Imatinib Binding to Human c-Src is Coupled to Inter-Domain Allostery and Suggest a Novel Kinase Inhibition Strategy

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One of non-receptor tyrosine kinases (nRTKs), c-Src, is a multi-modular protein kinase. Because c-Src is involved in numerous oncogenic signaling cascades, it is an important drug target to treat myeloid leukemia and gastrointestinal stromal tumors. Imatinib is a prototype nRTK inhibitor and binds to c-Src as well as another nRTK, c-Abl, with different affinities despite high sequence and structural conservations in their ATP/drug binding pockets. Structural dynamics of c-Src has been extensively investigated to elucidate underlying causes of the affinity difference between c-Src and c-Abl. One of such studies suggests the presence of a conformer that plays an important role in selective imatinib binding. However, because such a conformer accumulates slower than the timescale of conventional biophysical and computational approaches, its structural identity cannot be studied using those techniques. We used hydrogen-deuterium exchange and mass spectrometry (HDX-MS) to study structural dynamics of such a key c-Src conformer in the presence of imatinib. While most previous biochemical/biophysical studies used isolated c-Src kinase domain, we used purified human c-Src spanning residue 83-536 containing SH3, SH2, and kinase domains. This allowed us to visualize changes not only in the structural dynamics of the drug binding pocket in kinase domain but also of functional regulatory sites in distant SH3 and SH2 domain upon imatinib binding, suggesting an allosteric communication among those sites. Our results also indicate similarities in structural changes between imatinib binding and c-Src activation steps. Such changes include loosening of the interface between N- and C-lobes of kinase domain, loss of kinase domain anchoring to SH2-kinase connecting loop, and disassembly of the hydrophobic spine. Based on our findings, we propose a new cancer therapeutic strategy to improve binding of currently available nRTK inhibitors.