Human PBGS Quaternary Structure Dynamics, Drugs, and Half-of-the-sites Reactivity

描述（由申请人提供）：人胆色素原合酶（PBGS; E.C. 4.2.1.24）催化血红素生物合成途径中的基本生物吡咯、胆色素原的古老和基本形成。它是一种锌金属酶，是铅中毒的靶酶。人PBGS参与四级结构平衡，其包括高活性八聚体、低活性六聚体和二聚体的两种构象，每种构象决定进一步组装的化学计量。扰乱四级结构平衡的低活性六聚体的突变与ALAD卟啉症有关，ALAD卟啉症是一种罕见但严重的疾病状态。使用药物Levulan的皮肤病损的光动力疗法需要PBGS活性。提出了两个不同的目标。目的1研究药物和环境污染物是否可以调节人PBGS四级结构平衡，从而抑制或激活酶活性。将通过非变性PAGE迁移率变动试验评价获批药物和已知环境污染物的文库，以确定哪些组分可使人PBGS四级结构平衡向低或高活性状态转变。将使用已建立的方法来验证作为抑制剂或激活剂的命中。抑制剂是铅中毒或卟啉病患者应避免使用的分子;激活剂可缓解铅中毒的某些症状，作为ALAD卟啉病的治疗剂，或作为辅助剂以优化光动力疗法的结果。再加上对人类等位基因变异如何影响组装状态平衡的理解，这些研究为四级结构在个性化医疗中的应用引入了新的方法。它们为脱靶副作用提供了一种可能的解释，并建议使用标签外治疗。AIM 2使用人PBGS作为模型系统，用于理解半位点反应性（HSR）的结构基础。HSR类似于变构调节，因为蛋白质中一个位置处的结合现象深刻地影响同一蛋白质中远处位点处的结合/反应性行为。亚基间通讯是HSR所必需的，一般来说，所需通讯的分子基础知之甚少。我们对人类PBGS结构和机制的理解使我们能够调节各个亚基间的界面，确定哪些负责所需的通信，并探测各个残基以确定通信途径。定量席夫碱捕获将被用作HSR的诊断。我们已经确定，亚基间的相互作用时，发生的活性位点盖是封闭的活性八聚体的维护所需的。我们建议，基板/产品的结合，这是必不可少的活性位点盖关闭，因此也是必不可少的八聚体的维护和规定的要求HSR。拟议中的研究将提高我们的理解的作用，交替四级结构组件在人类疾病，并将提高一般知识的亚基间通信HSR和某些形式的变构至关重要。 公共卫生相关性：了解蛋白质的结构-功能关系对于了解无数治疗药物的作用机制至关重要。胆色素原合酶参与了一个新发现的涉及四级结构动力学的调控机制，它的研究为理解交替蛋白质组装体在人类疾病中的作用提供了一个新的结构基础，为理解药物副作用提供了一种机制，并提出了新的治疗方法。人类PBGS的失活涉及ALAD卟啉症和铅中毒的人类状况，后者仍然是美国和世界各地普遍存在的公共卫生问题。

DESCRIPTION (provided by applicant): Human porphobilinogen synthase (PBGS; E.C. 4.2.1.24) catalyzes the ancient and essential formation of the fundamental biological monopyrrole, porphobilinogen in the heme biosynthetic pathway. It is a zinc metalloenzyme and the target enzyme in lead poisoning. Human PBGS participates in a quaternary structure equilibrium that includes high activity octamers, low activity hexamers, and two conformations of a dimer, each of which dictates the stoichiometry of further assembly. Mutations that perturb the quaternary structure equilibrium toward the low activity hexamer are associated with ALAD porphyria, a rare but serious disease state. Photodynamic therapy of dermatologic lesions with the drug Levulan requires PBGS activity. Two distinct aims are proposed. AIM 1 investigates whether drugs and environmental contaminants can modulate the human PBGS quaternary structure equilibrium and thus inhibit or activate enzyme activity. Libraries of approved drugs and known environmental contaminants will be evaluated by a native PAGE mobility shift assay to determine which components can shift the human PBGS quaternary structure equilibrium toward either the low or high activity states. Established methods will be used to validate hits as inhibitors or activators. Inhibitors are molecules that should be avoided in patients with lead poisoning or porphyria; activators may alleviate some symptoms of lead poisoning, serve as a therapeutic for ALAD porphyria, or as adjuvant to optimize the outcome of photodynamic therapy. Coupled with the understanding of how human allelic variation affects the equilibrium of assembly states, these studies introduce a new approach to the application of quaternary structure to personalized medicine. They provide a possible explanation for off-target side effects, and also suggest off-label therapies. AIM 2 uses human PBGS as a model system for understanding the structural basis of half-of-the-sites reactivity (HSR). HSR is similar to allosteric regulation in that binding phenomena at one location in the protein profoundly affect the binding/reactivity behavior at a distant site in the same protein. Intersubunit communication is required for HSR and, in general, the molecular basis for the required communication is poorly understood. Our understanding of human PBGS structure and mechanism allows us to modulate the individual intersubunit interfaces, determine which is responsible for the required communication, and probe individual residues to determine the communication pathway. Quantitative Schiff base trapping will be used as a diagnostic for HSR. We have established that intersubunit interactions that occur when the active site lid is closed are required for maintenance of the active octamer. We propose that substrate/product binding, which is essential for active site lid closure, is therefore also essential for maintenance of the octamer and dictates the requirement for HSR. The proposed studies will improve our understanding of the role of alternate quaternary structure assemblies in human disease and will enhance general knowledge about intersubunit communication essential for HSR and some forms of allostery. PUBLIC HEALTH RELEVANCE: Understanding protein structure-function relationships is essential to understanding the mechanism of action of myriad therapeutic agents. Investigation of porphobilinogen synthase, which participates in a newly discovered regulation mechanism involving quaternary structure dynamics, provides a novel structural basis for understanding the role of alternate protein assemblies in human disease, provides a mechanism for understanding drug side effects, and suggests new therapies. Inactivation of human PBGS is involved in the human conditions of ALAD porphyria and lead poisoning, the latter of which remains a pervasive public health problem in the United States and around the world.