Functional Dissection of Escherichia coli Trigger Factor: Unraveling the Function of Individual Domains

Abstract

In Escherichia coli, the ribosome-associated chaperone Trigger Factor (TF) promotes the folding of newly synthesized cytosolic proteins. TF is composed of three domains: an N-terminal domain (N), which mediates ribosome binding; a central domain (P), which has peptidyl-prolyl cis/trans isomerase activity and is involved in substrate binding in vitro; and a C-terminal domain (C) with unknown function. We investigated the contributions of individual domains (N, P, and C) or domain combinations (NP, PC, and NC) to the chaperone activity of TF in vivo and in vitro. All fragments comprising the N domain (N, NP, NC) complemented the synthetic lethality of ⌬tig ⌬dnaK in cells lacking TF and DnaK, prevented protein aggregation in these cells, and cross-linked to nascent polypeptides in vitro. However, ⌬tig⌬dnaK cells expressing the N domain alone grew more slowly and showed less viability than ⌬tig⌬dnaK cells synthesizing either NP, NC, or full-length TF, indicating beneficial contributions of the P and C domains to TF's chaperone activity. In an in vitro system with purified components, none of the TF fragments assisted the refolding of denatured D-glyceraldehyde-3-phosphate dehydrogenase in a manner comparable to that of wild-type TF, suggesting that the observed chaperone activity of TF fragments in vivo is dependent on their localization at the ribosome. These results indicate that the N domain, in addition to its function to promote binding to the ribosome, has a chaperone activity per se and is sufficient to substitute for TF in vivo.

Structure of trigger factor binding domain in biologically homologous complex with eubacterial ribosome reveals its chaperone action

“…Consistently, when not associated with the ribosome, TFa does not display specific peptide binding in solution (26) and does not assist the refolding of denatured proteins (24). We therefore conclude that the observed TFa conformational change, which occurs upon association with the large ribosomal subunit and results in the exposure of a hydrophobic pocket, confers chaperone activity on TFa.…”

“…It was shown that TF can be crosslinked to an emerging nascent chain when bound to the ribosome (24). Furthermore, TF is known to delay the folding and͞or misfolding of nascent chains by recognizing hydrophobic regions in nascent polypeptides (25).…”

Regulation by a chaperone improves substrate selectivity during cotranslational protein targeting

“…The E. coli Ffh, FtsY, 4.5S RNA, and TF were expressed and purified using established protocols (76,77). Single cysteine mutations were introduced via Quikchange mutagenesis (Stratagene).…”

Molecular guardians for newborn proteins: ribosome-associated chaperones and their role in protein folding

“…The second TF domain (aa 149-245) carries a catalytic activity as a peptidyl-prolyl cis/trans isomerase (PPIase) and has homology to the FKBP (FK506 binding protein) type of PPIases. The contributions of this domain to TF's function in de novo folding of proteins is puzzling since mutations in this domain, either point mutations diminishing the catalytic PPIase activity or deletion of the entire domain, still display chaperone activity in vivo comparable to wild type TF [71,[77][78][79]. The C-terminal domain (aa 246-432), which constitutes nearly half of the TF protein, reveals no homology on the amino acid level to any other protein [80].…”

“…The C-terminal domain (aa 246-432), which constitutes nearly half of the TF protein, reveals no homology on the amino acid level to any other protein [80]. Deletion of this domain severely decreases TF chaperone activity in vitro and in vivo [71,77,78], indicating that this domain itself participates in substrate binding. Very recently, the crystal structure of E. coli TF [81][82][83][84], as well as that of an N-terminal TF fragment bound to Haloarcula marismortui 50S, were solved [81].…”