Conformational analysis of C -terminal Src kinase: Regulation of activity probed by hydrogen-deuterium exchange and mass spectrometry.

摘要

C-terminal Src kinase, Csk, is a key player in the regulation of cellular function due to its premier role as the negative regulator of Src family kinases. To understand more fully the mechanism of regulation for Csk, we employed enhanced methods of hydrogen-deuterium exchange-mass spectrometry (DXMS) to probe conformational changes on Csk in the presence of ligands. Previous kinetic studies demonstrated that nucleotide associated conformational changes regulate function in Csk. To provide an understanding for these phosphorylation-driven solution conformational changes, DXMS studies were performed on wildtype and mutant forms of Csk in the presence and absence of nucleotides and effector molecules. The results from these experiments suggested that delivery of the gamma phosphate group of ATP induces unique local and long range conformational changes in Csk that may influence regulatory motions in the catalytic pathway. These studies also suggested that a short peptide sequence, the SH2-kinase linker, provides a functional connection between the active site and distal SH2 domain. To understand the role of this linker, substitutions were made on a critical hydrophobic residue, Phe183, to tyrosine, alanine, and glycine and mutations were analyzed both biochemically and biophysically. The overall data imply that domain-domain interactions, controlled through the SH2-kinase linker, provide a dynamic balance within the Csk framework that is ideal for efficient phosphoryl transfer in the active site. Previous studies illustrated that a phosphopeptide from Csk binding protein, Cbp, enhances Csk activity. To determine if the communication between the active site and SH2 domain is bi-directional, Cbp phosphopeptide interactions with the SH2 domain of Csk were analyzed by DXMS. These studies showed that the phosphopeptide binding elicits both local and long-range conformational changes, as seen with the nucleotides. The results imply that communication between the two domains is bi-directional and occurs through a discrete pathway.