An interaction and an activation of ClpQ and ClpY protease in Escherichia coli
Date Issued
2006
Date
2006
Author(s)
Huang, Chi-Hsin
DOI
zh-TW
Abstract
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.
Subjects
蛋白酶
熱休克
大腸桿菌
交互作用
ClpY
ClpQ
protease
Escherichia coli
chaperone
HslU
HslV
ATP-dependent
interaction
Type
thesis
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