initial conformation. The RMSD calculation is performed using the C_α atoms of the two-turn helix (residues 857–863).

Fig. 5.

Conformations of the activation loop found in the simulations. (A) Active-like arrangement of the activation loop coupled with a DFG-out conformation. This conformation, with the β9 strand intact, resembles the Mig6-bound structure of EGFR kinase (red). (B) Substrate-competitive conformation, compared with the structure of imatinib-bound Abl kinase (red). This (DFG-out) conformation is an intermediate of the simulated DFG flip. (C) Detached activation loop with a dislodged αEF helix. This is a transient conformation at 6.12 µs into the simulation starting from the Src-like inactive structure. A similar conformation has been observed in crystal structures of other protein kinases [e.g., in the crystal structure of domain-swapped OSR1 (PDB ID code 3DAK)]. (D) Conformation in which the activation loop forms a helix that exposes the phosphorylation site Tyr845. The two-turn helix is intact in this conformation.