Identification of a potential hydrophobic peptide binding site in the C-terminal arm of trigger factor.
Trigger factor (TF) is the first chaperone to interact with nascent chains and facilitate their folding in bacteria. Escherichia coli TF is 432 residues in length and contains three domains with distinct structural and functional properties. The N-terminal domain of TF is important for ribosome binding, and the M-domain carries the PPIase activity. However, the function of the C-terminal domain remains unclear, and the residues or regions directly involved in substrate binding have not yet been identified. Here, a hydrophobic probe, bis-ANS, was used to characterize potential substrate-binding regions. Results showed that bis-ANS binds TF with a 1:1 stoichiometry and a K(d) of 16 microM, and it can be covalently incorporated into TF by UV-light irradiation. A single bis-ANS-labeled peptide was obtained by tryptic digestion and identified by MALDI-TOF mass spectrometry as Asn391-Lys392. In silico docking analysis identified a single potential binding site for bis-ANS on the TF molecule, which is adjacent to this dipeptide and lies in the pocket formed by the C-terminal arms. The bis-ANS-labeled TF completely lost the ability to assist GAPDH or lysozyme refolding and showed increased protection toward cleavage by alpha-chymotrypsin, suggesting blocking of hydrophobic residues. The C-terminal truncation mutant TF389 also showed no chaperone activity and could not bind bis-ANS. These results suggest that bis-ANS binding may mimic binding of a substrate peptide and that the C-terminal region of TF plays an important role in hydrophobic binding and chaperone function.
Shi
Yi
Y
National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
Fan
Dong-Jie
DJ
Li
Shu-Xin
SX
Zhang
Hong-Jie
HJ
Perrett
Sarah
S
Zhou
Jun-Mei
JM
D000817:Anilino Naphthalenesulfonates / Q000737:chemistry; D001665:Binding Sites; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D019696:Peptidylprolyl Isomerase / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding / Q000528:radiation effects; D017434:Protein Structure, Tertiary; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014466:Ultraviolet Rays
D000817:Anilino Naphthalenesulfonates; D029968:Escherichia coli Proteins; D010455:Peptides; C035763:5,5'-bis(8-(phenylamino)-1-naphthalenesulfonate); C403324:trigger factor, E coli; D019696:Peptidylprolyl Isomerase