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一种重要且广泛的生化机制：许多膜蛋白经过膜内蛋白水解而被激活用于信号转导，或转化为可能引起人类疾病的难溶性淀粉样肽。该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进行晶体学分析，更深入地了解这种机制。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和重要的药物靶标。目前的应用集中在GlpG，一个E.大肠杆菌菱形丝氨酸蛋白酶家族的整合膜蛋白。通过生物化学、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和其他菱形蛋白酶利用膜包埋的Ser-His二联体直接攻击底物的假说，并确定对催化作用重要的蛋白酶活性位点的特征;（2）GlpG如何通过两种蛋白质中复杂的结构重排与跨膜底物相互作用，以及哪些因素决定这种相互作用的特异性;和（3）一个感兴趣的结构基序调节膜中蛋白酶活性的机制。GlpG晶体结构测定的最新突破表明，这种细菌膜蛋白是研究细胞膜中酶作用的极好模型系统。

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.