Public Library of Science, Biology (2006) Vol. 4(5): e144   Local Copy

Summary / Figures / Table / Supporting Information / Dataset

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Summary: The improper activation of the Abl tyrosine kinase results in chronic myeloid leukemia (CML). The recognition of an inactive conformation of Abl, in which a catalytically important Asp-Phe-Gly (DFG) motif is flipped by approximately 180° with respect to the active conformation, underlies the specificity of the cancer drug imatinib, which is used to treat CML. The DFG motif is not flipped in crystal structures of inactive forms of the closely related Src kinases, and imatinib does not inhibit c-Src. We present a structure of the kinase domain of Abl, determined in complex with an ATP-peptide conjugate, in which the protein adopts an inactive conformation that resembles closely that of the Src kinases. An interesting aspect of the Src-like inactive structure, suggested by molecular dynamics simulations and additional crystal structures, is the presence of features that might facilitate the flip of the DFG motif by providing room for the phenylalanine to move and by coordinating the aspartate side chain as it leaves the active site. One class of mutations in BCR-Abl that confers resistance to imatinib appears more likely to destabilize the inactive Src-like conformation than the active or imatinib-bound conformations. Our results suggest that interconversion between distinctly different inactive conformations is a characteristic feature of the Abl kinase domain.

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Figures (Click on the small image to view the bigger one):

Figure 1. Distinct States of the c-Abl and c-Src Kinase Domains
Three key kinase domain conformations considered at length in the text are shown in (A-C). At the top, a schematic representation of each state and an enlarged schematic are shown, detailing the conformations of the DFG motif (red) and helix αC (blue). Below the schematics the crystal structure of each conformation is shown. The activation loop is colored red, helix αC blue, and the catalytic loop orange.

1. The conformation of inactive c-Abl, bound to imatinib (molecule A).
   
2. The conformation of the inactive Src family kinases. This conformation is now seen in Abl as well (molecule B, structure 1). Both the c-Src and Abl numbering are indicated.
   
3. Active Abl (molecule C, structure 2).
   
4. In cells the active conformation of Abl undergoes rapid autophosphorylation that is expected to trap the protein in the active conformation (indicated as C*). Similarly, imatinib only binds to Abl when the kinase domain adopts conformation A and forms a stable complex with the protein (A*). The interconversion between the different states of Abl is shown in the context of this competition.

Figure 2. Molecule B Closely Resembles the Structure of the Inactive Src Kinases

1. The kinase domain of Abl (molecule B) is shown as a cartoon with the bisubstrate analog inhibitor depicted as a stick model.
2. Structure of the ATP-peptide conjugate at the active site.
3. Comparison between molecule B (left) and the structure of a Src-family kinase (right).
4. Backbone torsion angles of Asp 381 of the DFG motif.
5. The backbone of Asp 381 can start to move from DFG-In to the DFG-Out conformations by three paths in torsion angle space.
6. Illustration of rotations about ϕ(Asp 381) and ψ(Asp 381) in the Src-like inactive versus the active structures of Abl.

Figure 3. Targeted Molecular Dynamics Simulations Suggest a Path for DFG Flipping

1. Coordination of Asp 381 during the DFG flip, shown in stereo, starting from the Src-like structure and following transition path A in backbone torsion angle space (see Figure 2E). The coordination of Asp 381 is almost identical for transition path B, except that the carbonyl of Asp 381 flips in the other direction (see Figure S3).
2. The side chain of Phe 382 passing through a hydrophobic pocket in the Src-like inactive molecule B.
3. The paths of Asp 381 and Phe 382 in backbone torsion angle space for two trajectories starting with the Src-like inactive structure. Shown in red are the torsion angles seen in the first quarter of a TMD time series, in green, the second quarter of the time series, in blue, the third quarter, and in purple, the last quarter.
4. Unfavorable interactions between the backbone carbonyl of Phe 382 and the carboxylate group of Glu 286 during TMD simulations starting from the active structure, where the Glu 286-Lys 271 salt bridge is present.

Figure 4. The Helical Turn following the DFG motif in Src-Like Inactive Structures

A. The helical turn in the activation loop of molecule B that immediately follows the DFG motif is a characteristic feature of the Src-like conformation and is conserved in four different kinase families.
B-C. The side chain of Arg 386, presented by the helical turn, forms a hydrogen bond to a backbone carbonyl of Ile 360, a salt bridge with Glu 286, and an amino-aromatic interaction with Phe 359 that positions it for interacting with Asp 381 during the DFG flip.
D. An intermediate structure during one of the TMD simulations, showing the capture of Asp 381 by Arg 386.

Figure 5. The Structure of Abl in an Intermediate Conformation Suggests a Path for the Transition between the Active and Src-Like Conformations

1. The intermediate structure of Abl (molecule E) is shown, with helix αC shown in blue, the activation loop in red, and the catalytic loop containing Arg 362 in orange.
2. The proposed pathway by which Abl makes the transition to the Src-like conformation.

Figure 6. Molecule B Helps Explain Mutations in the Kinase Domain of Abl That Confer Resistance to Imatinib

A. The side chains of residues implicated in imatinib resistance in the Azam et al. study are shown in blue in the context of molecule B. A large number of these mutations cluster in the interface between helix αC, the N-lobe, and the helical turn in the activation loop.
B-D. For three of these mutations we have shown the surface for all atoms within 6 Å of the mutated residue in the context of different structures. The Abl:imatinib complex is in green and the Src-like structure (molecule B) in yellow-orange. (B) Asp 276. (C) Leu 387. (D) Met 278.

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Table:   Table 1 Crystal Structures of the Kinase Domain of Abl

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Supporting Information: Figures

Figure S1. Sequence-Specific Interactions between the ATP-Peptide Conjugates and the Kinase Domain of Abl

1. The structure of the peptide-ATP conjugates.
2. Interactions between the ATP-peptide conjugate with the optimal peptide sequence and Abl molecule E.
3. Interactions between the ATP-peptide conjugate with the suboptimal peptide sequence and Abl molecule B.

Figure S2. Choice of Restraint Set and Force Constant for TMD

1. When only the residues ³⁷⁹VADF³⁸² are restrained, the deviation in the N-lobe is minimal. Gray: molecule B, starting structure; Yellow-orange: simulation final structure.
2. Restraint force (pN), and r.m.s. distance of the restraint from the target (Å), plotted versus time (picoseconds) for two low-force constant (K = 0.15 kcal mol⁻¹ Å⁻²) simulations starting with the active structure.

Figure S3. Targeted Molecular Dynamics Simulations Propose a Path for DFG Flipping

1. Coordination of Asp 381 during the DFG flip, starting from the Src-like inactive structure (molecule B) and following transition path B in backbone torsion angle space. The coordination of Asp 381 is almost identical for transition path A, except that the carbonyl of Asp 381 flips in the other direction.
2. The side chain of Phe 382 passing through a hydrophobic pocket in the Src-like molecule for transition path B.
3. The paths of Asp 381 and Phe 382 in backbone torsion angle space for two trajectories.

Figure S4. Molecule B Helps Explain Mutations in the Kinase Domain of Abl That Confer Resistance to Imatinib
For two of these mutations we have shown the surface for all atoms within 6 Å of the mutated residue in the context of different structures. The active structure is shown in pink, the Abl:imatinib complex in green, and the Src-like structure (molecule B) in yellow-orange. (A) Phe 359. (B) Tyr 253.

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Supporting Information: Tables

Table S1: Crystallographic Data and Refinement
Table S2: Hydrogen Bond Distances between Key Residues

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Supporting Information: Protocol

Protocol S1: Basis for the Specificity of the Kinase Domain of Abl for Peptide Substrates

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Dataset: (tar.gz files)

Dataset S1. Targeted Molecular Dynamics Trajectory following Transition 1 in Figure 1D
Dataset S2. Targeted Molecular Dynamics Trajectory following Transition 2 in Figure 1D