Structural Studies of GxGD Membrane Protease FlaK

GxGD 膜蛋白酶 FlaK 的结构研究

• 批准号: 8437932
• 负责人: YA HA
• 金额: $ 31.58万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-01-01 至 2016-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8437932
• 关键词: Achievement; Alzheimer' s Disease; Aspartate; Aspartic Endopeptidases; Binding; Biochemical; Biochemistry; Biology; Catalysis; Chemicals; Chimeric Proteins; Cleaved cell; Cocrystallography; Complex; Crystallization; Crystallography; Cysteine; Elements; Engineering; Family; Family member; Goals; Homologous Gene; Lateral; Length; Medical; Medicine; Membrane; Membrane Proteins; Modeling; Movement; Mutagenesis; Mutation; Nature; Pathogenesis; Peptide Hydrolases; Peptides; Play; Process; Research; Resolution; Role; Scanning; Site; Structure; Techniques; Testing; Transmembrane Domain; Urea; analog; base; crosslink; design; enzyme mechanism; familial Alzheimer disease; hydroxyethylene; inhibitor/antagonist; member; mutant; presenilin; protein folding; public health relevance; research study

DESCRIPTION (provided by applicant): The long-term objective of the application is to elucidate the catalytic mechanism of GxGD membrane protease, which differs in structure and mechanism from the soluble aspartyl proteases. Presenilin and type 4 prepilin peptidase (TFPP) are both members of the GxGD protease family: mutations in presenilin can cause familial Alzheimer's disease, whereas TFPPs are involved in bacterial pathogenesis. FlaK is an archaeal homolog of TFPP, and its crystal structure, solved in the preliminary study, is presently the only atomic resolution structure in the family. The crystal structure provides an overview of the membrane protein's fold, and suggests that the protease must undergo conformational changes upon substrate binding to move the uncoupled aspartyl residues together for catalysis. Three specific aims are proposed in the application. In specific aim 1, mutations that allosterically stimulate FlaK's enzymatic activity will be generated and studied by x-ray crystallography to help explain the nature of the conformational change. In specific aim 2, transition state analog inhibitors incorporating hydroxyethylene or (hydroxyethyl)urea isosteres will be synthesized to co- crystallize with FlaK. The substrate binding subsites and the allosteric model will be tested by mutagenesis. In specific aim 3, substrate-protease fusion proteins will be generated to evaluate the role of substrate's TM domain, which is downstream of the cleavage site, in the binding to the protease. Full-length substrate with an intact TM domain is more efficiently cleaved by FlaK than short peptide. Crystallographic and mutagenesis experiments are planned to characterize the fusion proteins.

描述（由申请人提供）：申请的长期目的是阐明GXGD膜蛋白酶的催化机制，该机制的结构和机制与可溶性白杨蛋白酶不同。 Presenilin和4型预钙蛋白肽酶（TFPP）都是GXGD蛋白酶家族的成员：Presenilin的突变会引起家族性阿尔茨海默氏病，而TFPP参与细菌发病机理。 FLAK是TFPP的古细菌同源物，其晶体结构在初步研究中解决了，目前是家族中唯一的原子分辨率结构。晶体结构提供了膜蛋白折叠的概述，并表明蛋白酶在底物结合时必须经历构象变化，以将未偶联的Aspartyl残基一起移动在一起进行催化。应用程序中提出了三个具体目标。在特定的目标1中，将通过X射线晶体学生成和研究刺激FLAK酶活性的突变，以帮助解释构象变化的性质。在特定的目标2中，将合成掺入羟基乙烯或（羟基乙基）尿素ISSOSTESTER的过渡状态模拟抑制剂合成与FLAK结晶。底物结合子站点和变构 模型将通过诱变测试。在特定的目标3中，将生成底物 - 蛋​​白酶融合蛋白，以评估与蛋白酶的结合中底物TM结构域的作用，后者是切割位点的下游。具有完整TM结构域的全长底物比短肽更有效地裂解。计划进行晶体学和诱变实验来表征融合蛋白。