UC San Diego
Functional Flexibility in the Regulation of Src Family Kinases /
- Author(s): Jamros, Michael Alan
- et al.
Structural studies of proteins have relied on the acquisition of high-resolution structure derived from X- ray crystallography and NMR and the use of these structures to guide hypothesis-driven research. While effective for compact, well-folded proteins, these techniques are not amenable to proteins displaying significant molecular flexibility. Macromolecular flexibility and intrinsic disorder are a vital aspect of the regulatory mechanisms of biological systems. Understanding the conformational dynamics within these systems is of great interest as novel modes of function may provide highly specific targets for therapeutics. Developments in solution structural methods and computational techniques have made characterization of these proteins and the various conformations that populate their functional landscape possible. The Src family of kinases (SFK) are a family of tyrosine kinases involved in numerous cellular processes including cell growth, differentiation, and proliferation. Improper regulation of SFKs leads to an excess of SFK activity, which is associated with the development of various cancers. Regulation of SFK activity occurs through the coordinated activities of another tyrosine kinase, Csk, and a scaffolding protein, Cbp. In the work presented here, we identify flexibility in these two regulatory proteins that is essential to their function. Using a combination of small-angle X-ray scattering (SAXS) and structure-based models, we identify two native basins containing two distinct conformational states of Csk, neither of which resemble the compact conformation of the X-ray crystal structure. A further analysis finds that Csk exists in solution an ensemble where both native basins are represented. Upon binding of nucleotide, we observe a shift in the ensemble towards more extended structures as well as the accession of a higher energy state, where the protein adopts an extremely extended state. Where Csk undergoes transitions between conformational states while intradomain contacts remain largely intact, Cbp displays an even greater degree of flexibility. Using a combination of structural prediction methods, analytical size- exclusion chromatography, hydrogen-deuterium exchange mass spectrometry, and circular dichroism we identify Cbp as an intrinsically disordered protein with limited secondary structure. This disorder is key to Cbp function, exposing a multitude of binding sites, not only for Csk and SFKs, but also for numerous other signaling proteins. These results display the inherent flexibility of Csk and Cbp and how it relates to their function