Binding specificity of Escherichia coli trigger factor

Abstract

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-cis͞trans 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 prolylindependent binding of peptide substrates and peptidyl-prolyl isomerization involve the same binding site.

Trigger Factor Retards Protein Export in Escherichia coli

“…The observation that TF overproduction delays OmpA export for up to several minutes strongly suggests that it can interact with its substrates post-translationally (presumably through multiple rounds of binding and dissociation). In addition, the observation that manipulation of the TF concentration did not affect the export of all proteins uniformly strongly suggests that its affinity for different nascent polypeptides varies considerably and corroborate the conclusion that it does not bind well to signal peptides (18,21).…”

“…In biochemical assays, TF can be cross-linked to virtually all nascent secretory and cytoplasmic proteins (18,19) and exhibits ATP-independent chaperone-like activities (20). Unlike typical molecular chaperones, however, TF does not appear to recognize exposed hydrophobic surfaces (21). TF is also homologous to FK506-binding proteins and displays a peptidyl-prolyl-cis-isomerase activity (19,22) of unknown significance.…”

Firefly luciferase mutants as sensors of proteome stress

“…TF is a 48 kDa protein that binds to ribosomes in a 1:1 stoichiometry and interacts with nascent chains in an ATP independent manner (Hesterkamp et al, 1996). Recognition of ribosome bound polypeptide by TF is mediated by short sequences enriched in hydrophobic (particularly aromatic) residues (Patzelt et al, 2001). …”

Interplay between trigger factor and other protein biogenesis factors on the ribosome

“…2; Table 1). TF bound very strongly (K d ¼ 2.5 nM) when translating ribosomes displayed the nascent chain of proOmpA, which contains a TF-binding motif consisting of an extended hydrophobic patch with flanking positive charges 6 . Furthermore, TF bound to HemK75-RNCs rather strongly, consistent with the presence of a similar TF-binding motif in nascent HemK.…”

“…TF is present in the cell in excess over ribosomes and potentially interacts with many nascent proteins 4,5 , with a preference for hydrophobic stretches flanked by positively charged amino acids 6 . Structural and mechanistic aspects of TF function have been reviewed recently 4,7,8 .…”