Mapping and Exploiting the Internal Wiring of Structurally Dynamic Phosphatases

PROJECT SUMMARY/ABSTRACT Proteins recognize signals from their cellular environments and respond by altering their function in a phenomenon known as allostery. However, the atomic-level mechanisms underlying allostery, including the key role of shifts in protein conformational ensembles, remain poorly understood. In particular, we lack an understanding of how allostery differs between related proteins, such as the biomedically important Protein Tyrosine Phosphatases (PTPs), which all share a common ground-state 3D structure yet exhibit many distinct cellular roles and human disease associations. Research in my lab focuses on developing experimental and computational methods to unveil allosteric mechanisms from protein conformational ensembles, and applying these methods to decipher and exploit differences in allosteric wiring between PTP family members. We have pushed the frontiers of experimentally modulating protein conformational landscapes using multidataset X-ray crystallography and developed algorithms to model coupled protein conformational heterogeneity. Using these tools for two distinct PTPs, we have revealed coupled conformations that link distal sites to the dynamic active site and new small-molecule ligands at promising allosteric sites. With renewed funding for this award, we will work to understand how allosteric wiring differs across the larger family of PTPs, and how such differences can be exploited to modulate the functions of specific individual PTP drug targets. We will develop and apply an expanded repertoire of avant-garde X-ray crystallography experiments and computational tools for modeling and biasing protein ensembles, and use them to elucidate allosteric circuitry that is unique to specific PTP enzyme architectures. To target these allosteric weak points in selected PTPs of high biomedical interest, we will leverage methodological improvements in high-throughput crystallographic ligand screening and complementary solution biophysics methods like high-resolution HDX-MS to identify and characterize novel small-molecule allosteric modulators. Overall, the proposed research will open new doors for the challenging task of achieving specific functional modulation of individual PTP drug targets, and will have broader implications for our understanding of the mechanisms by which evolution has sculpted a menagerie of structurally dynamic, functionally distinct enzymes given the constraints of modular structural building blocks.