利用尾端專一性蛋白酶Prc靜止態之結構探討對基質辨別及依賴PDZ結構域活化之機制

Abstract

由於細菌細胞壁之周質(periplasm)中缺乏三磷酸腺苷(ATP)，細菌必須透過具有特殊調節機制及不依賴水解三磷酸腺苷(ATP-independent)的蛋白酶(protease)來降解錯誤或廢棄不用的蛋白。在大腸桿菌中，具有 PDZ 結構域(PDZ domain)的蛋白酶 Prc 參與蛋白質品質管控(protein quality control)及調節肽聚醣(peptidoglycan)型細胞壁合成。本研究以晶體學(crystallography)得到靜止態(resting state)的 Prc 蛋白酶結構，與先前發表酶受質結合之活化狀態(substrate-bound activated state)不同。在Prc 蛋白酶未活化狀態下，其高度可變性(flexibility)的 PDZ 結構域位於碗狀的單體中，並且其肽結合凹槽(peptide-binding cleft)朝向開放構形的蛋白酶結構域(protease domain)。與 PDZ 結構域相連之第九螺旋(helix h9)形成向外延伸之結構。當 PDZ 結構域捕捉到特定多肽之 C 端(C terminus)，蛋白酶結構域內部會與第九螺旋形成狹窄的通道，並將多肽拉入水解位點中心(proteolytic site)。本研究還以晶體結構學得知 Prc 鉸鏈區的胺基酸(hinge residues)會與多肽之 C 端結合，來介導 PDZ 結構域與 蛋白酶結構域之間的相互作用。藉由酶受質所引起 Prc 構形變化，可合理解釋酶受質如何被 Prc 識別與活化蛋白酶的功能。

As the periplasm lacks ATP, periplasmic proteases must implement distinct mechanisms for regulation of degradation activity against defective and inactive proteins. The Escherichia coli periplasmic PDZ-protease Prc is involved in protein quality control and the turnover of peptidoglycan during cell wall growth. This thesis shows that crystal structures of Prc in a unliganded resting conformation differ considerably from the previous published stucture of Prc in a liganded activated state. In a resting conformation, the flexible PDZ domain is located inside the bowl-shape body, and its peptide-binding celft is exposed to an open active center cleft. The partially unfolded helix h9 linking the PDZ domain adopts an extended conformation. The PDZ domain captures C terminus of a specific polypeptide substrate, and subsequently pulls the cleavage site into a narrow passage formed by the protease platform and the helix h9. These crystal structures further demonstrate hinge residues mediate the interplay of the PDZ domain and the protease platform. The thesis provides structural insight for understanding the mechanism of substrate recognition and Prc activation.