Characterization of the Conformational Ensemble in the C-Terminal Src Tyrosine Kinases.

The Src family of tyrosine kinases [SFKs] are important enzymes involved with cell motility and differentiation and are associated with various cancers when their control mechanisms are disrupted. Thus, understanding how Src is regulated is critically important for the study of proliferative human diseases. Csk (C-terminal Src kinase) down-regulates all SFKs by phosphorylating a single tyrosine in the C-terminus of its substrate. This reaction leads to repression of the catalytic activity of SFKs. Csk contains a catalytic kinase domain and two regulatory domains, the SH2 and SH3. Deletion mutants of these domains showed they are necessary to maintain Csk activity, but these regulatory domains do not play a role in substrate recognition and binding. X-ray diffraction studies have shown two conformations of Csk. In one conformation the SH2 domain and N-terminal lobe of the kinase domain are compact and the active site residues are aligned in a catalytically active conformation. In the other, the SH2 domain and N-terminal lobe are in a more open conformation where active site residues are misaligned. We developed small angle X-ray scattering [SAXS] methods to show that the multidomain tyrosine kinase Csk adopts an extended conformation in solution compared to the compact crystal structures. In all cases, theoretical scattering curves generated from mixed populations of Csk structures fit the experimental SAXS data better than any rigid model (Figure 6). These methods will be invaluable in investigating the multimeric complexes in general and will help us further our understanding of conformationally regulated signaling.