PPIase domain of trigger factor acts as auxiliary chaperone site to assist the folding of protein substrates bound to the crevice of trigger factor.
Trigger factor (TF) is the first chaperone encountered by nascent chains in bacteria, which consists of two modules: peptidyl-prolyl-cis/trans-isomerase (PPIase) domain and a crevice built by both N- and C-terminal domains. While the crevice is suggested to provide a protective space over the peptide exit site of ribosome for nascent polypeptides to fold, it remains unclear whether PPIase domain is directly involved in assisting protein folding. Here, we introduced structural change into different regions of TF, and investigated their influence on the chaperone function of TF in assisting the folding of various substrate proteins, including oligomeric glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and monomeric carbonic anhydrase II (CA II) and lysozyme. Results showed that structural disturbances by site-specific mutations in the PPIase active site or by deletion of the PPIase domain from TF affected the chaperone activity of TF toward CA II and GAPDH but had no effect on TF-assisted lysozyme refolding, suggesting PPIase domain is involved in assisting the folding of substrates larger than lysozyme. Mutants with the structural disturbances in the crevice totally lost the chaperone activity toward all the substrates we used in this investigation. These results provide further evidence to confirm that the crevice is the major chaperone site of TF, and the hydrophobic pocket in PPIase domain acts as an auxiliary site to assist the folding of substrate proteins bound to the crevice in a substrate-dependent manner, which is beneficial for TF to provide appropriate assistance for protein folding by changing protective space and binding affinity.
Liu
Chuan-Peng
CP
Bio-X Center, Academy of Fundamental and Interdisciplinary Sciences, Harbin Institute of Technology, 2 Yikuang Street, Harbin 150080, China. liucp74@hotmail.com
Zhou
Qi-Ming
QM
Fan
Dong-Jie
DJ
Zhou
Jun-Mei
JM
D000595:Amino Acid Sequence; D000818:Animals; D024402:Carbonic Anhydrase II / Q000378:metabolism; D020134:Catalytic Domain / Q000235:genetics; D002417:Cattle; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / Q000378:metabolism; D006801:Humans; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D019696:Peptidylprolyl Isomerase / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering; D017510:Protein Folding; D011817:Rabbits; D013329:Structure-Activity Relationship; D013552:Swine
D029968:Escherichia coli Proteins; D018832:Molecular Chaperones; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating); D024402:Carbonic Anhydrase II; C403324:trigger factor, E coli; D019696:Peptidylprolyl Isomerase