The role of intrinsic disorder in the allosteric regulation of human UGDH

项目摘要 葡萄糖醛酸化通常是不利的药代动力学或药效学的来源， 药物在临床试验中失败，因此，药物开发迫切需要一种工具， 对照葡萄糖醛酸化。我们的长期目标是通过限制葡萄糖醛酸化， 底物可用性为此，我们将确定控制人UDP-葡萄糖的变构机制 葡萄糖醛酸脱氢酶（hUGDH）是产生葡萄糖醛酸化的必需底物的酶。在我们 在先前的研究中，我们发现了30个残基固有无序的C-末端（ID-尾）如何修饰 酶的结构，以有利于反馈抑制剂UDP-Xyl，下游代谢物的结合。我们也 发现了一个隐藏的变构位点来抑制这种酶。简言之，反馈抑制剂UDP-木糖 与底物竞争活性位点;结合后，UDP-Xyl诱导酶缓慢异构化为 一种不活跃的复合物叫EW变构转变将活性位点转化为两个新的变构位点 叫做SBSW和NBSW SBSW位点对UDP-Xyl抑制剂是特异性的，但NBSW位点可以结合 UDP-Xyl或底物UDP-Glc。我们推测NBSW和SBSW的变构位点协同作用 稳定EW，NBSW的双重特异性是一个重要的特征， UDP-Glc以增强SBSW中丰度较低的抑制剂UDP-Xyl的结合亲和力。这种假设是 基于我们的初步数据，（i）底物UDP-Glc可以结合NBSW位点并抑制hUGDH，和 (ii)抑制剂UDP-Xyl可以结合SBSW和NBSW两者来抑制。这一假设将由 以下具体目标：1）我们将确定NBSW和SBSW变构位点如何相互作用以增强 UDP-Xyl的变构抑制; 2）我们将确定推定的低势垒氢之间的关系， 键（LBHB）和NBSW和SBSW变构位点的稳定性; 3）我们将确定结构 将hUGDH的固有无序C末端（ID-尾）与EW的有利形成偶联的特征。 本申请中提出的研究具有创新性，因为它关注hUGDH的变构抑制 作为控制葡萄糖醛酸化的全球机制，并使用我们最近的发现：（i）新的NBSW 变构位点;（ii）在变构机制中假定的低势垒氢键;和（iii）熵力 由本质上无序的C-末端产生。因为这些特征是我实验室最近发现的， 该研究与以前试图控制葡萄糖醛酸化的尝试不同。的预期成果 这项工作意义重大。hUGDH的变构机制的详细描述将作为本发明的一个方面。 为设计一类变构抑制剂奠定了基础，这些变构抑制剂将作为葡萄糖醛酸化的全局调节剂。 更广泛地说，由于长（>30个残基）的固有无序片段在 人类蛋白质组（>44%的所有人类蛋白质），学习如何熵力产生的无序 hUGDH末端修饰蛋白功能将产生广泛的影响。

PROJECT SUMMARY Glucuronidation is often the source of unfavorable pharmacokinetics or pharmacodynamics that lead to the failure of drugs during clinical trials, and as such, there is a critical need in drug development for a tool to control glucuronidation. Our long-term goal is to develop strategies to control glucuronidation by limiting its substrate availability. To do this, we will determine the allosteric mechanism that controls human UDP-glucose dehydrogenase (hUGDH), the enzyme that produces the essential substrate for glucuronidation. In our previous grant, we discovered how the 30-residue intrinsically disordered C-terminus (the ID-tail) modifies the structure of the enzyme to favor binding of the feedback inhibitor UDP-Xyl, a downstream metabolite. We also discovered a cryptic allosteric site for inhibiting the enzyme. Briefly, the feedback inhibitor UDP-Xylose competes with substrate for the active site; upon binding, UDP-Xyl induces the enzyme to slowly isomerize into an inactive complex called EW. The allosteric transition converts the active site into two novel allosteric sites called SBSW and NBSW. The SBSW site is specific for the UDP-Xyl inhibitor, but the NBSW site can bind either UDP-Xyl or the substrate UDP-Glc. We hypothesize that the NBSW and SBSW allosteric sites cooperatively stabilize EW, and the dual-specificity of the NBSW is an important feature that allows the abundant substrate UDP-Glc to enhance the binding affinity of the less abundant inhibitor UDP-Xyl in the SBSW. This hypothesis is based on our preliminary data that (i) the substrate UDP-Glc can bind to the NBSW site and inhibit hUGDH, and (ii) the inhibitor UDP-Xyl can bind to both the SBSW and NBSW to inhibit. This hypothesis will be tested by the following specific aims: 1) we will determine how the NBSW and SBSW allosteric sites interact to enhance the allosteric inhibition by UDP-Xyl; 2) we will determine the relationship between a putative low barrier hydrogen bond (LBHB) and the stability of the NBSW and SBSW allosteric sites; and 3) we will identify the structural features that couple the intrinsically disordered C-terminus (ID-tail) of hUGDH to the favorable formation of EW. The research proposed in this application is innovative because it focuses on the allosteric inhibition of hUGDH as a global mechanism for controlling glucuronidation, and uses our recent discoveries of: (i) the novel NBSW allosteric site; (ii) a putitive low barrier hydrogen bond in the allosteric mechanism; and (iii) the entropic force generated by the intrinsically disordered C-terminus. Since these features are recent discoveries from my lab, this research is distinct from previous attempts that tried to control glucuronidation. The expected outcomes of this work are significant. A detailed description of the allosteric mechanism of hUGDH will serve as a foundation for the design of a class of allosteric inhibitors that will act as global regulators of glucuronidation. And more broadly, because of the persistence of long (>30 residues) intrinsically disordered segments in the human proteome, (>44% of all human proteins), learning how the entropic force generated by the disordered terminus of hUGDH modifies the protein function will have a broad impact.