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.