Hydrogen Exchange Mass Spectrometry Demonstrates a Role for the Kinase Domain in Src‐family Kinase Regulation

The highly conserved SH3 and SH2 domains present in all Src‐family kinases participate in regulation of the kinase domain via intramolecular interactions. To elucidate the role of the kinase domain in SH3/SH2 interactions, hydrogen exchange was used to compare dynamics and binding in full‐length Hck and various constructs of SH3 and SH2. Hck constructs were overexpressed in E. coli or Sf9 cells and purified with ion‐exchange and gel filtration chromatography. Proteins were labeled with D2O for various amounts of time and the location and amount of deuterium incorporation measured using electrospray mass spectrometry. Online pepsin digestion was used to digest the labeled proteins into peptides and increase the spatial resolution. In a construct containing SH3, SH2, and the SH2‐kinase linker (SH32L), the linker did not associate with SH3. However, when the natural linker was mutated to a high‐affinity form (SH32HAL), the linker readily associated with the SH3 domain. Full length Hck, minus the N‐terminal ~80 residues, had similar SH3 unfolding behavior as SH32HAL implying that the presence of the kinase domain resulted in SH3 binding tightly to the SH2 linker. These experiments suggest that the kinase domain plays a role in the structuring of the SH2‐kinase linker. In the absence of the kinase domain, the native linker is likely unstructured and has little or no affinity for the SH3 domain. Funded by NIH GM70590, CA81398, AI57083 and RR16480.