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c-Src Binds to the Cancer Drug Imatinib with an Inactive Abl/c-Kit Conformation and a Distributed Thermodynamic Penalty

Markus A. Seeliger, Bhushan Nagar, Filipp Frank, Xiaoxian Cao, M. Nidanie Henderson, and John Kuriyan

Structure 15(3): 299-311.   Local Copy

Summary / Figures / Table / Supplemental Data / PDB Coordinates


The cancer drug imatinib inhibits the tyrosine kinases c-Abl, c-Kit, and the PDGF receptor. Imatinib is less effective against c-Src, which is difficult to understand because residues interacting with imatinib in crystal structures of Abl and c-Kit are conserved in c-Src. The crystal structure of the c-Src kinase domain in complex with imatinib closely resembles that of Abl•imatinib and c-Kit•imatinib, and differs significantly from the inactive "Src/CDK" conformation of the Src family kinases. Attempts to increase the affinity of c-Src for imatinib by swapping residues with the corresponding residues in Abl have not been successful, suggesting that the thermodynamic penalty for adoption of the imatinib-binding conformation by c-Src is distributed over a broad region of the structure. Two mutations that are expected to destabilize the inactive Src/CDK conformation increase drug sensitivity 15-fold, suggesting that the free-energy balance between different inactive states is a key to imatinib binding.

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

Figure 1. The Imatinib-Binding Pocket
(A) Chemical structure of imatinib.
(B) The imatinib-binding pocket in Abl (PDB code 1OPJ). The Cα atoms of residues that interact with imatinib are depicted as spheres. Blue, residues that are invariant among Abl, c-Src, Lck, Hck, PDGFR, and c-Kit; yellow, residues that are identical between Abl and c-Src, but not between Abl and c-Kit or PDGFR; red, residues unique to Abl.
(C) Sequence alignment of Abl, c-Src, Hck, Lck, PDGFR, and c-Kit kinase domains. Residues that are the same as in Abl are yellow. Residues that interact with imatinib in the structure of Abl are in orange. Stars are color coded according to the spheres in (B).

Figure 2. The DFG Motif Flip
The surface of imatinib bound to Abl (PDB code 1OPJ [Nagar et al., 2003]) is shown and is superposed upon inactive c-Src in the Src/CDK conformation (PDB code 2SRC [Xu et al., 1999]), active Lck (PDB code 3LCK [Yamaguchi et al., 1996]), the c-Src structure in the imatinib complex (this work, PDB code 2OIQ), and the Abl•imatinib complex (PDB code 1OPJ). For the c-Src•imatinib complex, electron density for imatinib is shown calculated before imatinib was included in the refinement and contoured at 3σ. Protein structures were aligned on the C? atoms of the displayed Abl fragment (Abl residues 380-403, PDB code 1OPJ).

Figure 3. Inhibition of Protein Kinase Activity by Imatinib
(A) Comparison of inhibitory constants (Ki) for wild-type kinase domains. Error bars indicate the uncertainties of curve fits used to calculate the drug concentration at 50% enzyme inhibition. For sample inhibition curves, see Figure S1.
(B) Comparison of imatinib sensitivities of mutant c-Src kinase domain proteins. Mutants were grouped according to the location of the mutation (activation loop, β3-αC loop, phosphate-binding P loop, swap of the 388RAA390 motif in c-Src to the Abl 365AAR 367 motif) or destabilization of the Src/CDK inactive conformation. The bars in blue correspond to mutations that were aimed at converting sequence motifs in c-Src into the corresponding sequences in c-Abl. Bars in green correspond to mutations aimed at destabilizing the Src/CDK inactive state. The value of Ki for the wild-type c-Src kinase domain is indicated by a vertical, red line.

Figure 4. Autophosphorylation of the c-Abl and c-Src Kinase Domains Detected by Staining with an Anti-Phosphotyrosine Antibody
(A) c-Abl kinase domain at 1 μM kinase concentration. The primary antibody was detected by a secondary antibody-alkaline phosphatase stain.
(B) Autophosphorylation of c-Src at 1 μM and 30 nM kinase concentrations.

Figure 5. Isothermal Titration Calorimetry of Imatinib Binding to Src and Abl
(A) Sample titration curve for imatinib binding to the Abl kinase domain.
(B) Sample titration curve of imatinib binding to the c-Src kinase domain. The lack of an upper and lower baseline makes the determination of the binding parameters less reliable in the case of c-Src.
(C) Comparison of KD values obtained by ITC for Abl kinase domain constructs. Leu384 in Abl corresponds to Leu407 in c-Src. Abl was phosphorylated on the activation loop by using Hck and was purified (pAbl). Error bars indicate the uncertainties of the least mean square fit of the binding isotherm used to calculate the dissociation constant, KD.
(D) Comparison of KD values obtained by ITC for the c-Src kinase domain. Error bars indicate the uncertainties of the least mean square fit of the binding isotherm used to calculate the dissociation constant, KD.

Figure 6. Monitoring Imatinib Binding by Fluorescence
(A) Time dependence of normalized fluorescence emission intensity upon mixing of equal volumes of 100 nM Abl kinase domain with 25 μM imatinib at 25°C. The transient was fit to a single equation with a sloping baseline to yield the observed rate constant, ko, of 1.84 s−1. The insert shows the residuals of the curve fit.
(B) Kinetic transient of imatinib binding upon mixing 100 nM c-Src kinase domain with 25 μM imatinib. The fit to a single exponential with a sloping baseline yields a value of ko of 0.125 s−1; the residuals are shown in the insert.
(C) The pseudo first-order rate constants were plotted against the imatinib concentration and were fit to a linear equation (Fersht, 1999). The slope of the fitted line yields the association rate constant, kon, and the intercept yields the dissociation rate constant, koff.
(D) Comparison of association rate constants for c-Src and c-Abl kinase domain constructs. The dissociation constants calculated from the on and off rates together with the errors are shown in Table S1. Error bars indicate the uncertainties of the least mean square fit to the experimental data in the ko versus imatinib plot.

Figure 7. Comparison of Imatinib-Bound Structures
(A) Structure of c-Src•imatinib (this work, PDB code 2OIQ). The activation loop is disordered from residue 408 to residue 420, and the phosphate-binding P loop is extended, leaving the phenylalanine side chain solvent exposed.
(B) Structure of Abl•imatinib (PDB code 1OPJ [Nagar et al., 2002]). The phosphate-binding loop is kinked toward the C lobe of the kinase, allowing for hydrogen bonds between Tyr253 and Asn322 (not shown).
(C) Structure of c-Kit•imatinib (PDB code 1T46 [Mol et al., 2004]). The phosphate-binding loop is extended, as seen in the structure of c-Src, and Phe600 is solvent exposed.

Figure 8. Distance Difference Matrices
(A) Left: structure of the kinase domain of c-Src in the inactive Src/CDK conformation (PDB code 2SRC). Regions that undergo structural changes upon binding to imatinib are colored. Right: distance difference matrix between c-Src in the inactive Src/CDK conformation and the c-Src•imatinib complex (Abl/c-Kit conformation). Each entry in this symmetrical matrix represents the difference in the distance between the corresponding Cα atoms in the two structures. Cα atoms that come closer together upon going from the inactive Abl/c-Kit conformation to the inactive Src/CDK conformation are indicated in yellow/red. Distances that do not change much are green, and distances that increase are blue. he tips of four β strands in the N lobe of the kinase domain and the adjacent loops are colored in red (P loop: residues 274–281), orange (β3αC loop: residues 326–337), and yellow (β4-β5 loop: residues 326–337). These strands close down on the C lobe of the kinase domain.
(B) Left: structure of Abl•imatinib; loops that change their structure relative to Src•imatinib are colored. Distance difference matrix between c-Src•imatinb and Abl•imatinib (PDB code 1OPJ).

Figure 9. Conformational Equilibria in c-Src and Abl
The hallmark of the Abl/c-Kit inactive state is the inward conformation of the C helix, the salt bridge between a C helix glutamate and an N lobe lysine, and the outward orientation of the aspartate of the DFG motif. The characteristics of the Src/CDK state are the outward rotation of the C helix, which disrupts the salt bridge with the N lobe lysine, and the inward orientation of the DFG aspartate. Imatinib binding favors the inactive Abl/c-Kit state (A), and activation-loop phosphorylation favors the active phosphorylated state (C).

Table 1. Data Collection and Refinement Statistics

Supplemental Data:
Three figures and one table

PDB Coordinates:
Download PDB file: 2OIQ
Coordinates in the Protein Data Bank: 2OIQ