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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 of the ABL-T315I and EGFR-T790M mutants.(A) Superposition of the predicted structural model of the ABL-T315I mutant (in blue) with the crystal structure of ABL-T315I (active form, pdb entry 2Z60, in green). (B) Superposition of the predicted structural model of the EGFR-T790M mutant (in blue) with the crystal structure of EGFR-T790M (active form, pdb entry 2JIT, in green). The initial ABL and EGFR structures that converged during homology modeling refinement to the crystallographic active conformations of the mutants correspond to the Src-like inactive ABL (pdb entry 2G1T) and Src-like inactive EGFR (pdb entry 2GS7).
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pcbi-1000487-g001: The predicted structural models of the ABL-T315I and EGFR-T790M mutants.(A) Superposition of the predicted structural model of the ABL-T315I mutant (in blue) with the crystal structure of ABL-T315I (active form, pdb entry 2Z60, in green). (B) Superposition of the predicted structural model of the EGFR-T790M mutant (in blue) with the crystal structure of EGFR-T790M (active form, pdb entry 2JIT, in green). The initial ABL and EGFR structures that converged during homology modeling refinement to the crystallographic active conformations of the mutants correspond to the Src-like inactive ABL (pdb entry 2G1T) and Src-like inactive EGFR (pdb entry 2GS7).

Mentions: An important initial insight into structural effects of activating mutations was obtained from homology modeling of the conserved gatekeeper mutants ABL-T315I and EGFR-T790M. Structural analysis of ABL-T315I was attempted from the Imatinib-bound inactive and Src-like inactive conformations. Similarly, homology refinement of EGFR-T790M was carried out using the Src/Cdk-like inactive EGFR conformations. The predicted structural models conform closely to the crystallographic conformations of the activation loop in the mutant structures within the root mean square deviation (RMSD) of 2.0 Å–2.2 Å (Figure 1). It is worth noting that the target conformations of the activation loop in the mutant crystal structures assume the active form and were vastly different (RMSD ∼8 Å–10Å) from the starting conformations corresponding to the Src-like kinase state. Consequently, homology modeling of mutational effects in the ABL and EGFR kinase domains is rather challenging, albeit at a high sequence identity. While homology refinement could not accurately reproduce structural changes in the remote peripheral regions of the N-terminal and C-terminal lobes, simulations were capable of depicting the correct repositioning of the functionally important P-loop, αC-helix and the activation loop (Figure 1). A close structural inspection of the predicted ABL-T315I (Figure S3A) and EGFR-T790M models (Figure S3B) revealed a specific intermediate position of the Phe residue in the DFG motif that was favored by the enhanced hydrophobic interactions with the respective gatekeeper residue. Interestingly, while one copy of the EGFR-T790M crystal structure was in the active state with the characteristic “DFG-in” conformation, the second molecule displayed a rather unique intermediate DFG conformation [52]. The DFG conformation in the predicted model of EGFR-T790M closely overlapped with the one observed in the second molecule of the EGFR-T790M crystal structure. A convergent structural effect of the ABL-T315I and EGFR-T790M mutants (Figure S3C) seen in the predicted models may have functional relevance in facilitating conformational transition of the activation loop. The bulkier mutant residues at the gatekeeper position may unlock the DFG motif from its autoinhibitory conformation by strengthening the hydrophobic interactions with the Phe residue in the intermediate conformation. A specific DFG conformation, recruited by the mutated gatekeeper residues, may act as a conformational trigger in facilitating transition from the inactive DFG-out to the active DFG-in state. The proposed structural mechanism may thus initiate the disruption of the autoinhibitory interactions and cause imbalance in the dynamic equilibrium between inactive and active conformational states regulating kinase activity. Hence, homology modeling of the ABL-T315I and EGFR-T790M mutants could provide an initial insight into molecular basis of activation by cancer mutations.


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 of the ABL-T315I and EGFR-T790M mutants.(A) Superposition of the predicted structural model of the ABL-T315I mutant (in blue) with the crystal structure of ABL-T315I (active form, pdb entry 2Z60, in green). (B) Superposition of the predicted structural model of the EGFR-T790M mutant (in blue) with the crystal structure of EGFR-T790M (active form, pdb entry 2JIT, in green). The initial ABL and EGFR structures that converged during homology modeling refinement to the crystallographic active conformations of the mutants correspond to the Src-like inactive ABL (pdb entry 2G1T) and Src-like inactive EGFR (pdb entry 2GS7).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2722018&req=5

pcbi-1000487-g001: The predicted structural models of the ABL-T315I and EGFR-T790M mutants.(A) Superposition of the predicted structural model of the ABL-T315I mutant (in blue) with the crystal structure of ABL-T315I (active form, pdb entry 2Z60, in green). (B) Superposition of the predicted structural model of the EGFR-T790M mutant (in blue) with the crystal structure of EGFR-T790M (active form, pdb entry 2JIT, in green). The initial ABL and EGFR structures that converged during homology modeling refinement to the crystallographic active conformations of the mutants correspond to the Src-like inactive ABL (pdb entry 2G1T) and Src-like inactive EGFR (pdb entry 2GS7).
Mentions: An important initial insight into structural effects of activating mutations was obtained from homology modeling of the conserved gatekeeper mutants ABL-T315I and EGFR-T790M. Structural analysis of ABL-T315I was attempted from the Imatinib-bound inactive and Src-like inactive conformations. Similarly, homology refinement of EGFR-T790M was carried out using the Src/Cdk-like inactive EGFR conformations. The predicted structural models conform closely to the crystallographic conformations of the activation loop in the mutant structures within the root mean square deviation (RMSD) of 2.0 Å–2.2 Å (Figure 1). It is worth noting that the target conformations of the activation loop in the mutant crystal structures assume the active form and were vastly different (RMSD ∼8 Å–10Å) from the starting conformations corresponding to the Src-like kinase state. Consequently, homology modeling of mutational effects in the ABL and EGFR kinase domains is rather challenging, albeit at a high sequence identity. While homology refinement could not accurately reproduce structural changes in the remote peripheral regions of the N-terminal and C-terminal lobes, simulations were capable of depicting the correct repositioning of the functionally important P-loop, αC-helix and the activation loop (Figure 1). A close structural inspection of the predicted ABL-T315I (Figure S3A) and EGFR-T790M models (Figure S3B) revealed a specific intermediate position of the Phe residue in the DFG motif that was favored by the enhanced hydrophobic interactions with the respective gatekeeper residue. Interestingly, while one copy of the EGFR-T790M crystal structure was in the active state with the characteristic “DFG-in” conformation, the second molecule displayed a rather unique intermediate DFG conformation [52]. The DFG conformation in the predicted model of EGFR-T790M closely overlapped with the one observed in the second molecule of the EGFR-T790M crystal structure. A convergent structural effect of the ABL-T315I and EGFR-T790M mutants (Figure S3C) seen in the predicted models may have functional relevance in facilitating conformational transition of the activation loop. The bulkier mutant residues at the gatekeeper position may unlock the DFG motif from its autoinhibitory conformation by strengthening the hydrophobic interactions with the Phe residue in the intermediate conformation. A specific DFG conformation, recruited by the mutated gatekeeper residues, may act as a conformational trigger in facilitating transition from the inactive DFG-out to the active DFG-in state. The proposed structural mechanism may thus initiate the disruption of the autoinhibitory interactions and cause imbalance in the dynamic equilibrium between inactive and active conformational states regulating kinase activity. Hence, homology modeling of the ABL-T315I and EGFR-T790M mutants could provide an initial insight into molecular basis of activation by cancer mutations.

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