Structural Studies of GxGD Membrane Protease FlaK

描述（由申请人提供）：本申请的长期目标是阐明GxGD膜蛋白酶的催化机制，其在结构和机制上不同于可溶性乙酰基蛋白酶。早老素和4型前菌毛蛋白肽酶（TFPP）都是GxGD蛋白酶家族的成员：早老素的突变可引起家族性阿尔茨海默病，而TFPP参与细菌的发病机制。Flak是TFPP的古同系物，其晶体结构在初步研究中得到解决，是目前家族中唯一的原子分辨率结构。晶体结构提供了一个概述的膜蛋白的折叠，并表明，蛋白酶必须经历构象变化后，底物结合移动未偶联的乙酰基残基一起催化。在申请中提出了三个具体目标。在具体目标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性质。在具体的目标2中，将合成掺入羟基磷灰石或（羟乙基）脲电子等排体的过渡态类似物抑制剂以与FlaK共结晶。底物结合亚位点和变构 模型将通过诱变进行检测。在具体目标3中，将产生底物-蛋白酶融合蛋白以评估底物的TM结构域（其在切割位点的下游）在与蛋白酶结合中的作用。具有完整TM结构域的全长底物比短肽更有效地被FlaK切割。计划进行晶体学和诱变实验以表征融合蛋白。

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.