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).

项目摘要 我们已经发现，单个水透明会引起整体变化的结构和动力学的变化 酶家族的多个成员，在酶结构，动态和 变构对整个氧化还原酶超家族具有影响。具体而言，BLVRB家族是 在多种生物中存在NADPH依赖性的还原大量，它们通过 减少双脂蛋白到双脂蛋白和各种黄素底物。虽然我们最近的出版物有 揭示了辅酶结合与全局构象和动态变化耦合，我们现在已经 发现与辅酶的氧化状态相连的大规模变化至23Å 离开。那， 结构 催化 该应用的中心前提是辅酶的氢化物在全球范围内耦合 酶家族中的动态变化，这种全球耦合与 功能。 这里的新颖性是，我们将明确确定单个氢化物的方式，即 NADPH/NADP+是全局链接的（AIM 1）以及该全局耦合如何控制酶功能（AIM 2）。 进一步的创新包括以下内容。首先，我们发现氢化物耦合的网络可以是 由直接针对酶/辅酶界面的突变调节，但也分别耦合的位点 为我们提供了确定这些网络在功能中的作用的独特机会。第二，我们有 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功能的进化作用的显着洞察力。进化 因此，差异将被利用以识别与氧化状态结合的变构网络 辅酶并简单地揭示了它们在功能中的进化作用。基于我们的初步数据 包括NMR，X射线晶体学和生化研究，我们假设辅酶氧化 诱导其自身的会议变化，通过多种酶在全球范围内进一步传播 BLVRB家庭成员（称为“内部Out”耦合），并且网络与这些更改相连 调制函数（称为“外部”耦合）。我们将通过以下来解决这一假设： AIM 1）确定单个Hydrode如何调节BLVRB内的全局动力学和结构 酶家族。使用CSP，松弛研究和合奏方法的NMR解决方案研究将是 用于确定单个Hydrode如何使用三个进行动态和结构的全局调节 具有主动部位和明显差异的独特的BLVRB家族成员（人，Hyrax和蚊子）。 目标2）确定与辅酶的氧化状态相连的网络的功能作用。 生化和生物物理方法将用于确定Hydrode介导的全局的功能作用 调节，其中包括与辅酶的氢化物的直接相互作用的作用以及 通信网络连接到辅酶（变构）。