Proceedings of the National Academy of Sciences - PNAS, 2001-12, Vol.98 (25), p.14244-14249
2001
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Details
- Autor(en) / Beteiligte
- Titel
- Binding Specificity of Escherichia coli Trigger Factor
- Ist Teil von
- Proceedings of the National Academy of Sciences - PNAS, 2001-12, Vol.98 (25), p.14244-14249
- Ort / Verlag
- United States: National Academy of Sciences
- Erscheinungsjahr
- 2001
- Quelle
- Free E-Journal (出版社公開部分のみ）
- Beschreibungen/Notizen
- 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 FKBP(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 prolyl-independent binding of peptide substrates and peptidyl-prolyl isomerization involve the same binding site.
- Sprache
- Englisch
- Identifikatoren
- ISSN: 0027-8424eISSN: 1091-6490DOI: 10.1073/pnas.261432298Titel-ID: cdi_pnas_primary_98_25_14244
- Format
- –
- Schlagworte
- Amino Acid Sequence, Amino acids, Amino Acyl-tRNA Synthetases - metabolism, Bacteria, Binding sites, Binding Sites - genetics, Biochemistry, Biological Sciences, Crosslinking, Datasets, Escherichia coli, Escherichia coli - genetics, Escherichia coli - metabolism, Escherichia coli Proteins - chemistry, Escherichia coli Proteins - genetics, Escherichia coli Proteins - metabolism, Fluorescence, Isomerization, Libraries, Macromolecular Substances, Models, Molecular, Nucleic Acid Conformation, Peptide Elongation Factor Tu - metabolism, Peptides, Peptidylprolyl Isomerase - chemistry, Peptidylprolyl Isomerase - genetics, Peptidylprolyl Isomerase - metabolism, Point Mutation, Protein Binding, Protein Conformation, Protein Structure, Tertiary, Proteins, Ribosomes, RNA, Bacterial - chemistry, RNA, Bacterial - metabolism, RNA, Transfer - chemistry, RNA, Transfer - metabolism, Studies, Substrate specificity, trigger factor