UC Berkeley

The amino acid sequence of a protein dictates its function. This function is informed not only by the folded shape(s) adopted by the protein but also the dynamic equilibria between these different shapes, or conformations. Evolution modulates the conformational dynamics of proteins in order to optimize existing functions or select for new functions. Here I characterize the conformational dynamics of the regulatory tandem SH2 domain in the Syk family of kinases. I find that in the T-cell specific Syk family kinase, ZAP-70, this domain is predominantly in an ‘inactive’ conformation while its B-cell paralog, Syk, is found in the ‘active’ conformation, even in the absence of the activating ITAM peptide. The long- and short-time scale dynamics of the C-terminal SH2 domains are found to be particularly different between the two proteins. Residues which form stabilizing interactions in the C-SH2 are found in Syk but not in ZAP-70. Significantly, these residues are highly conserved in the Syk lineage but variable in the ZAP-70 linage. These data suggest that the differences in the conformational dynamics of the regulatory tandem SH2 domain are a result of evolutionary selection in both Syk and ZAP-70. In order to explore how the conformational dynamics of the catalytic domains in Syk family kinases may also have diverged I developed a high-throughput assay to probe kinase activity in bacteria. This assay, based on a bacterial two-hybrid, allowed for the screening of the relative activities of a saturation mutagenesis library of a bacterially expressed Syk family kinase. The loss of function mutations identified in this screen correlate well to residues identified in other kinases as critical to function and/or structure. Activating mutations in the regulatory hydrophobic spine, catalytic hydrophobic spine, and activation loop suggest that single point mutants can shift the conformational equilibria of the kinase. These positions may be critical to the tuning of the catalytic activity of Syk and ZAP-70. The data presented in this thesis describe how the conformational dynamics of the paralogous Syk family of kinases have diverged and specialized in order to fulfill their related, yet distinct roles in the adaptive immune system.