Investigating the relationship between allostery and substrate specificity in protein tyrosine phosphatases

蛋白酪氨酸磷酸酶(Ptp)是调节大量生物过程的酶。 通过调节它们的目标蛋白(底物)。PTP通过催化去除一种 从其靶标中的氨基酸酪氨酸中分离出磷酸基团，从而改变靶标酶的活性。 PTPs的失调与许多疾病有关，包括II型糖尿病、肥胖症、癌症和 炎症和PTP因此被视为潜在的药物靶点。给PTP下药的一个主要障碍是 它们的酶作用部位(活性部位)几乎相同，从而针对特定的PTP酶 通过一种专注于活性部位的药物一直没有取得成效。解决这个问题的一个办法是瞄准 该药物结合到PTP上远离活性部位的部位，即所谓的变构部位，这些部位不是 在PTP家族中都是相同的。这种方法的一个困难是识别和识别 表征这些变构位置，以更好地了解它们是如何相互作用的 分子距离，与活性中心。我们假设不同的底物改变了蛋白质的结构。 一种不同的方式，并导致不同的变构部位暴露。我们计划测试这一假设，并 通过溶液核磁共振的强大组合来表征这些变构位置 两种医学上重要的人类酶的(核磁共振)光谱、生化和计算方法， 蛋白酪氨酸磷酸酶1B(PTP1B)和痘苗H1相关(VHR)磷酸酶。我们的目标是： 用不同底物鉴定和鉴定PTP1B和VHR中底物依赖的变构位点 来自这些PTP的天然体内靶标的多肽。我们将通过监视以下方面的变化来确定这些站点 核磁共振化学位移和动力学参数。在补充性实验中，我们将使用新的计算 由我们的研究团队开发的方法，以进一步了解这些酶中的变构机制。 随后，我们将通过突变和功能分析来验证我们新确定的变构位点 更好地了解变构位置对底物特异性影响的计算方法 PTP1B和VHR中的酶活性。

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.