Conformational Selection and Induced Fit Mechanisms in the Binding of an Anticancer Drug to the c-Src Kinase

Abstract

Understanding the conformational changes associated with the binding of small ligands to their biological targets is a fascinating and meaningful question in chemistry, biology and drug discovery. One of the most studied and important is the so-called "DFG-flip" of tyrosine kinases. The conserved three amino-acid DFG motif undergoes an "in to out" movement resulting in a particular inactive conformation to which "type II" kinase inhibitors, such as the anti-cancer drug Imatinib, bind. Despite many studies, the details of this prototypical conformational change are still debated. Here we combine various NMR experiments and surface plasmon resonance with enhanced sampling molecular dynamics simulations to shed light into the conformational dynamics associated with the binding of Imatinib to the proto-oncogene c-Src. We find that both conformational selection and induced fit play a role in the binding mechanism, reconciling opposing views held in the literature. Moreover, an external binding pose and local unfolding (cracking) of the aG helix are observed.

Figure 1. Structure of the kinase domain of c-Src with a detailed view of the DFG motif in the DFG-in apo conformation (DFG represented as green sticks; PDB ID: 2SRC).

The inset shows the DFG-out Imatinib-bound form (the DFG and Imatinib are shown as green and blue sticks, respectively; PDB ID: 2OIQ).

Figure 2. c-Src Dynamics, (left) difference in the Cα RMSF (Δ_rmsf) between the apo and ligand-bound forms of c-Src averaged over 1 μs-long MD simulations.

Low (blue) values correspond to regions that are more rigid (lower RMSF) in the bound form, high (red) values correspond to regions that are more flexible in the bound form. (right) Experimentally determined order parameters S² for the apo (blue) and bound (red) forms.

Figure 3. Difference of combined ¹H, ¹⁵N chemical shift Δδ for Src upon binding of Imatinib (left) and Dasatinib (right).

Ligands are shown as blue sticks. Residues for which data is not available are shown in dark grey.

Figure 4. Imatinib binding free energy profile along the S_path variable, which defines an optimal association coordinate.

The most relevant minima and the transition state are labelled and discussed in the text. The area of the transition state is represented as a dashed line due to a larger uncertainty. The two profiles in the region to (secondary pocket to the unbound state) correspond to the conformational selection mechanism (red line, “flip-bind”) and to an induced fit mechanism (yellow line, “bind-flip”). The 2D FES in the inset shows the dependence of the free energy from the DFG-flip coordinate.

Figure 5. Free energies for local unfolding.

The values are derived from the k_intr and k_exch parameters obtained from H/D exchange NMR measurements. Red circles indicate αD and αG, the regions with increased exposure to the solvent in the bound structure.