Structural studies of intramembrane protease GlpG

描述（由申请人提供）：膜内蛋白水解是细胞生物学中重要而广泛的生化机制：许多膜蛋白通过膜内蛋白水解激活信号转导，或转化为可引起人类疾病的难溶性淀粉样肽。本提案的长期目标是通过对催化反应的特定膜蛋白及其与抑制剂和底物的复合物的晶体学分析，对这一机制有更深入的了解。许多膜内蛋白酶已被认为是治疗感染性疾病和年龄相关疾病的新的重要药物靶点。目前的应用主要集中在GlpG，大肠杆菌菱形丝氨酸蛋白酶家族的整体膜蛋白。计划进行生化、诱变和晶体学实验研究：(1)GlpG如何与类特异性抑制剂相互作用，以验证GlpG和其他菱形蛋白酶使用膜嵌入Ser-His二偶体直接攻击底物的假设，并确定蛋白酶活性位点对催化作用的重要特征；(2) GlpG如何通过两种蛋白的复杂结构重排与跨膜底物相互作用，哪些因素决定了这种相互作用的特异性；(3)膜中一个有趣的结构基序调节蛋白酶活性的机制。近年来在GlpG晶体结构测定方面的突破表明，这种细菌膜蛋白是研究细胞膜酶作用的一个很好的模型系统。

DESCRIPTION (provided by applicant): Intramembrane proteolysis is an important and widespread biochemical mechanism in cell biology: many membrane proteins undergo intramembrane proteolysis to become activated for signal transduction, or to be converted to poorly soluble amyloidal peptides that may cause human disease. The long-term objective of this proposal is to gain a deeper understanding of this mechanism through crystallographic analysis of specific membrane proteins that catalyze the reaction and of their complexes with inhibitors and substrates. Many intramembrane proteases have been recognized as novel and important drug targets for treating infectious and age-related diseases. The current application focuses on GlpG, an E. coli integral membrane protein of the rhomboid serine protease family. Biochemical, mutagenesis and crystallographic experiments are planned to study: (1) how GlpG interacts with class specific inhibitors in order to examine hypothesis that GlpG and other rhomboid proteases use a membrane-embedded Ser-His dyad to directly attack substrate, and to determine features of the protease active site that are important for catalysis; (2) how GlpG interacts with transmembrane substrates through complex structural rearrangements in both proteins, and which factors determine the specificity of this interaction; and (3) the mechanism by which an interesting structural motif regulates protease activity in the membrane. Recent breakthroughs in crystal structure determination of GlpG suggest that this bacterial membrane protein is an excellent model system for studying enzyme action in cell membranes.