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

项目总结 葡萄糖醛酸化通常是不利的药代动力学或药效学的来源，从而导致 临床试验期间药物失败，因此，在药物开发中迫切需要一种工具来 控制葡萄糖醛酸化反应。我们的长期目标是开发控制葡萄糖醛酸化的策略，通过限制其 底物利用率。要做到这一点，我们将确定控制人类UDP-葡萄糖的变构机制 脱氢酶(HUGDH)，产生葡萄糖醛酸化的基本底物的酶。在我们的 在之前的授予中，我们发现了30个残基本质上无序的C末端(ID-ail)是如何修改 酶的结构有利于与反馈抑制剂UDP-XYL的结合，UDP-XYL是下游代谢产物。我们也 发现了一个隐秘的变构位点来抑制这种酶。简而言之，反馈抑制剂UDP-木糖 与底物竞争活性部位；结合后，UDP-Xyl诱导酶缓慢异构化为 一种叫做EW的不活跃的复合体。变构转变将活性中心转化为两个新的变构中心 称为SBSW和NBSW。SBSW位点专用于UDP-XYL抑制剂，但NBSW位点可以结合以下任一项 UDP-XYL或底物UDP-GLC。我们假设NBSw和SBSw变构中心协同作用 稳定的EW，NBSW的双重特异性是允许丰富底物的重要特征 UDP-GLC以增强SBSW中含量较少的抑制剂UDP-XYL的结合亲和力。这一假设是 基于我们的初步数据，(I)底物UDP-GLC可以与NBSW位点结合并抑制hUGDH，以及 (2)抑制剂UDP-XYL可与SBSW和NBSW结合抑制。这一假设将由 以下具体目标如下：1)我们将确定NBSW和SBSW变构中心如何相互作用，以增强 UDP-Xyl的变构抑制；2)我们将确定假定的低能垒氢与 键(LBHB)和NBSW和SBSW变构中心的稳定性；以及3)我们将确定结构 将hUGDH的固有无序的C-末端(ID-尾)耦合到有利的EW形成的特征。 本申请中提出的研究具有创新性，因为它集中在hUGDH的变构抑制上 作为一种控制葡萄糖醛酸化的全球机制，并利用我们最新的发现：(I)新的NBSW 变构位置；(Ii)变构机制中的一种活泼的低势垒氢键；以及(Iii)熵力 由本质上无序的C-末端产生。由于这些特征是我实验室的最新发现， 这项研究不同于以前试图控制葡萄糖醛酸化的尝试。的预期结果 这项工作意义重大。对hUGDH的变构机制的详细描述将作为 为设计一类变构抑制剂奠定了基础，该抑制剂将作为全球葡萄糖醛酸化反应的调节剂。 更广泛地说，由于长的(&gt；30残基)固有的无序片段在 人类蛋白质组，(占所有人类蛋白质的44%)，学习无序的 HUGDH末端修饰蛋白质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.