Publikation: Binding specificity of Escherichia coli trigger factor
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The ribosome-associated chaperone trigger factor (TF) assists the folding of newly synthesized cytosolic proteins in Escherichia coli. Here, we determined the substrate specificity of TF by examining its binding to 2842 membrane-coupled 13meric peptides. The binding motif of TF was identified as a stretch of eight amino acids, enriched in basic and aromatic residues and with a positive net charge. Fluorescence spectroscopy verified that TF exhibited a comparable substrate specificity for peptides in solution. The affinity to peptides in solution was low, indicating that TF requires ribosome association to create high local concentrations of nascent polypeptide substrates for productive interaction in vivo. Binding to membrane-coupled peptides occurred through the central peptidyl-prolyl-cis/trans isomerase (PPIase) domain of TF, however, independently of prolyl residues. Crosslinking experiments showed that a TF fragment containing the PPIase domain linked to the ribosome via the N-terminal domain is sufficient for interaction with nascent polypeptide substrates. Homology modeling of the PPIase domain revealed a conserved FKB (FK506-binding protein)- like binding pocket composed of exposed aromatic residues embedded in a groove with negative surface charge. The features of this groove complement well the determined substrate specificity of TF. Moreover, a mutation (E178V) in this putative substrate binding groove known to enhance PPIase activity also enhanced TF s association with a prolyl-free model peptide in solution and with nascent polypeptides. This result suggests that both prolylindependent binding of peptide substrates and peptidyl-prolyl isomerization involve the same binding site.
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PATZELT, Holger, Stefan RÜDIGER, Dirk BREHMER, Günter KRAMER, Sonja VORDERWÜLBECKE, Elke SCHAFFITZEL, Andreas WAITZ, Thomas HESTERKAMP, Liying DONG, Jens SCHNEIDER-MERGENER, Bernd BUKAU, Elke DEUERLING, 2001. Binding specificity of Escherichia coli trigger factor. In: Proceedings of the National Academy of Sciences of the United States of America. 2001, 98(25), pp. 14244-14249. ISSN 0027-8424. eISSN 1091-6490. Available under: doi: 10.1073/pnas.261432298BibTex
@article{Patzelt2001Bindi-7324, year={2001}, doi={10.1073/pnas.261432298}, title={Binding specificity of Escherichia coli trigger factor}, number={25}, volume={98}, issn={0027-8424}, journal={Proceedings of the National Academy of Sciences of the United States of America}, pages={14244--14249}, author={Patzelt, Holger and Rüdiger, Stefan and Brehmer, Dirk and Kramer, Günter and Vorderwülbecke, Sonja and Schaffitzel, Elke and Waitz, Andreas and Hesterkamp, Thomas and Dong, Liying and Schneider-Mergener, Jens and Bukau, Bernd and Deuerling, Elke} }
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