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，单个氢化物会引起内部结构和动力学的全局变化， 酶家族的多个成员，提供酶结构、动力学和 这对整个氧化还原酶超家族都有影响。具体来说，BLVRB家族是 NADPH依赖性还原酶存在于多种生物体中，其中它们通过氧化还原酶调节细胞氧化还原。 将胆绿素还原为胆红素和多种黄素底物。虽然我们最近的出版物 发现辅酶结合与全局构象和动力学变化相耦合，我们现在已经 发现，有大规模的变化耦合到辅酶的氧化态高达23 距离因此，在本发明中， 结构 催化 本申请的中心前提是辅酶的氢化物整体上与 以及酶家族内的动态变化，并且这种全局耦合与 功能 这里的新奇在于，我们将明确确定单个氢化物，即，的区别 NADPH/NADP+是全局连接的（目标1），以及这种全局偶联如何控制酶功能（目标2）。 进一步的创新包括以下内容。首先，我们已经发现，解耦网络可以 通过突变直接调节酶/辅酶界面，但也调节远端偶联位点， 给了我们一个独特的机会来确定这些网络在功能中的作用。二是 发现进化上变化的残基调节氢化物偶联网络和功能， 这是一个对糖蛋白介导的偶联和功能的进化作用的卓越见解。进化 因此，将利用这些差异来鉴定与氧化态偶联的变构网络。 辅酶，同时揭示其功能的进化作用。根据我们的初步数据， 包括核磁共振，X射线晶体学和生物化学研究，我们假设辅酶氧化 诱导其自身的构象变化，该构象变化进一步通过酶在多个区域中在全球传播。 BLVRB家族成员（简称“内异外”耦合）和网络耦合这些变化 调制功能（称为“外部耦合”）。我们将通过以下方式来解决这一假设： 目的1）确定单个氢化物如何调节BLVRB内的全局动力学和结构 酵素家族将使用CSP、弛豫研究和系综方法进行NMR溶液研究。 用于确定单个氢化物如何使用三个氢化物对动力学和结构进行全局调节 具有活性位点和远端差异的不同BLVRB家族成员（人、蹄兔和蚊子）。 目的2）确定与辅酶氧化态偶联的网络的功能作用。 生物化学和生物物理学方法将用于确定β-葡糖苷酶介导的全球性的功能作用， 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 通讯网络耦合到辅酶（变构）。