The global regulation of dynamics and structure mediated by single hydride in a family of reductases

还原酶家族中单个氢化物介导的动力学和结构的全局调节

• 批准号: 10296136
• 负责人: ELAN Z EISENMESSER
• 金额: $ 30.28万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-15 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10296136
• 关键词: Active Sites; Address; Affinity; Amides; Bilirubin; Biliverdin reductase; Biliverdine; Binding; Biochemical; Biological; Cells; Chemicals; Coenzyme A; Coenzymes; Communication; Coupled; Coupling; Culicidae; Data; Distal; Enzymes; Enzymes and Coenzymes; Family; Family member; Flavins; Global Change; Hematopoietic; Human; Link; Maps; Mediating; Methods; Molecular Conformation; Monitor; Mutate; Mutation; NADP; Nature; Organism; Oxidation-Reduction; Oxides; Oxidoreductase; Plant Roots; Protein Engineering; Proteins; Publications; Regulation; Relaxation; Resolution; Roentgen Rays; Role; Site; Structure; System; X-Ray Crystallography; base; biophysical techniques; enzyme structure; human pathogen; innovation; insight; member; oxidation; stereochemistry; therapeutic target; tumor

PROJECT SUMMARY We have discovered that a single hydride induces global changes to both structure and dynamics within multiple members of an enzyme family, providing a fundamental link between enzyme structure, dynamics, and allostery that has implications to the entire oxidoreductase superfamily. Specifically, the BLVRB family are NADPH-dependent reductases present in multiple organisms where they regulate cellular redox through the reduction of biliverdin-to-bilirubin and a wide array of flavin substrates. While our recent publications have revealed that coenzyme binding is coupled to global conformational and dynamic changes, we have now discovered that there are largescale changes coupled to the oxidation state of the coenzyme as far as 23 Å away. Thus, structural catalytic the central premise of this application is that a coenzyme's hydride is globally coupled to both and dynamic changes within an enzyme family and that such global coupling is integrally related to function. The novelty here is that we will explicitly determine how a single hydride, i.e., the difference between NADPH/NADP+, is globally linked (Aim 1) and how this global coupling controls enzyme function (Aim 2). Further innovation includes the following. First, we have discovered that hydride-coupled networks can be modulated by mutations directly to the enzyme/coenzyme interface but also to distally coupled sites, which gives us the unique opportunity to determine the role of these networks in function. Second, we have discovered that evolutionarily changing residues modulate hydride coupled networks and function, providing remarkable insight into the evolutionary role of hydride-mediated coupling and function. Evolutionary differences will therefore be exploited to identify allosteric networks coupled to the oxidative state of the coenzyme and simultaneously reveal their evolutionary roles in function. Based on our preliminary data that includes NMR, X-ray crystallographic, and biochemical studies, we hypothesize that the coenzyme oxidation induces its own conformational change that is further propagated globally through the enzyme in multiple BLVRB family members (referred to as “insideout” coupling) and that networks coupled to these changes modulate function (referred to as “outsidein” coupling). We will address this hypothesis through the following: Aim 1) Determine how a single hydride modulates the global dynamics and structure within the BLVRB family of enzymes. NMR solution studies using CSPs, relaxation studies, and ensembles methods will be used to determine how a single hydride imparts its global regulation to dynamics and structure using three distinct BLVRB family members with both active site and distal differences (human, hyrax, and mosquito). Aim 2) Determine the functional role of networks coupled to the oxidative state of the coenzyme. Biochemical and biophysical methods will be used to determine the functional role of hydride-mediated global regulation, which include both the role of direct interactions with the coenzyme's hydride as well as the role of networks of communication coupled to the coenzyme (allostery).

项目概要 我们发现单个氢化物会引起内部结构和动力学的整体变化 酶家族的多个成员，提供结构、动力学和酶之间的基本联系 对整个氧化还原酶超家族有影响的变构，特别是 BLVRB 家族。 NADPH 依赖性还原酶存在于多种生物体中，它们通过 胆绿素到胆红素和多种黄素底物的减少，而我们最近的出版物有。 揭示辅酶结合与整体构象和动态变化耦合，我们现在有 发现辅酶氧化态发生了高达 23 Å 的大规模变化 离开。 结构性的 催化 该应用的中心前提是辅酶的氢化物与两者全局偶联 和酶家族内的动态变化，并且这种全局耦合与 功能。 这里的新颖之处在于，我们将明确确定单个氢化物如何，即 NADPH/NADP+ 是全局关联的（目标 1），以及这种全局耦合如何控制酶功能（目标 2）。 进一步的创新包括以下内容：首先，我们发现氢化物耦合网络可以是。 通过直接突变到酶/辅酶界面以及远端偶联位点来调节， 为我们提供了确定这些网络在功能中的作用的独特机会。 发现进化变化的残基调节氢化物耦合网络和功能，提供 对氢化物介导的耦合和进化功能的进化作用的非凡见解。 因此，将利用差异来识别与氧化状态相关的变构网络。 根据我们的初步数据，辅酶同时揭示了它们在功能中的进化作用。 包括核磁共振、X射线晶体学和生化研究，我们认为辅酶氧化 诱导其自身的构象变化，并通过多种酶在全球范围内进一步传播 BLVRB 家族成员（称为“内→外”耦合）以及与这些变化耦合的网络 调制函数（称为“外→内”耦合）我们将通过以下方式解决这个假设： 目标 1) 确定单个氢化物如何调节 BLVRB 内的整体动力学和结构 使用 CSP、松弛研究和集成方法进行 NMR 解决方案研究。 用于确定单个氢化物如何使用三个参数将其全局调节传递给动力学和结构 不同的 BLVRB 家族成员具有活性位点和远端差异（人类、蹄兔和蚊子）。 目标 2) 确定与辅酶氧化状态耦合的网络的功能作用。 将使用生物化学和生物物理方法来确定氢化物介导的全局的功能作用 调节，其中包括与辅酶氢化物直接相互作用的作用以及 与辅酶（变构）耦合的通讯网络。