Research Project
10369808
Abstract The overall objective of the project is to contribute fundamental understanding of the exquisitely selective recognition of protein tyrosine kinases for their protein substrates. Selectivity is generated from multiple factors. One factor is spatial localization of the kinase to the proper regions in the cell through docking interactions often mediated by domains independent of the catalytic domain. A second factor is amino-acid sequence recognition of the substrate polypeptide chain near the cleavage site. Protein kinases play a major role in disease and are second to only G-protein coupled receptors for the most targeted protein group for drug development. Among all protein kinases, tyrosine kinases occur most frequently on a list of cancer-driver genes, and are the most clinically targeted to date. The immediate objectives of this project are to 1) achieve a physical rationale for a new entropy-driven allosteric phosphorylation mechanism that regulates tandem SH2 domains of Syk that dock Syk to membrane immunoreceptors; 2) determine if the tandem SH2 domains also serve to localize Syk to cytoplasmic regions in conjunction with newly discovered functionalities of Syk; 3) define the structure of the substrate polypeptide chain existing the C-terminal side of the catalytic site of Src kinase and several other tyrosine kinases in order to evaluate a new perspective on kinase substrate recognition determinants; and 4) identify new tyrosine kinase ligands that are substrate like and interact in the shallow groove where the substrate polypeptide chain binds. To accomplish these objectives, a variety of solution NMR studies will be conducted to characterize molecular interactions, define three-dimensional structure and estimate thermodynamic parameters of tyrosine kinase-peptide substrate and tyrosine kinase- protein substrate complexes. The NMR and biophysical analyses will be tightly integrated with computer simulation methods selected to effectively explore large regions of protein conformational space. To broaden impact, the knowledge obtained from using purified samples and computer simulations will be complemented and validated with cell-based experiments to inform on cellular functionality. Finally, chemical screening using a phosphorylation-based selection with DNA-encoded libraries of high complexity, will identify new substrate-like inhibitor molecules of Src. The expected outcomes will advance scientific knowledge on the physical mechanisms determining selective substrate phosphorylation by these key enzymes in human health. Successful execution of the research plan would also identify small-molecule ligands with potent and selective cellular activity, and impact future efforts to develop molecules useful for studying disease in animal models or for cancer drug development.
