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

描述(由申请人提供)：人胆红素原合成酶(PBGS；E.C.4.2.1.24)催化血红素生物合成途径中最基本的生物单吡咯的形成。它是一种锌金属酶，也是铅中毒的靶酶。人PBGS参与了一个四元结构平衡，其中包括高活性八聚体、低活性六聚体和二聚体的两种构象，每一种构象都决定了进一步组装的化学计量比。扰乱四级结构平衡而倾向于低活性六聚体的突变与ALAD卟啉症有关，这是一种罕见但严重的疾病状态。使用药物Levulan进行皮肤病皮损的光动力疗法需要PBGS活性。提出了两个截然不同的目标。目的1研究药物和环境污染物是否能调节人PBGS的四级结构平衡，从而抑制或激活酶的活性。已批准的药物和已知环境污染物的库将通过天然PAGE迁移率变化分析进行评估，以确定哪些成分可以将人类PBGS的四元结构平衡向低活性状态或高活性状态转变。已建立的方法将用于验证HITS是否为抑制剂或激活剂。抑制剂是铅中毒或卟啉症患者应该避免的分子；激活剂可以缓解铅中毒的某些症状，作为治疗ALAD卟啉症的药物，或者作为优化光动力疗法结果的佐剂。结合对人类等位基因变异如何影响组装状态平衡的理解，这些研究引入了一种将四元结构应用于个性化药物的新方法。他们为非靶向副作用提供了可能的解释，也建议了非标签疗法。目的2使用人PBGS作为一个模型系统来理解半部位反应性(HSR)的结构基础。HSR类似于变构调节，因为蛋白质中一个位置的结合现象深刻地影响同一蛋白质中远处位置的结合/反应行为。HSR需要亚基间的通讯，一般来说，人们对所需通讯的分子基础知之甚少。我们对人PBGS结构和机制的了解使我们能够调节单个亚基之间的界面，确定哪个负责所需的通信，并探测单个残基以确定通信途径。定量Schiff碱基捕获将被用于HSR的诊断。我们已经确定，当活性部位盖关闭时发生的亚基间相互作用是维持活性八聚体所必需的。因此，我们认为底物/产物结合是关闭活性部位盖所必需的，也是维持八聚体所必需的，这决定了对HSR的要求。建议的研究将提高我们对交替的四级结构组件在人类疾病中的作用的理解，并将增强关于HSR和某些形式的变构所必需的亚基间通信的一般知识。 公共卫生相关性：了解蛋白质结构-功能关系对于了解各种治疗剂的作用机制至关重要。胆红素原合成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组合在人类疾病中的作用提供了新的结构基础，为理解药物副作用提供了机制，并提出了新的治疗方法。人类PBGS的失活与ALAD卟啉症和铅中毒的人类状况有关，后者在美国和世界各地仍然是一个普遍的公共卫生问题。

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.