Structural Studies of GxGD Membrane Protease FlaK

描述（由申请人提供）：申请的长期目标是阐明GxGD膜蛋白酶的催化机制，该酶在结构和机制上不同于可溶性天冬氨酸蛋白酶。早老素和4型prepilin肽酶（TFPP）都是GxGD蛋白酶家族的成员：早老素突变可导致家族性阿尔茨海默病，而TFPP参与细菌发病。FlaK是TFPP的古细菌同源物，其晶体结构在初步研究中得到解决，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，并表明蛋白酶在底物结合时必须经历构象变化，以将未偶联的天冬氨酸残基移动到一起进行催化。在应用程序中提出了三个具体目标。在特定目标1中，将产生变构刺激FlaK酶活性的突变，并通过x射线晶体学进行研究，以帮助解释构象变化的本质。在特定目标2中，将合成含有羟乙基或（羟乙基）尿素同分异构体的过渡态类似抑制剂，以与FlaK共结晶。底物结合亚位和变构

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.