Determinants of Abl kinase stability and drug binding thermodynamics: Role of regulatory domains and phosphorylation.

Activity dysregulation of Abl kinase is the primary cause for chronic myeloid leukemia (CML). Despite extensive characterization, the thermodynamic basis of Abl stability and inhibitor recognition remains incompletely understood. To characterize the cooperative folding organization of Abl and the communication between its structural units, we comprehensively characterized three Abl constructs: the isolated catalytic domain (AblCD), the three-domain regulatory construct (AblSH3SH2CD), and its phosphorylated form (PO4AblSH3SH2CD), using high-resolution calorimetric techniques. DSC revealed that the catalytic domain behaves as a single cooperative unfolding unit, with the SH3-SH2 module in AblSH3SH2CD exerting a modest stabilizing effect. The catalytic domain decoupled into two transitions in PO4AblSH3SH2CD, an observation rationalized by the disruption of the interlobe structural bridge formed by the A-loop in the unphosphorylated forms. Imatinib and dasatinib had opposite effects on this decoupling, providing calorimetric evidence for the distinct intradomain reorganizations induced by each inhibitor type. ITC showed that both inhibitors bind with distinct thermodynamic signatures, with a modest effect of the regulatory domains on inhibitor recognition. Imatinib binding involved net proton uptake, consistent with Asp381 protonation required for the DFG-out transition, while dasatinib showed no proton linkage. Phosphorylation-induced reduction in imatinib affinity was driven by a large loss of favorable enthalpy reflecting the energetic cost of displacing the ordered A-loop, while dasatinib affinity remained largely unaffected. These results provide a thermodynamic rationale for the superior potency of dasatinib against activated Abl in CML cells, revealing mechanistic features of kinase regulation not accessible from structural data alone.