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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-SH2-SH3 regulatory complex in the active form.Upper Panel: Color-coded mapping of the averaged protein flexibility profiles (RMSF values) from MD simulations in the active form (pdb entry 1OPL). The mapping is presented for ABL-WT (A) and ABL-T315I (B). The color-coded sliding scheme is the same as was adopted for Figure 3. Lower Panel: The RMSD fluctuations of Cα Atoms (C) and the RMSF values of Cα Atoms (D) from MD simulations. MD simulations of ABL-WT (in blue), and ABL-T315I (in red) were performed using the active ABL form (“top-hat”) (pdb entry 1OPL).
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pcbi-1000487-g010: MD simulations of the ABL-SH2-SH3 regulatory complex in the active form.Upper Panel: Color-coded mapping of the averaged protein flexibility profiles (RMSF values) from MD simulations in the active form (pdb entry 1OPL). The mapping is presented for ABL-WT (A) and ABL-T315I (B). The color-coded sliding scheme is the same as was adopted for Figure 3. Lower Panel: The RMSD fluctuations of Cα Atoms (C) and the RMSF values of Cα Atoms (D) from MD simulations. MD simulations of ABL-WT (in blue), and ABL-T315I (in red) were performed using the active ABL form (“top-hat”) (pdb entry 1OPL).

Mentions: Although ABL and EGFR share a common kinase domain, they can be regulated through different mechanisms. Indeed, activation of the ABL kinases can be linked with the formation of multi-protein regulatory complexes with the SH2 and SH3 domains [13],[17], whereas EGFR may be activated through dimerization mechanism [24]. These regulatory interactions have a major role in determining the conformational dynamics of the kinase domain and activation mechanisms. Crystallographic studies [13],[17] demonstrated that the ABL regulatory complex in the downregulated, inactive can form a tightly assembled conformation in which the SH3-SH2 unit docked onto the back of the kinase domain, considerably restricting its conformational flexibility (Figure 9). Moreover, a critical N-terminal cap segment connecting the myristoyl group to the SH3 domain contributed to the stability of the inactive ABL state (pdb entry 2FO0) [89],[90]. Small angle X-ray scattering (SAXS) analysis of the activated ABL form revealed that the release of the inhibitory interactions could allow the SH2 and SH3 domains to switch to a vastly different structural arrangement [17]. In this structure (Figure 10), the SH2 domain was docked directly on the top of the N-lobe (“top-hat” conformation) (pdb entry 1OPL), while the SH3 domain became disordered along with the linker connecting the SH2 and the kinase domains. To gain dynamic and mechanistic insight into allosteric effects of the ABL-T315I mutant, we performed MD simulations of the ABL-SH2-SH3 regulatory complexes in the inactive (“side-to-side”) and active (“top-hat”) states. The objectives of these simulations were the following: (a) to determine the effect of activated ABL-T315I mutation on the conformational dynamics in different functional states of ABL; (b) to test a hypothesis whether upregulation of kinase activity, caused by ABL-T315I, may be related to the dynamic preferences in the “top-hat” conformation of the ABL-SH2-SH3 complex. The results of simulations were analyzed using a direct comparison with the recent HX MS experiments of ABL regulatory complexes in the normal and mutational forms [58].


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-SH2-SH3 regulatory complex in the active form.Upper Panel: Color-coded mapping of the averaged protein flexibility profiles (RMSF values) from MD simulations in the active form (pdb entry 1OPL). The mapping is presented for ABL-WT (A) and ABL-T315I (B). The color-coded sliding scheme is the same as was adopted for Figure 3. Lower Panel: The RMSD fluctuations of Cα Atoms (C) and the RMSF values of Cα Atoms (D) from MD simulations. MD simulations of ABL-WT (in blue), and ABL-T315I (in red) were performed using the active ABL form (“top-hat”) (pdb entry 1OPL).
© Copyright Policy
Related In: Results  -  Collection

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

pcbi-1000487-g010: MD simulations of the ABL-SH2-SH3 regulatory complex in the active form.Upper Panel: Color-coded mapping of the averaged protein flexibility profiles (RMSF values) from MD simulations in the active form (pdb entry 1OPL). The mapping is presented for ABL-WT (A) and ABL-T315I (B). The color-coded sliding scheme is the same as was adopted for Figure 3. Lower Panel: The RMSD fluctuations of Cα Atoms (C) and the RMSF values of Cα Atoms (D) from MD simulations. MD simulations of ABL-WT (in blue), and ABL-T315I (in red) were performed using the active ABL form (“top-hat”) (pdb entry 1OPL).
Mentions: Although ABL and EGFR share a common kinase domain, they can be regulated through different mechanisms. Indeed, activation of the ABL kinases can be linked with the formation of multi-protein regulatory complexes with the SH2 and SH3 domains [13],[17], whereas EGFR may be activated through dimerization mechanism [24]. These regulatory interactions have a major role in determining the conformational dynamics of the kinase domain and activation mechanisms. Crystallographic studies [13],[17] demonstrated that the ABL regulatory complex in the downregulated, inactive can form a tightly assembled conformation in which the SH3-SH2 unit docked onto the back of the kinase domain, considerably restricting its conformational flexibility (Figure 9). Moreover, a critical N-terminal cap segment connecting the myristoyl group to the SH3 domain contributed to the stability of the inactive ABL state (pdb entry 2FO0) [89],[90]. Small angle X-ray scattering (SAXS) analysis of the activated ABL form revealed that the release of the inhibitory interactions could allow the SH2 and SH3 domains to switch to a vastly different structural arrangement [17]. In this structure (Figure 10), the SH2 domain was docked directly on the top of the N-lobe (“top-hat” conformation) (pdb entry 1OPL), while the SH3 domain became disordered along with the linker connecting the SH2 and the kinase domains. To gain dynamic and mechanistic insight into allosteric effects of the ABL-T315I mutant, we performed MD simulations of the ABL-SH2-SH3 regulatory complexes in the inactive (“side-to-side”) and active (“top-hat”) states. The objectives of these simulations were the following: (a) to determine the effect of activated ABL-T315I mutation on the conformational dynamics in different functional states of ABL; (b) to test a hypothesis whether upregulation of kinase activity, caused by ABL-T315I, may be related to the dynamic preferences in the “top-hat” conformation of the ABL-SH2-SH3 complex. The results of simulations were analyzed using a direct comparison with the recent HX MS experiments of ABL regulatory complexes in the normal and mutational forms [58].

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