大腸桿菌ClpQ蛋白質的C端為負責其單元體間交互作用的區段

The ClpQ C-domain is responsible for subunit interaction in E. coli

余建泓(Chien-Hung Yu)

指導教授 ： 吳蕙芬

國立臺灣大學/生物資源暨農學院/農業化學研究所/碩士(2004年)

https://doi.org/10.6342/NTU.2004.02174

摘要

clpQY ( hslVU )是大腸桿菌熱休克效應基因，其所生產的ClpQY蛋白酶( protease )在細菌中扮演分解錯誤摺疊蛋白質或是調控其他蛋白質生成，以維持細菌正常生理狀況的重要角色。由膠體過濾分析及電子顯微鏡研究發現，ClpQ及ClpY在細胞中皆是由六元體所組成。進一步由X-ray結晶結構分析，發現ClpQ及ClpY各形成六元環狀結構，由一個或是兩個ClpY的六元環堆疊在兩個ClpQ六元環的兩側，形成具有活性的protease。另外，ClpQY 的複合體被認為是真核細胞中proteasome的同源蛋白，因為兩者在生物體中具有類似的功能以及桶柱狀構形。ClpY負責基質的辨識，基質摺疊肢解開，以及利用能量將基質送入ClpQ中，由ClpQ負責基質之分解。
本篇研究焦點放在ClpQ單元體間如何進行交互作用，以形成六元環狀結構(oligomerization)。因為當ClpQ的六個單元體形成環狀結構時，才能將催化中心以及與基質結合的疏水性區域包覆在其構形當中，也才具有催化的能力。因此，我們先利用yeast-two-hybrid的方法檢視ClpQ是用那個區段進行單元體之間的相互作用，並利用綠膿桿菌(P. aeruginosa)的ClpQ進行作用區段的確認，得知ClpQ為利用其C-端進行單元體之間的交互作用。與結晶結構比對，我們選定C-端的第一段helix﹝O-helix﹞，進行點突變。並建構缺失突變及前人所挑到的突變一起放入大腸桿菌系統中進行確認。
在大腸桿菌的分析中，利用RcsA為ClpQY的基質及RcsA正調控cpsB的關係，以lacZ為報導基因，或是以MMS誘導ClpQY基質SulA的產生等方式，測試ClpQ的突變株是否能在大腸桿菌當中形成六元環而分解基質。結果顯示，C-端20個胺基酸刪除的ClpQ突變蛋白在三個系統中不僅影響其環狀結構之形成，且都可以看到其分解基質能力的下降。因此，ClpQ蛋白質之羧基端序列負責其本身形成六元環結構之重要區塊。

關鍵字

蛋白酶 ； 單元體交互作用

並列摘要

In response to heat shock and other stressful conditions, cells increase the synthesis of a group of proteins known as heat shock proteins (Hsps). The heat shock proteins function either as molecular chaperons in the refolding of misfolded peptides or as proteases which catalyse the degradation of such abnormal proteins. ClpQY (HslVU) is one of the Hsps in E. coli which is a multimeric complex of two components encoded in the hslVU operon: the proteolytic component ClpQ (HslV) is about 19kDa and the ATPase ClpY (HslU) is about 50kDa.
Negatively staining electron microscopy of purified ClpQ and ClpY shows a uniform field of particles, most of which are ring-shaped, shared a six-fold symmetry. Crystallographic studies also reveal that the ClpQ is a “double donut” hexameric rings and ClpY locates outside the ClpQ particle with a vis-à-vis orientation. Otherwise, ClpQY complex is a bacterial homolog of the eukaryotic proteasome. They both have ring structures, providing for compartmentalization of substrate protein inside a central cavity. General view is that ClpY, an ATPase is responsible for the substrate recognition, unfolding of substrate, and translocation of unfolded protein to the peptidase region, ClpQ. The ability of hydrolysing short hydrophobic peptide by ClpYQ complex is thousand-fold greater than ClpQ alone in vitro and only ClpQY complex can degrade intact protein subtrate.
We are interested in how does ClpQ subunit interaction occur and becomes a functional hexameric ring. Therefore, we use the yeast-two-hybrid system to examine which region of the E. coli ClpQ is responsible for the ClpQ/ClpQ interaction and also use the P. aeruginosa ClpQ as a contrast model. Accordingly, the P. aeruginosa ClpQ can not interact with E.coli ClpQ in our system. We define the ClpQ C-domain (S125-A176) is important in the ClpQ/ClpQ interaction. While compared this data to the crystal structure, we made ClpQ with several point mutations in the O-helix （S125-E139） region and it’s deletion mutants screened in the Yeast-two-hybrid system.
Several meaningful mutants with altering ClpQ interactive abilities were verified . Based on Cps expression, RcsA*-LacZ expression and MMS test, we found that when the C-terminal last 20 amino acids were deleted, ClpQ lost it’s interaction activity i.e. it could not constitute oligomer. Taken together, we think that the C-terminal ends of ClpQ is responsible for ClpQ self-oligomerization.

並列關鍵字

subunit interaction ； RcsA ； ClpQ ； SulA

參考文獻

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