Mechanistic studies of intramembrane protease GlpG

膜内蛋白酶GlpG的机理研究

• 批准号: 9193640
• 负责人: YA HA
• 金额: $ 33.5万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9193640
• 关键词: Active Sites; Address; Adopted; Binding; Biochemical; Biological Models; Biological Process; Biology; Biophysics; C-terminal; Catalysis; Cells; Complex; Crystallization; Cysteine; Disulfides; Docking; Enzymes; Escherichia coli; Evolution; Family; Free Energy; Health; Human; Integral Membrane Protein; Malaria; Maps; Medicine; Membrane; Modeling; Molecular Conformation; Mutagenesis; Mutation; Nature; Organic Synthesis; Parasites; Peptide Hydrolases; Peptides; Plasmodium falciparum; Play; Proteolysis; Reaction; Research; Roentgen Rays; Role; Site; Specificity; Spectrum Analysis; Structure; Substrate Specificity; Testing; Transmembrane Domain; base; biophysical techniques; constriction; crosslink; experimental study; fluorophosphate; inhibitor/antagonist; laser tweezer; mutant; nanodisk; novel strategies; peptidomimetics; phosphonate; public health relevance; rhomboid; single molecule; success

 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.

 描述(由申请人提供)：我们研究的一个长期目标是阐明菱形膜内酶的机制。菱形是一种完整的膜蛋白，代表着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都很重要。例如，在导致疟疾的恶性疟原虫中，菱形蛋白水解酶在人类宿主细胞的入侵中起着至关重要的作用。我们以前已经测定了大肠杆菌菱形GlpG及其与机制特异性抑制剂的络合物的X射线结构。在这个应用中，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上的外显子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TM结构域之间的复合体将被解决，以解释变构转变的本质。在目标2中，计划进行半胱氨酸可及性和光钳实验，以研究底物TM结构域的展开，这是膜内切割反应的先决条件。在目标3，在发现二异丙基氟磷酸盐作为GlpG共价抑制剂的基础上，合成了两种含有反应性膦酸基的肽类化合物，以制备稳定的GlpG配合物，用于X-射线分析。还计划进行诱变和功能实验来解释菱形的底物专一性。