Protein tyrosine phosphatases (PTPs) are enzymes that regulate an enormous number of biological processes through the modulation of their target proteins (substrates). PTPs do this by catalyzing the removal of a phosphate group from the amino acid tyrosine in their targets, which alters the activity of the target enzyme. The misregulation of PTPs are linked to a number of diseases including type II diabetes, obesity, cancer, and inflammation and therefore PTPs are viewed as potential drug targets. A primary obstacle to drugging PTPs is that the sites of their enzymatic action (active sites) are nearly identical, thus targeting a specific PTP enzyme by a drug focused on the active site has not been a productive endeavor. A solution to the problem is to target the drug to sites on PTPs that are distant from the active site, the so-called allosteric sites, which are not identical across the family of PTPs. A difficulty with this approach is the challenge of identification and characterization of these allosteric sites to obtain a better understanding of how they interact, from long molecular distances, with the active site. We hypothesize that different substrates alter the protein structure in a distinct manner and cause different allosteric sites to be exposed. We plan to test this hypothesis and characterize these allosteric sites through a powerful combination of solution nuclear magnetic resonance (NMR) spectroscopy, biochemical, and computational approaches in two medically important human enzymes, protein tyrosine phosphatase 1B (PTP1B) and Vaccinia H1-related (VHR) phosphatase.
Our aims are: To identify and characterize substrate dependent allosteric sites in PTP1B and VHR using different substrate peptides from the natural in vivo targets of these PTPs. We will identify these sites by monitoring changes in NMR chemical shift and dynamics parameters. In complementary experiments we will use novel computational methods developed by our research team to further understand the mechanism of allostery in these enzymes. Subsequently we will validate our newly identified allosteric sites by mutation followed by functional assays and computational methods to better understand the impact of the allosteric sites on the substrate specific enzymatic activity in PTP1B and VHR.
The control of protein function by modulation of its structure and dynamics is of significant therapeutic benefit to human health. One of the hurdles to such control is a more complete understanding of how different regions in a protein architecture interact with one another. This proposal will fill this void through a collaborative effort combining biophysical experiments and computational methods.
| Cui, Danica S; Beaumont, Victor; Ginther, Patrick S et al. (2017) Leveraging Reciprocity to Identify and Characterize Unknown Allosteric Sites in Protein Tyrosine Phosphatases. J Mol Biol 429:2360-2372 |
| Lipchock, James M; Hendrickson, Heidi P; Douglas, Bonnie B et al. (2017) Characterization of Protein Tyrosine Phosphatase 1B Inhibition by Chlorogenic Acid and Cichoric Acid. Biochemistry 56:96-106 |
| Lisi, George P; Loria, J Patrick (2016) Using NMR spectroscopy to elucidate the role of molecular motions in enzyme function. Prog Nucl Magn Reson Spectrosc 92-93:1-17 |
