Unfolding in the SH3 Domain of the Abelson Tyrosine Kinase is Inhibited by Intramolecular Binding

The core region of the Abelson tyrosine kinase is structurally similar to Src‐family kinases. In both cases, kinase activity is regulated by intramolecular interactions involving the SH3 and SH2 domains. SH3 interactions may play a more important role in Abl than in Src‐family kinases because Abl lacks a C‐terminal tail for SH2 domain engagement. The dynamics of the Abl SH3 domain and its ability to associate with the SH2‐kinase linker were investigated with hydrogen exchange mass spectrometry. Constructs of Abl SH3, Abl SH3+SH2 (Abl32) and SH3+SH2+linker (Abl32L) were overexpressed in E. coli and purified to homogeneity. Proteins were labeled with D 2 O for various amounts of time and the location and incorporation of deuterium measured with electrospray mass spectrometry. Constructs were analyzed alone or when bound to a strong SH3 ligand (BP1) or the natural SH2‐kinase linker. Isolated Abl SH3 was dynamic and partially unfolded with a half‐life of ~5 minutes. Binding to the BP1 peptide completely abolished unfolding in both Abl SH3 and Abl SH32. SH3 unfolding was partially inhibited in the SH32L construct implying that the natural linker was able to associate with the SH3 domain. This result is in stark contrast to similar studies on the Src‐family kinase Hck in which the natural linker has no affinity for the SH3 domain in the absence of the kinase domain. These results support the hypothesis that the SH2‐kinase linker has a dominant role in the regulation of c‐Abl structure and kinase activity. Funded by NIH GM70590, CA81398, AI57083 and RR16480.