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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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The predicted structural models and interactions of the EGFR-L858R mutant.(A) Superposition of the crystal structures for the inactive EGFR-WT structure (initial structure in homology refinement) (pdb entry 2GS7, in green), EGFR-L858R mutant crystal structure (target structure in homology refinement) (pdb entry 2ITT, in blue) and computationally predicted EGFR-L858R model (in red). (B) A close-up comparison between activation loop conformations in the crystal structures of inactive EGFR-WT (pdb entry 2J6M, in green), EGFR-L858R mutant crystal structure (pdb entry 2ITT, in blue) and the predicted mutant conformation (in red). The lowest energy mutant model is within RMSD = 1.98 Å from the crystal structure of EGFR-L858R. (C) A close-up of functionally important residues and key interactions stabilizing the active conformation of EGFR-L858R. The ion pairs between Lys-745 and Glu-762 are shown for the crystal structure of the EGFR-L8585R (in blue) and the predicted mutant conformation (in red).
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pcbi-1000487-g002: The predicted structural models and interactions of the EGFR-L858R mutant.(A) Superposition of the crystal structures for the inactive EGFR-WT structure (initial structure in homology refinement) (pdb entry 2GS7, in green), EGFR-L858R mutant crystal structure (target structure in homology refinement) (pdb entry 2ITT, in blue) and computationally predicted EGFR-L858R model (in red). (B) A close-up comparison between activation loop conformations in the crystal structures of inactive EGFR-WT (pdb entry 2J6M, in green), EGFR-L858R mutant crystal structure (pdb entry 2ITT, in blue) and the predicted mutant conformation (in red). The lowest energy mutant model is within RMSD = 1.98 Å from the crystal structure of EGFR-L858R. (C) A close-up of functionally important residues and key interactions stabilizing the active conformation of EGFR-L858R. The ion pairs between Lys-745 and Glu-762 are shown for the crystal structure of the EGFR-L8585R (in blue) and the predicted mutant conformation (in red).

Mentions: Homology refinement successfully reconstructed the active crystallographic form of EGFR-L858R (pdb entry 2ITT) (Figure 2A). A considerable conformational rearrangement in the activation loop was predicted within RMSD = 1.9 Å–2.0 Å from the mutant crystal structure (Figure 2B). Moreover, homology refinement correctly reproduced coordinated structural changes of the activation loop and αC-helix (Figure 2A). The ability of homology modeling refinement to reproduce a large conformational change in EGFR-L858R may be partly attributed to a considerable incompatibility of Arg858 with the Src/Cdk-like inactive EGFR structure. In agreement with the assertion originally made by Kuriyan and coworkers [24], a local hydrophobic environment seemed to disfavor a polar side-chain of EGFR-L858R in the Src/Cdk-like inactive structure, and this may have facilitated a transition to a different local minimum corresponding to the active kinase form. A close inspection of the predicted structural model indicated that important interactions formed in the crystal structure of EGFR-L858R could be adequately reproduced, including a stable K745-E762 ion pair, known as a critical attribute of the active kinase form (Figure 2C).


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)

The predicted structural models and interactions of the EGFR-L858R mutant.(A) Superposition of the crystal structures for the inactive EGFR-WT structure (initial structure in homology refinement) (pdb entry 2GS7, in green), EGFR-L858R mutant crystal structure (target structure in homology refinement) (pdb entry 2ITT, in blue) and computationally predicted EGFR-L858R model (in red). (B) A close-up comparison between activation loop conformations in the crystal structures of inactive EGFR-WT (pdb entry 2J6M, in green), EGFR-L858R mutant crystal structure (pdb entry 2ITT, in blue) and the predicted mutant conformation (in red). The lowest energy mutant model is within RMSD = 1.98 Å from the crystal structure of EGFR-L858R. (C) A close-up of functionally important residues and key interactions stabilizing the active conformation of EGFR-L858R. The ion pairs between Lys-745 and Glu-762 are shown for the crystal structure of the EGFR-L8585R (in blue) and the predicted mutant conformation (in red).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2722018&req=5

pcbi-1000487-g002: The predicted structural models and interactions of the EGFR-L858R mutant.(A) Superposition of the crystal structures for the inactive EGFR-WT structure (initial structure in homology refinement) (pdb entry 2GS7, in green), EGFR-L858R mutant crystal structure (target structure in homology refinement) (pdb entry 2ITT, in blue) and computationally predicted EGFR-L858R model (in red). (B) A close-up comparison between activation loop conformations in the crystal structures of inactive EGFR-WT (pdb entry 2J6M, in green), EGFR-L858R mutant crystal structure (pdb entry 2ITT, in blue) and the predicted mutant conformation (in red). The lowest energy mutant model is within RMSD = 1.98 Å from the crystal structure of EGFR-L858R. (C) A close-up of functionally important residues and key interactions stabilizing the active conformation of EGFR-L858R. The ion pairs between Lys-745 and Glu-762 are shown for the crystal structure of the EGFR-L8585R (in blue) and the predicted mutant conformation (in red).
Mentions: Homology refinement successfully reconstructed the active crystallographic form of EGFR-L858R (pdb entry 2ITT) (Figure 2A). A considerable conformational rearrangement in the activation loop was predicted within RMSD = 1.9 Å–2.0 Å from the mutant crystal structure (Figure 2B). Moreover, homology refinement correctly reproduced coordinated structural changes of the activation loop and αC-helix (Figure 2A). The ability of homology modeling refinement to reproduce a large conformational change in EGFR-L858R may be partly attributed to a considerable incompatibility of Arg858 with the Src/Cdk-like inactive EGFR structure. In agreement with the assertion originally made by Kuriyan and coworkers [24], a local hydrophobic environment seemed to disfavor a polar side-chain of EGFR-L858R in the Src/Cdk-like inactive structure, and this may have facilitated a transition to a different local minimum corresponding to the active kinase form. A close inspection of the predicted structural model indicated that important interactions formed in the crystal structure of EGFR-L858R could be adequately reproduced, including a stable K745-E762 ion pair, known as a critical attribute of the active kinase form (Figure 2C).

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