大腸桿菌ClpQ與ClpY之交互作用與其蛋白酶之活化

Abstract

大腸桿菌的ClpY/HslU為一種AAA 蛋白質（ATPase associated with various cellular activities），是具有ATPase活性的伴隨蛋白（chaperone）；ClpQ/HslV則是一種具胜肽酶（peptidase）活性的蛋白質。ClpY能與ClpQ形成具有蛋白酶活性的的多元體ClpYQ，其中ClpY扮演辨識基質並藉著消耗能量將基質解開送入ClpQ的角色；再經由ClpQ胜肽酶分解基質；前人發現ClpYQ與真核生物中的26S proteasome是具有同源性的。在結構上ClpY是以六元體環狀的形式，與兩個同為環狀的六元體ClpQ所構成的十二元體結合來作用。 在近年來的研究中指出，從已有的結晶結構來預測ClpYQ的作用區域可能在ClpY的羧基端以及ClpQ六元環之間形成的空間。而在2002年亦有實驗發現，若將ClpY最後十個胺基酸逐漸去除時，ClpYQ蛋白酶會失去其分解胜肽的能力；並且也會逐漸失去形成ClpYQ多元體的能力；更進一步甚至影響ClpY其本身形成六元環的能力；導致ClpY無法與胜肽酶 ClpQ結合而進行作用，因此失去ClpYQ蛋白酶的活性。 本實驗從已有的結晶結構預測可能之ClpQ與ClpY之交互作用點：ClpQ E61及K28及各自對應點ClpY R440及L443。並使用分子生物技術的方法，分別在胺基酸位置E61及K28建構全長為175個胺基酸之ClpQ點突變株，先使用RcsA及SulA相關系統來分析建構之突變株是否能與ClpY或是前人建構之ClpY突變株形成ClpYQ蛋白酶的活性，再分別使用酵母菌雙雜交系統 （Yeast two-hybrid system）觀察ClpY與ClpQ之間的作用力，以期望了解ClpY 羧基端結合與活化ClpQ功能的關係。發現ClpY之羧基端就如同一把鑰匙，插入ClpQ與相鄰ClpQ間產生的之孔隙，並開啟 ClpQ 的活性。藉著 ClpQ 結構改變的作用力可以幫助其緊密結合。其ClpY羧基端本身於長度、電荷以及其胺基酸構形上皆有適當的要求，且ClpQ孔隙由大量正電荷胺基酸所構成。

ATP-dependent proteolysis plays an essential role in controlling the levels of key regulatory proteins and in the elimination of abnormal polypeptides. These tasks are carried out by architecturally related ATP-dependent proteases such as the 26S proteasome in eukaryotes and two component protease, the ClpAP, ClpXP, and ClpYQ (HslUV) in archea and eubacteria. The clpYQ/hslVU operon in Escherichia coli encodes two heat shock proteins, the HslV/ClpQ peptidase and HslU/ClpY ATPase. Both ClpY and ClpQ self-assemble into a hexameric rings. Until now, two substrates RcsA and SulA of another ATP-dependent protease Lon, and RpoH, can be recognized by ClpY. In the clpYQ complex, the ClpY and ClpQ central pores are aligned, and the proteolytic active sites are sequestered in an internal chamber of ClpQ, with access to this chamber restricted to small axial pore. The roles of translocate peptides from ClpY to ClpQ and how interaction of ClpY and ClpQ to initiate ClpQ activity are not clear. In the research of Seong et al, an insertion of ClpY C-terminal tails into pockets at the ClpQ-ClpQ interface in the ATP-bound state might cause an opening of the central pore of ClpQ peptidase for an access of unfolded polypeptide substrates into the ClpQ proteolytic chamber. The ClpY C-terminal tail is essential for its interaction with ClpQ and for an activation of the peptidase. With ClpY molecule, the last 7 amino acids sequence of ClpY is highly conserved, and the lacking of the last four amino acids, ClpY leads to ClpYQ inactive. In this study, from ClpYQ crystal structure, R440 and L443 in ClpY C-terminal are opposited to E61 and K28 in ClpQ. Moreover, ClpQ point mutants in the position E61, K28 were constructed. And these positions were substituted with other 19 Amino acids. Subsequently, two methods were used to funtionally assay the activation between ClpQ mutants and ClpY as well as ClpY C-terminal mutants in E.coli. The yeast two-hybrid system was used to analyze oligomerlization of mutants between ClpQ-ClpY and ClpQ-ClpQ. The aim of this study is to find the significance of E61 and K28 in ClpQ, and a relationship between an interaction and an activation of ClpY and ClpQ protease.