Characterization of the Escherichia coli ClpY (HslU) substrate recognition site in the ClpYQ (HslUV) protease using the yeast two-hybrid system.
In Escherichia coli, ClpYQ (HslUV) is a two-component ATP-dependent protease in which ClpQ is the peptidase subunit and ClpY is the ATPase and the substrate-binding subunit. The ATP-dependent proteolysis is mediated by substrate recognition in the ClpYQ complex. ClpY has three domains, N, I, and C, and these domains are discrete and exhibit different binding preferences. In vivo, ClpYQ targets SulA, RcsA, RpoH, and TraJ molecules. In this study, ClpY was analyzed to identify the molecular determinants required for the binding of its natural protein substrates. Using yeast two-hybrid analysis, we showed that domain I of ClpY contains the residues responsible for recognition of its natural substrates, while domain C is necessary to engage ClpQ. Moreover, the specific residues that lie in the amino acid (aa) 137 to 150 (loop 1) and aa 175 to 209 (loop 2) double loops in domain I of ClpY were shown to be necessary for natural substrate interaction. Additionally, the two-hybrid system, together with random PCR mutagenesis, allowed the isolation of ClpY mutants that displayed a range of binding activities with SulA, including a mutant with no SulA binding trait. Subsequently, via methyl methanesulfonate tests and cpsB::lacZ assays with, e.g., SulA and RcsA as targets, we concluded that aa 175 to 209 of loop 2 are involved in the tethering of natural substrates, and it is likely that both loops, aa 137 to 150 and aa 175 to 209, of ClpY domain I may assist in the delivery of substrates into the inner core for ultimate degradation by ClpQ.
Lien
Hsiang-Yun
HY
Department of Agricultural Chemistry, National Taiwan University, Taipei, Taiwan, Republic of China.
Shy
Ru-Shan
RS
Peng
Sheng-Shiang
SS
Wu
Yuei-Long
YL
Weng
Yu-Ting
YT
Chen
Hsuan-He
HH
Su
Pin-Chih
PC
Ng
Wei-Fu
WF
Chen
Yu-Chun
YC
Chang
Pei-Yi
PY
Wu
Whei-Fen
WF
D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D049071:Endopeptidase Clp / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D020798:Two-Hybrid System Techniques
C483566:ClpYQ protease, E coli; D029968:Escherichia coli Proteins; D049071:Endopeptidase Clp