Molecular dynamics simulations show that conformational selection governs the binding preferences of imatinib for several tyrosine kinases

Abstract

Tyrosine kinases transmit cellular signals through a complex mechanism, involving their phosphorylation and switching between inactive and active conformations. The cancer drug imatinib binds tightly to several homologous kinases, including Abl, but weakly to others, including Src. Imatinib specifically targets the inactive, so-called "DFG-out" conformation of Abl, which differs from the preferred, "DFG-in" conformation of Src in the orientation of a conserved Asp-Phe-Gly (DFG) activation loop. However, recent x-ray structures showed that Src can also adopt the DFG-out conformation and uses it to bind imatinib. The Src/Abl-binding free energy difference can thus be decomposed into two contributions. Contribution i measures the different protein-imatinib interactions when either kinase is in its DFG-out conformation. Contribution ii depends on the ability of imatinib to select or induce this conformation, i.e. on the relative stabilities of the DFG-out and DFG-in conformations of each kinase. Neither contribution has been measured experimentally. We use molecular dynamics simulations to show that contribution i is very small, 0.2 +/- 0.6 kcal/mol; imatinib interactions are very similar in the two kinases, including long range electrostatic interactions with the imatinib positive charge. Contribution ii, deduced using the experimental binding free energy difference, is much larger, 4.4 +/- 0.9 kcal/mol. Thus, conformational selection, easy in Abl, difficult in Src, underpins imatinib specificity. Contribution ii has a simple interpretation; it closely approximates the stability difference between the DFG-out and DFG-in conformations of apo-Src. Additional calculations show that conformational selection also governs the relative binding of imatinib to the kinases c-Kit and Lck. These results should help clarify the current framework for engineering kinase signaling.

FIGURE 1.

A, Abl, Src, Lck, and c-Kit sequences; residues less than 5 Å from imatinib and part of the A-loop are included. B, three-dimensional structures, DFG-out inactive conformation; Abl is red; Src is cyan; Lck is green; and c-Kit is orange; residue selection as in A. C, Src three-dimensional structure, comparing the DFG-in (blue and violet) and DFG-out (red and pink) conformations. The A loop, selected backbone portions, and side chains are shown. The viewpoint is perpendicular to B (B viewed from above).

FIGURE 2.

Thermodynamic cycle to study imatinib (Ima) binding to the DFG-out (Abl-like) and DFG-in (Src-like) conformations (labeled In and Out) of a given kinase. Horizontal arrows correspond to conformational changes; vertical arrows correspond to ligand binding. States and transitions are labeled with the corresponding free energies.