Mechanistic studies of intramembrane protease GlpG

 描述（由申请人提供）：我们研究的长期目标是阐明菱形膜内蛋白酶的机制。菱形蛋白是膜蛋白的一部分，是一个古老而广泛的酶家族。在进化过程中，菱形获得了许多不同的生物学功能，其中许多在生物学和医学上很重要。例如，在引起疟疾的寄生虫恶性疟原虫中，菱形蛋白酶在人类宿主细胞的入侵中发挥重要作用。我们以前测定了E.大肠杆菌菱形GlpG及其与机制特异性抑制剂的复合物。在本申请中，我们继续使用GlpG作为模型系统来研究菱形如何与TM底物相互作用。提出了三个具体目标。在目标1中，我们测试了底物的TM结构域对接到GlpG上的外位点上并诱导激活蛋白酶的构象变化的假设。活性增强突变体的晶体结构和GlpG与底物的TM结构域之间的复合物将被解析以解释变构转变的性质。在目标2中，计划进行半胱氨酸可及性和光镊实验以研究底物的TM结构域的解折叠，这是膜内切割反应的先决条件。在目标3中，基于氟磷酸二异丙酯作为GlpG的共价抑制剂的发现，将合成两种类型的引入反应性膦酸酯基团的肽模拟物，以制备用于X射线分析的与GlpG的稳定复合物。本论文还计划通过诱变和功能实验来解释rhomboid的底物特异性。

 DESCRIPTION (provided by applicant): A long-term objective of our research is to clarify the mechanism of rhomboid intramembrane proteases. Rhomboids are integral membrane proteins and represent an ancient and widespread enzyme family. During the course of evolution rhomboids have acquired many different biological functions, many of which are important in biology and medicine. For example, in P. falciparum, the parasite that causes malaria, rhomboid proteases play an essential role in the invasion of human host cells. We have previously determined the x-ray structures of E. coli rhomboid GlpG and its complexes with mechanism-specific inhibitors. In this application we continue to use GlpG as a model system to investigate how rhomboid interacts with TM substrate. Three specific aims are proposed. In aim 1, we test the hypothesis that substrate's TM domain docks onto an exosite on GlpG and induces a conformational change that activates the protease. Crystal structures of activity- enhancing mutants and the complex between GlpG and substrate's TM domain will be solved to explain the nature of the allosteric transition. In aim 2, cysteine accessibility and optical tweezers experiments are planned to investigate the unfolding of substrate's TM domain, a prerequisite for the intramembrane cleavage reaction. In aim 3, building on the discovery of diisopropyl fluorophosphate as a covalent inhibitor for GlpG, two types of peptidomimetics incorporating reactive phosphonate groups will be synthesized to prepare stable complexes with GlpG for x-ray analysis. Mutagenesis and functional experiments are also planned to explain rhomboid's substrate specificity.