Investigation of ClpXP Protease Mechanism of Function and its Interaction with the Folding Chaperone Trigger Factor

• Main Author: Yu, Angela Yeou Hsiung
• Other Authors: Houry, Walid A.
• Language: en_ca
• Published: 2013
• Subjects: chaperones; proteases; cotranslational; ClpP dynamics; 0487
• Online Access: http://hdl.handle.net/1807/36076

The major chaperones identified in Escherichia coli that assist in protein folding include trigger factor (TF), DnaK/DnaJ/GrpE and GroEL/GroES systems. The main ATP-dependent proteases are ClpXP, ClpAP, HslUV, Lon, and FtsH. From detailed sequence analysis, we found that tig (gene for TF), clpX, and clpP genes co-localize next to each other in most examined bacteria. We hypothesized that TF and ClpXP are functionally associated. TF is a ribosome-associated folding chaperone whereas ClpXP is a degradation complex. ClpX serves as the regulatory ATPase that recognizes substrates, unfolds and translocates polypeptides into ClpP for degradation. I found that TF physically interacts with ClpX, and that they collaborate to enhance degradation of certain ClpXP substrates. It is estimated that TF enhances the degradation of about 2% of newly synthesized E. coli proteins. One of the ClpXP substrates with degradation enhanced by TF was λO, the λ phage replication protein. Furthermore, TF also enhanced the degradation of ribosome-stalled λO nascent chains. Experiments suggest that TF transfers ribosome-stalled λO to ClpX for degradation by ClpP, demonstrating the existence of co-translational protein degradation in E. coli. To understand ClpXP mechanism, we had previously proposed that the degraded peptides are released from ClpP through transient equatorial side pores. To further understand ClpP dynamics, we determined the structure of ClpP(Ala153Cys) in its oxidized state. The structure shows that each opposing pair of protomers is linked by a disulfide bond. Unexpectedly, this structure resembles the compact structures of Streptococcus pneumoniae, Mycobacterium tuberculosis, and Plasmodium falciparum ClpPs, rather than the extended states seen in previous E. coli ClpP structures. Normal mode analysis of ClpP structures suggested that the iii compact structure is a naturally sampled conformation of WT ClpP. My findings provide insights for understanding ClpP dynamics as well as reveal a novel association between ClpXP protease and TF folding chaperone.