Figure 1.
Structural Characteristics of the ErbB Kinases.
The crystal structures of the ErbB kinase family in different functional states are depicted using a comparison of key regulatory regions in the catalytic domain. The three regulatory elements of the kinase domain shown are the αC-helix, the DFG-Asp motif (DFG-Asp in, active; DFG-Asp out, inactive), and the activation loop (A-loop open, active; A-loop closed, inactive). In Cdk/Src inactive structures the αC-helix is displaced outwards the N-terminal lobe adopting a αC-out (swung-out) conformation that inhibits the formation of the active enzyme form. The R-spine residues (M766, L777, H835, F856, and D896) and the DFG motif are shown in colored sticks. Note that the R-spine residues in a different sequence numbering of the EGFR kinase domain correspond to M742, L753, H811, F832, and D872 residues. Left Upper Panel. Structural differences in the functional regions of the EGFR-WT crystal structures: Cdk/Src-IF1 state (in blue), DFG-in/αC-helix-out (pdb id 1XKK, 2GS7); Cdk/Src-IF2 conformation (in red), DFG-out/αC-helix-out (pdb id 2RF9); and the active conformation (in green), DFG-in/αC-helix-in (pdb id 2ITX, 2J6M). Right Upper Panel. Structural similarities in the functional regions of the Cdk/Src-IF2 EGFR-WT conformation (in blue), DFG-out/αC-helix-out (pdb id 2RF9); Cdk/Src-IF2 EGFR-L858R conformation (in red), DFG-out/αC-helix-out (pdb id 4I20); and Cdk/Src-IF2 EGFR-L858R/T790M double mutant conformation (in green), DFG-out/αC-helix-out (pdb id 4I21). Left Lower Panel. Structural similarities in the functional regions of the active EGFR-WT conformation (in blue), DFG-in/αC-helix-in (pdb id 2ITX, 2J6M); the active EGFR-L858R conformation (in red), DFG-in/αC-helix-in (pdb id 2ITV); and the active EGFR-T790M conformation (in green), DFG-in/αC-helix-in (pdb id 2JIT). Right Lower Panel. Structural differences in the functional regions of Cdk/Src-IF3 ErbB2-WT conformation (in blue), DFG-in/αC-helix-out, A-loop open (pdb id 3PP0); Cdk/Src-IF1 ErbB3-WT conformation (in red), DFG-in/αC-helix-out, A-loop closed (pdb id 3KEX, 3LMG); and Cdk/Src-IF1 ErbB4-WT conformation (in green), DFG-in/αC-helix-out, A-loop closed (pdb id 3BBT).
Table 1.
The Functional Regions of the ErbB Kinases.
Figure 2.
Residue-Based Equilibrium Fluctuations of the ErbB Kinases.
(A) The computed B-factors describe time-averaged residue fluctuations obtained from simulations of Cdk/Src-IF1 EGFR-WT (pdb id 1XKK, in blue), Cdk/Src-IF2 EGFR-WT structure (pdb id 2RF9, in red), and the active EGFR-WT structure (pdb id 2ITX, in green). (B) The computed B-factors obtained from simulations of the active structures of EGFR-WT (pdb id 2ITX, in blue), EGFR-L858R (pdb id 2ITV, in red), and EGFR-T790M (pdb id 2JIT, in green). C) The computed B-factors for Cdk/Src-IF2 structures of EGFR-WT (pdb id 2RF9, in blue), EGFR-L858R (pdb id 4I20, in red), and EGFR-L858R/T790M (pdb id 4I21, in green). (D) The computed B-factors obtained from simulations of Cdk/Src-IF3 ErbB2-WT structure (pdb id 3PP0, in blue), Cdk/Src-IF1 ErbB3-WT structure (pdb id 3LMG, in red), Cdk/Src-IF1 ErbB4-WT structure (pdb id 3BBT, in green), and the active ErbB4-WT structure (pdb id 3BCE, in maroon).
Figure 3.
Conformational Mobility Analysis of the EGFR-WT and EGFR-L858R Kinases.
Conformational mobility profiles of EGFR-WT are shown for the inactive Cdk/Src-IF1 form (pdb id 1XKK, left upper panel), the inactive Cdk/Src-IF2 state (pdb id 2RF9, middle upper panel) and the active conformation (pdb id 2ITX, right upper panel). Conformational mobility of EGFR-L858R is shown for the Cdk/Src-IF2 form (left lower panel) and the active conformation (right lower panel). The backbone heavy atoms (N,Cα,Cβ,C,O) were employed for the PCA computations. Conformational dynamics profiles were computed by averaging protein motions in the space of three lowest frequency modes. The color gradient from blue to red indicates the decreasing structural rigidity (or increasing conformational mobility) of the protein residues and refers to an average value over the backbone atoms in each residue. The functional kinase regions αC-helix, αC-β4-loop, and αE-helix as well as the R-spine residues are annotated and their positions are indicated by arrows. The R-spine residues are also highlighted in spheres and colored according to their degree of structural stability. Conformational mobility profiles were obtained from simulations of complete structures, where unresolved segments and disordered loops were modeled with the ModLoop server [127], [128]. These profiles were mapped onto the original crystal structures of EGFR for clarity of presentation.
Figure 4.
Conformational Mobility Analysis of the ErbB Kinases.
Conformational mobility mapping of ErbB2-WT in the inactive Cdk/Src-IF3 form (left upper panel), ErbB3-WT in the inactive Cdk/Src-IF1 conformation (right upper panel), ErbB4-WT in the Cdk/Src-IF1 form (left lower panel) and the active form (right lower panel). The backbone heavy atoms (N, Cα, Cβ, C, O) were employed for the PCA calculations. Conformational dynamics profiles were computed by averaging protein motions in the space of three lowest frequency modes. The color gradient from blue to red indicates the decreasing structural rigidity (or increasing conformational mobility) of the protein residues and refers to an average value over the backbone atoms in each residue. The key functional regions αC-helix, αC-β4-loop, and αE-helix as well as the R-spine residues are annotated and their positions are indicated by arrows as in Figure 3. The conformational mobility profiles were mapped onto the original crystal structures of ErbB kinases.
Figure 5.
Conformational Mobility Profile of the Active EGFR Dimer.
Structural distribution of conformational mobility in the asymmetric active dimer of EGFR-WT. In an asymmetric dimer arrangement a donor monomer interacts with an acceptor through interactions involving the αH-helix and αI-helix of the donor as well as the JM-B segment and the αC-helix of the acceptor. The key functional regions are annotated and pointed to by arrows as in Figures 3, 4. Note the increased stability of the acceptor monomer, particularly a uniform stabilization of the R-spine residues in the acceptor subunit. The conformational mobility profiles were mapped onto the original crystal structure of the active EGFR dimer.
Figure 6.
The Force Constant Profiles of the Kinase Catalytic Domain.
Dynamics-based analysis of structural stability in the ErbB crystal structures. (A) The residue-based force constant profiles of the EGFR-WT crystal structures: Cdk/Src-IF1 conformation (in blue), Cdk/Src-IF2 conformation (in red), and the active conformation (in green). A close-up view of the EGFR force constant profile in the αC-helix (residues 752–768) and the adjacent αC-β4-loop regions (residues 769–777) is provided as an inset. (B) The force constant profile of Cdk/Src-IF3 ErbB2 structure. (C) The force constant profile of Cdk/Src-IF1 ErbB3 structure. (D) The force constant profiles of Cdk/Src-IF1 ErbB4 conformation (in blue) and the active ErbB4 conformation (in green). A close-up view of the ErbB4 force constant profile in the αC-helix (residues 735–749) and the adjacent αC-β4-loop (residues 750–758) is provided as an inset. The annotated functional regions included P-loop, αC-helix, hinge, αE-helix, HRD motif, DFG motif, substrate binding P+1 loop, αF-helix, αH, and αI helix. The R-spine residues are indicated by filled maroon-colored diamond symbols. Note that the R-spine residues corresponded to the peaks in the distributions.
Figure 7.
The Force Constant Profiles of the Active EGFR Dimers.
Dynamics-based analysis of structural stability in the active asymmetric dimers of EGFR-WT (A, B) and EGFR-L858R/T790M double mutant (C, D). Note that a different EGFR sequence numbering was adopted in these crystal structures and we adhered to the original numbering to streamline the discussion and comparison with the experimental data. The force constant profiles are shown separately for the acceptor monomer (A, C) and donor monomers (B, D). The annotated functional regions included JM-B region, P-loop, αC-helix, hinge, αE-helix, HRD motif, DFG motif, substrate binding P+1 loop, αF-helix, αH, and αI helix. The annotated peaks in the profiles reflecting structural stability of the EGFR-WT dimer included L680 (JM-B region), M742, L753, H811, F832, D872 (R-spine residues), and W856 (P+1 substrate loop). The respective peaks in the profile of the EGFR-L858R/T790M dimer corresponded to L704 (JM-B region), M766, L777, H835, F856, and D896 (R-spine residues), and W880 (P+1 substrate loop). The R-spine residues are indicated by filled maroon-colored diamond symbols. The position of JM-B peaks (L680 in EGFR-WT, L704 in EGFR- L858R/T790M) and P+1 loop peaks (W856 in EGFR-WT, W880 in EGFR- L858R/T790M) are indicated by arrows.
Figure 8.
A Comparative Analysis of Residue Connectivity Parameters in the Functional States of the Kinase Domains and Active EGFR Dimers.
The scatter graphs between the force constant (a dynamics-based residue connectivity measure) and residue closeness (a network-based residue connectivity measure) values are shown for Cdk/Src-IF1 EGFR-WT (A), Cdk/Src-IF2 EGFR-WT (B), active EGFR-WT (C), Cdk/Src-IF1 ErbB4-WT (D), active ErbB4-WT (E), acceptor monomer of the EGFR-WT dimer (F), donor monomer of the EGFR-WT dimer (G), acceptor monomer of the EGFR-L858R/T790M dimer (H), and donor monomer of the EGFR-L858R/T790M dimer (I). The positions of the R-spine residues are indicated by filled squares colored in red.
Figure 9.
Community Analysis of the EGFR Kinase.
The distribution of residue interaction communities (A) and stabilization centers (B) in different functional states of the EGFR kinase. The analysis is based on structurally stable residue interaction networks that were maintained in more than 75% of the simulation samples. The principal interaction communities were mapped onto conformational dynamics profiles of Cdk/Src-IF1 EGFR conformation (C), Cdk/Src-IF2 EGFR conformation (D) and the active EGFR conformation. The communities that are characteristic of different functional states are highlighted in spheres and colored according to structural stability of protein residues. A larger number of stable communities were observed in Cdk/Src-IF1 (C) and active EGFR forms (E).
Figure 10.
Community Analysis of the ErbB Kinases.
The distribution of residue interaction communities (A) and stabilization centers (B) is shown for different functional states of ErbB2, ErbB3, and ErbB4 kinases. The principal interaction communities were mapped onto conformational dynamics profiles of the inactive Cdk/Src-IF3 ErbB2 (C), inactive Cdk/Src-IF1 ErbB3 (D), inactive Cdk/Srdc-IF1 ErbB4 (E), and active ErbB4 (F). The communities that are characteristic of different functional states are highlighted in spheres and colored according to structural stability of protein residues. A larger number of stable communities were observed in the functional states of ErbB4.
Figure 11.
Centrality Analysis of the EGFR and ErbB4 Kinase Domains.
(A) The residue-based betweenness profiles of the EGFR-WT structures are shown for Cdk/Src-IF1 (in blue), Cdk/Src-IF2 (in red) and the active conformation (in green). (B) The betweenness profiles of Cdk/Src-IF2 EGFR structures (WT in blue, L858R in red, and L858R/T790M in green). (C) The betweenness profiles of the active EGFR structures (WT in blue, L858R in red, and T790M in green). In (A–C) a close-up view of the EGFR force constant profile in the αC-helix (residues 752–768) and the adjacent αC-β4-loop regions (residues 769–777) is provided as an inset. (D) The betweenness profiles of Cdk/Src-IF1 and active ErbB4 structures are shown in blue and green respectively. A close-up view of the EGFR force constant profile in the αC-helix (residues 752–768) and the adjacent αC-β4-loop regions (residues 769–777) is provided as an inset. The annotated EGFR residues and respective functional regions corresponding to the peaks in the profiles (A–C) included: F723 (P-loop), catalytic pair K745 and E762, M766, L777 (αC-helix), hinge, αE-helix, H835(HRD motif), F856 (DFG motif), W880 (P+1 substrate loop), D896 (αF-helix), αH, and αI helix. The R-spine EGFR residues (M766, L777, H835, F856, D896) are shown by filled maroon-colored diamond symbols. The annotated ErbB4 residues and functional regions in (D) included F704 (P-loop), catalytic pair K726 and E743, M747, L758 (αC-helix), hinge, αE-helix, H816 (HRD motif), F837 (DFG motif), W861 (P+1 substrate loop), D877 (αF-helix), αH, and αI helix. The R-spine ErbB4 residues (M747, L758, H816, F837, and D877) are shown by filled maroon-colored diamond symbols.
Figure 12.
Centrality Analysis of the Active EGFR Dimers.
The residue-based betweenness profiles of the active EGFR dimer are shown for EGFR-WT (A, B) and L858R/T790M (C, D). The profiles are shown for the acceptor (left panels A, C) and donor monomers (right panels B, D). The annotated functional regions included JM-B region, P-loop, αC-helix, hinge, αE-helix, HRD motif, DFG motif, substrate binding P+1 loop, αF-helix, αH, and αI helix. The annotated peaks in the profiles reflecting structural stability of the EGFR-WT dimer included L680 (JM-B region), M742, L753, H811, F832, D872 (R-spine residues), and W856 (P+1 substrate loop). The respective peaks in the profile of the EGFR-L858R/T790M dimer corresponded to L704 (JM-B region), M766, L777, H835, F856, and D896 (R-spine residues), and W880 (P+1 substrate loop). The R-spine residues are annotated as maroon-colored diamond symbols and the oncogenic mutation sites are indicated as orange-colored diamond symbols. Note that a different EGFR sequence numbering was adopted in the original crystal structures of the EGFR-WT dimer and L858R/T790M double mutant dimer. We kept the original numbering to avoid confusion in comparisons with the experimental data. In the EGFR-WT structure (A, B); the oncogenic sites correspond to L834 and T766. The R-spine residues in EGFR-WT are M742, L753, H811, F832, and D872. In the crystal structure of the EGFR oncogenic mutant (C, D), the mutated residues correspond to L858R and T790M. The R-spine residues in the mutant structure are M766, L777, H835, F856, and D896.
Figure 13.
Conformational Allosteric Pathways in the ErbB Kinases.
Conformational allosteric pathways between P-loop of the N-terminal lobe and P+1 substrate loop of the C-terminal lobe are shown for the EGFR-WT Kinase domain (Upper Left Panel) and ErbB4 kinase domain (Upper Right Panel). The allosteric pathways are based on the constructed protein structure networks and are determined as the shortest paths between two given residues: F723 in the P-loop and W880 in the P+1 substrate loop for EGFR (Upper left Panel); and between F704 in the P-loop and W861 in the P+1 substrate loop for ErbB4 (Upper Right Panel). The allosteric pathway in the EGFR-WT dimer was obtained as the shortest path in the ensemble of pathways connecting the P-loop of the N-terminal donor monomer with the P+1 substrate loop of the C-terminal acceptor monomer (W856). The residues are colored according to their conformational mobility as in Figures 3–5. The color gradient from blue to red indicates the decreasing structural rigidity (or increasing conformational mobility) of the protein residues. The allosteric pathways are annotated with the contributing residues shown in filled spheres.
Figure 14.
Conformational Mobility and Centrality Profiles: A Comparative Analysis of Apo-EGFR and ATP-bound EGFR Structures.
(A, B) Joint distributions of conformational mobility (computed B-factors) and relative solvent accessibility (RSA) are shown for Apo EGFR form (pdb id 2GS2) and ATP-bound active EGFR structure (pdb id 2ITX). Joint distributions of network centrality and RSA parameters are shown for Apo EGFR form (C) and ATP-bound active EGFR structure (D). The MD-based distributions indicated similarity in the conformational mobility and important differences in the network centrality profiles.
Figure 15.
Centrality Analysis of the Apo and ATP-bound EGFR Structures.
(A) The residue-based closeness profiles are shown for the Apo form of EGFR-WT (in blue) and ATP-bound EGFR (in green). (B) The residue-based closeness in the Apo-EGGFR (blue bars) and ATP-bound EGFR (green bars) are highlighted for key functional and nucleotide binding site residues, including the catalytic pair (K745, E762), R-spine (H835, F856, and D896), W880 (P+1 substrate loop), P-loop residues (L718,G719,A722,F723), hinge residues (T790,Q791,L792,M793), HRD motif (H835,R836,D837), DFG motif (D855,F856,G857). (C) The residue-based betweenness profiles are shown for the Apo form of EGFR-WT (in blue) and ATP-bound EGFR (in green). (D) The residue-based betweenness in the Apo-EGFR (blue bars) and ATP-bound EGFR (green bars) are highlighted for key functional residues including the R-spine and ATP-binding site residues.