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Hierarchical modeling of activation mechanisms in the ABL and EGFR kinase domains: thermodynamic and mechanistic catalysts of kinase activation by cancer mutations.

Dixit A, Verkhivker GM - PLoS Comput. Biol. (2009)

Bottom Line: We have also simulated the activating effect of the gatekeeper mutation on conformational dynamics and allosteric interactions in functional states of the ABL-SH2-SH3 regulatory complexes.Collectively, the results of this study have revealed thermodynamic and mechanistic catalysts of kinase activation by major cancer-causing mutations in the ABL and EGFR kinase domains.The results of this study reconcile current experimental data with insights from theoretical approaches, pointing to general mechanistic aspects of activating transitions in protein kinases.

View Article: PubMed Central - PubMed

Affiliation: Graduate Program in Bioinformatics and Center for Bioinformatics, The University of Kansas, Lawrence, Kansas, United States of America.

ABSTRACT
Structural and functional studies of the ABL and EGFR kinase domains have recently suggested a common mechanism of activation by cancer-causing mutations. However, dynamics and mechanistic aspects of kinase activation by cancer mutations that stimulate conformational transitions and thermodynamic stabilization of the constitutively active kinase form remain elusive. We present a large-scale computational investigation of activation mechanisms in the ABL and EGFR kinase domains by a panel of clinically important cancer mutants ABL-T315I, ABL-L387M, EGFR-T790M, and EGFR-L858R. We have also simulated the activating effect of the gatekeeper mutation on conformational dynamics and allosteric interactions in functional states of the ABL-SH2-SH3 regulatory complexes. A comprehensive analysis was conducted using a hierarchy of computational approaches that included homology modeling, molecular dynamics simulations, protein stability analysis, targeted molecular dynamics, and molecular docking. Collectively, the results of this study have revealed thermodynamic and mechanistic catalysts of kinase activation by major cancer-causing mutations in the ABL and EGFR kinase domains. By using multiple crystallographic states of ABL and EGFR, computer simulations have allowed one to map dynamics of conformational fluctuations and transitions in the normal (wild-type) and oncogenic kinase forms. A proposed multi-stage mechanistic model of activation involves a series of cooperative transitions between different conformational states, including assembly of the hydrophobic spine, the formation of the Src-like intermediate structure, and a cooperative breakage and formation of characteristic salt bridges, which signify transition to the active kinase form. We suggest that molecular mechanisms of activation by cancer mutations could mimic the activation process of the normal kinase, yet exploiting conserved structural catalysts to accelerate a conformational transition and the enhanced stabilization of the active kinase form. The results of this study reconcile current experimental data with insights from theoretical approaches, pointing to general mechanistic aspects of activating transitions in protein kinases.

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MD simulations of the ABL kinase domain in the Src-like inactive form.Upper Panel: (A) The RMSD fluctuations of Cα atoms and (B) the RMSF values of Cα atoms from MD simulations of ABL-WT (in blue), ABL-T315I (in red), and ABL-L387M (in green). MD simulations were performed using the Src-like inactive form of the ABL kinase domain. Legends inside the figure panels refer to the pdb entries used in MD simulations. Lower Panel: Color-coded mapping of the averaged protein flexibility profiles (RMSF values) from MD simulations of the ABL-WT and ABL mutants. This mapping is presented for ABL-WT (C), ABL-T315I (D) and ABL-L387M (E). The color-coded sliding scheme is the same as was adopted for Figure 3.
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pcbi-1000487-g004: MD simulations of the ABL kinase domain in the Src-like inactive form.Upper Panel: (A) The RMSD fluctuations of Cα atoms and (B) the RMSF values of Cα atoms from MD simulations of ABL-WT (in blue), ABL-T315I (in red), and ABL-L387M (in green). MD simulations were performed using the Src-like inactive form of the ABL kinase domain. Legends inside the figure panels refer to the pdb entries used in MD simulations. Lower Panel: Color-coded mapping of the averaged protein flexibility profiles (RMSF values) from MD simulations of the ABL-WT and ABL mutants. This mapping is presented for ABL-WT (C), ABL-T315I (D) and ABL-L387M (E). The color-coded sliding scheme is the same as was adopted for Figure 3.

Mentions: MD simulations of the ABL and EGFR kinase domains were performed to address the following objectives: (a) to determine whether thermal motions in structurally different conformational states of ABL and EGFR may tolerate structural impact of activating mutations; (b) to study equilibrium dynamics of the ABL and EGFR kinase domain in the normal and oncogenic forms; and (c) to investigate the impact of activating mutations on the thermodynamic stability of structurally different conformational states of ABL and EGFR. MD trajectories of the ABL kinase domain (Figures 3–5) and EGFR kinase domain (Figures 6,7) displayed important similarities and differences in the equilibrium profile of the WT and mutant forms. The RMSD fluctuations of the Src-like ABL (Figures 4) and Src/Cdk-like EGFR (Figure 6) displayed a similar convergence to an equilibrium plateau within first 2 ns–3 ns and remained stable for the remainder of simulations. Characteristically, mutations can induce larger thermal fluctuations in the inactive kinase state (RMSD = 2.2 Å–2.5 Å), especially for ABL-L387M and EGFR-L858R. Overall, there were no significant differences between the RMSD profiles of ABL and EGFR in structurally different kinase states, although thermal fluctuations of the EGFR variants were somewhat smaller (1.5Å–2.0Å) (Figures 6,7). We also analyzed protein flexibility variations computed from the root mean square fluctuation (RMSF) of the backbone residues. To facilitate the analysis, the average protein flexibility profiles were mapped onto the respective conformational states of ABL and EGFR using a color-coded sliding scheme. In agreement with the structural factors, the regions of larger thermal fluctuations and the increased conformational flexibility corresponded to P-loop, αC-helix and the activation loop. During simulations in the Src-like inactive states, we observed a consistently increased level of thermal fluctuations for the ABL-L387M (Figure 4) and EGFR-L858R mutants (Figure 6) that are strategically located in the middle of the activation loop and may thus cause larger local perturbations. In contrast, EGFR-L858R mutant displayed the decreased thermal fluctuations and smaller RMSF values in the active state (Figure 7). This may partly reflect the activating nature of the EGFR-L858R mutation that is known to shift the thermodynamic equilibrium away from the Src/Cdk-like form towards a more stable active kinase structure.


Hierarchical modeling of activation mechanisms in the ABL and EGFR kinase domains: thermodynamic and mechanistic catalysts of kinase activation by cancer mutations.

Dixit A, Verkhivker GM - PLoS Comput. Biol. (2009)

MD simulations of the ABL kinase domain in the Src-like inactive form.Upper Panel: (A) The RMSD fluctuations of Cα atoms and (B) the RMSF values of Cα atoms from MD simulations of ABL-WT (in blue), ABL-T315I (in red), and ABL-L387M (in green). MD simulations were performed using the Src-like inactive form of the ABL kinase domain. Legends inside the figure panels refer to the pdb entries used in MD simulations. Lower Panel: Color-coded mapping of the averaged protein flexibility profiles (RMSF values) from MD simulations of the ABL-WT and ABL mutants. This mapping is presented for ABL-WT (C), ABL-T315I (D) and ABL-L387M (E). The color-coded sliding scheme is the same as was adopted for Figure 3.
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pcbi-1000487-g004: MD simulations of the ABL kinase domain in the Src-like inactive form.Upper Panel: (A) The RMSD fluctuations of Cα atoms and (B) the RMSF values of Cα atoms from MD simulations of ABL-WT (in blue), ABL-T315I (in red), and ABL-L387M (in green). MD simulations were performed using the Src-like inactive form of the ABL kinase domain. Legends inside the figure panels refer to the pdb entries used in MD simulations. Lower Panel: Color-coded mapping of the averaged protein flexibility profiles (RMSF values) from MD simulations of the ABL-WT and ABL mutants. This mapping is presented for ABL-WT (C), ABL-T315I (D) and ABL-L387M (E). The color-coded sliding scheme is the same as was adopted for Figure 3.
Mentions: MD simulations of the ABL and EGFR kinase domains were performed to address the following objectives: (a) to determine whether thermal motions in structurally different conformational states of ABL and EGFR may tolerate structural impact of activating mutations; (b) to study equilibrium dynamics of the ABL and EGFR kinase domain in the normal and oncogenic forms; and (c) to investigate the impact of activating mutations on the thermodynamic stability of structurally different conformational states of ABL and EGFR. MD trajectories of the ABL kinase domain (Figures 3–5) and EGFR kinase domain (Figures 6,7) displayed important similarities and differences in the equilibrium profile of the WT and mutant forms. The RMSD fluctuations of the Src-like ABL (Figures 4) and Src/Cdk-like EGFR (Figure 6) displayed a similar convergence to an equilibrium plateau within first 2 ns–3 ns and remained stable for the remainder of simulations. Characteristically, mutations can induce larger thermal fluctuations in the inactive kinase state (RMSD = 2.2 Å–2.5 Å), especially for ABL-L387M and EGFR-L858R. Overall, there were no significant differences between the RMSD profiles of ABL and EGFR in structurally different kinase states, although thermal fluctuations of the EGFR variants were somewhat smaller (1.5Å–2.0Å) (Figures 6,7). We also analyzed protein flexibility variations computed from the root mean square fluctuation (RMSF) of the backbone residues. To facilitate the analysis, the average protein flexibility profiles were mapped onto the respective conformational states of ABL and EGFR using a color-coded sliding scheme. In agreement with the structural factors, the regions of larger thermal fluctuations and the increased conformational flexibility corresponded to P-loop, αC-helix and the activation loop. During simulations in the Src-like inactive states, we observed a consistently increased level of thermal fluctuations for the ABL-L387M (Figure 4) and EGFR-L858R mutants (Figure 6) that are strategically located in the middle of the activation loop and may thus cause larger local perturbations. In contrast, EGFR-L858R mutant displayed the decreased thermal fluctuations and smaller RMSF values in the active state (Figure 7). This may partly reflect the activating nature of the EGFR-L858R mutation that is known to shift the thermodynamic equilibrium away from the Src/Cdk-like form towards a more stable active kinase structure.

Bottom Line: We have also simulated the activating effect of the gatekeeper mutation on conformational dynamics and allosteric interactions in functional states of the ABL-SH2-SH3 regulatory complexes.Collectively, the results of this study have revealed thermodynamic and mechanistic catalysts of kinase activation by major cancer-causing mutations in the ABL and EGFR kinase domains.The results of this study reconcile current experimental data with insights from theoretical approaches, pointing to general mechanistic aspects of activating transitions in protein kinases.

View Article: PubMed Central - PubMed

Affiliation: Graduate Program in Bioinformatics and Center for Bioinformatics, The University of Kansas, Lawrence, Kansas, United States of America.

ABSTRACT
Structural and functional studies of the ABL and EGFR kinase domains have recently suggested a common mechanism of activation by cancer-causing mutations. However, dynamics and mechanistic aspects of kinase activation by cancer mutations that stimulate conformational transitions and thermodynamic stabilization of the constitutively active kinase form remain elusive. We present a large-scale computational investigation of activation mechanisms in the ABL and EGFR kinase domains by a panel of clinically important cancer mutants ABL-T315I, ABL-L387M, EGFR-T790M, and EGFR-L858R. We have also simulated the activating effect of the gatekeeper mutation on conformational dynamics and allosteric interactions in functional states of the ABL-SH2-SH3 regulatory complexes. A comprehensive analysis was conducted using a hierarchy of computational approaches that included homology modeling, molecular dynamics simulations, protein stability analysis, targeted molecular dynamics, and molecular docking. Collectively, the results of this study have revealed thermodynamic and mechanistic catalysts of kinase activation by major cancer-causing mutations in the ABL and EGFR kinase domains. By using multiple crystallographic states of ABL and EGFR, computer simulations have allowed one to map dynamics of conformational fluctuations and transitions in the normal (wild-type) and oncogenic kinase forms. A proposed multi-stage mechanistic model of activation involves a series of cooperative transitions between different conformational states, including assembly of the hydrophobic spine, the formation of the Src-like intermediate structure, and a cooperative breakage and formation of characteristic salt bridges, which signify transition to the active kinase form. We suggest that molecular mechanisms of activation by cancer mutations could mimic the activation process of the normal kinase, yet exploiting conserved structural catalysts to accelerate a conformational transition and the enhanced stabilization of the active kinase form. The results of this study reconcile current experimental data with insights from theoretical approaches, pointing to general mechanistic aspects of activating transitions in protein kinases.

Show MeSH
Related in: MedlinePlus