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.

说明(申请人提供)：本申请的长期目标是阐明GxGD膜蛋白水解酶的催化机理，该酶在结构和机理上与可溶性天冬氨酸蛋白酶不同。早老素和TFPP都是GxGD蛋白水解酶家族的成员：早老素基因突变可导致家族性阿尔茨海默病，而TFPP参与细菌致病。FLAK是TFPP的远古同系物，其晶体结构在初步研究中被破解，是目前该家族中唯一的原子分辨结构。晶体结构提供了膜蛋白折叠的概述，并表明蛋白酶必须在底物结合时经历构象变化，才能将未偶联的天冬氨酸残基移动到一起进行催化。在申请中提出了三个具体目标。在特定的目标1中，将产生变构刺激FLAK酶活性的突变，并通过X射线结晶学进行研究，以帮助解释构象变化的性质。在具体目标2中，将合成含有羟乙基或(羟乙基)尿素异构体的过渡态类似物，使其与LAK共结晶。底物结合亚基与变构 模型将通过诱变进行测试。在特定目标3中，将产生底物-蛋白酶融合蛋白，以评估底物的TM结构域在与蛋白酶结合中的作用。底物TM结构域位于裂解位点下游。具有完整TM结构域的全长底物比短肽更有效地被FLAK切割。计划进行结晶学和诱变实验来表征融合蛋白。