Comparative mutational analysis of peptidyl prolyl <i>cis/trans</i> isomerases: active sites of <i>Escherichia coli</i> trigger factor and human FKBP12

103

The ribosome-associated Trigger Factor (TF) cooperates with the DnaK system to assist the folding of newly synthesized polypeptides in Escherichia coli. TF unifies two functions in one to promote proper protein folding in vitro. First, as a chaperone it binds to unfolded protein substrates, thereby preventing aggregation and supporting productive folding. Second, TF catalyzes the cis/trans isomerization of peptidyl-prolyl bonds, which can be a rate-limiting step in protein folding. Here, we investigated whether the peptidyl-prolyl cis/trans isomerase (PPIase) function is essential for the folding activity of TF in vitro and in vivo by separating these two TF activities through site-directed mutagenesis of the PPIase catalytic center. Of the four different TF variants carrying point mutations in the PPIase domain, only the exchange of the conserved residue Phe-198 to Ala (TF F198A) abolished the PPIase activity of TF toward both a tetrapeptide and the model protein substrate RNase T1 in vitro. In contrast, all other activities of TF F198A tested were comparable with wild type TF. TF F198A retained a similar binding specificity toward membrane-bound peptides, assisted the refolding of denatured D-glyceraldehyde-3-phosphate dehydrogenase in vitro, and associated with nascent polypeptides in an in vitro transcription/translation system. Importantly, expression of the TF F198A encoding gene complemented the synthetic lethality of ⌬tig⌬dnaK cells and prevented global protein misfolding at temperatures between 20 and 34°C in these cells. We conclude that the PPIase activity is not required for the function of TF in folding of newly synthesized proteins.Trigger Factor (TF) 1 was first discovered in Escherichia coli as a protein that triggered the translocation of the precursor of pro-OmpA into membrane vesicles (1). Although this activity indicated a secretion-specific chaperone function for TF, E. coli cells depleted for TF were subsequently shown to lack any secretion defect (2). Later, TF was rediscovered as a ribosomeassociated chaperone and peptidyl-prolyl cis/trans isomerase (PPIase) (3, 4).TF is located on the large ribosomal subunit near the peptide exit channel and binds to virtually all nascent polypeptides (4 -6). It was therefore proposed that TF is the first chaperone that interacts with nascent chains and assists cotranslational protein folding. Genetic evidence indicates that TF cooperates with the DnaK system to ensure proper folding of cytosolic proteins. E. coli cells deleted for the TF-encoding tig gene show no growth defects at temperatures between 15 and 42°C, and mutants lacking the dnaK gene are viable between 20°C and 37°C. However, the combined deletion of the tig and dnaK genes causes aggregation of at least 340 species of newly synthesized cytosolic proteins and synthetic lethality at 37°C (7-9). In vitro TF chaperone activity prevents aggregation and promotes refolding of denatured rabbit D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (10).In contrast to DnaK, TF, in addition to its chape...

Section: Discussionsupporting

confidence: 93%

Section: Resultssupporting

confidence: 76%

See 1 more Smart Citation

Trigger Factor Peptidyl-prolyl cis/trans Isomerase Activity Is Not Essential for the Folding of Cytosolic Proteins in Escherichia coli

Krämer¹,

Patzelt²,

Rauch

et al. 2004

103

“…We then performed pulse-chase studies to examine the effect of TF overproduction on protein export in more detail. In these experiments HDB38 (HDB37 tigϪ) was transformed with pJH42 or a nearly identical plasmid (pHL22) encoding a TF mutant (F233Y) that has almost no peptidylprolyl-cis-isomerase activity (40). OmpA was exported very slowly over a 10-min period in cells that overproduced wildtype TF (Fig.…”

Section: Fig 2 Disruption Of Tig Enhances Bla Export In Srp-deficiementioning

confidence: 99%

Trigger Factor Retards Protein Export in Escherichia coli

Lee

Bernstein

2002

Trigger factor (TF) is a ribosome-associated protein that interacts with a wide variety of nascent polypeptides in Escherichia coli. Previous studies have indicated that TF cooperates with DnaK to facilitate protein folding, but the basis of this cooperation is unclear. In this study we monitored protein export in E. coli that lack or overproduce TF to obtain further insights into its function. Whereas inactivation of genes encoding most molecular chaperones (including dnaK) impairs protein export, inactivation of the TF gene accelerated protein export and suppressed the need for targeting factors to maintain the translocation competence of presecretory proteins. Furthermore, overproduction of TF (but not DnaK) markedly retarded protein export. Manipulation of TF levels produced similar effects on the export of a cytosolic enzyme fused to a signal peptide. The data strongly suggest that TF has a unique ability to sequester nascent polypeptides for a relatively prolonged period. Based on our results, we propose that TF and DnaK promote protein folding by distinct (but complementary) mechanisms.Protein biogenesis in Escherichia coli is aided by a diverse set of specialized factors that interact with nascent polypeptides chains. Several of these factors ("molecular chaperones") promote the folding of cytoplasmic proteins by binding promiscuously to exposed hydrophobic surfaces and preventing aggregation (reviewed in Ref.

“…Peptides were characterized by electrospray ionization-mass spectrometry. Recombinant human Cyp18, human FKBP12, and human Pin1 were prepared as described elsewhere (27,28). The catalytic subunit of bovine heart cAMP-dependent protein kinase (protein kinase A) was obtained from Roche (Mannheim, Germany).…”

Section: Methodsmentioning

confidence: 99%

Reversible Inhibition of Calcineurin by the Polyphenolic Aldehyde Gossypol

Baumgrass

Weiwad

Erdmann

et al. 2001

Self Cite

The reversible inhibition of calcineurin (CaN), which is the only Ca 2؉ /calmodulin-dependent protein Ser/Thr phosphatase, is thought to be a key functional event for most cyclosporin A (CsA)-and tacrolimus (FK506)-mediated biological effects. In addition to CaN inhibition, however, CsA and FK506 have multiple biochemical effects because of their action in a gain-of-function model that requires prior binding to immunophilic proteins. We screened a small molecule library for direct inhibitors of CaN using CaN-mediated dephosphorylation of 33 P-labeled 19-residue phosphopeptide substrate (RII phosphopeptide) as an assay and found the polyphenolic aldehyde gossypol to be a novel CaN inhibitor. Unlike CsA and FK506, gossypol does not require a matchmaker protein for reversible CaN inhibition with an IC 50 value of 15 M. Gossypolone, a gossypol analog, showed improved inhibition of both RII phosphopeptide and p-nitrophenyl phosphate dephosphorylation with an IC 50 of 9 and 6 M, respectively. In contrast, apogossypol hexaacetate was inactive. Gossypol acts noncompetitively, interfering with the binding site for the cyclophilin 18⅐CsA complex in CaN. In contrast to CsA and FK506, gossypol does not inactivate the peptidyl-prolyl-cis/ trans-isomerase activity of immunophilins. Similar to CsA and FK506, T cell receptor signaling induced by phorbol 12-myristate 13-acetate/ionomycin is inhibited by gossypol in a dose-dependent manner, demonstrated by the inhibition of nuclear factor of activated T cell (NFAT) c1 translocation from the cytosol into the nucleus and suppression of NFAT-luciferase reporter gene activity.In vivo inhibition by membrane-penetrable low molecular mass compounds plays an important role in evaluating the biological function of enzymes involved in protein phosphorylation/dephosphorylation. The functional discrimination with specific inhibitors of the four major protein Ser/Thr phosphatases, protein phosphatase 1 (PP1), 1 protein phosphatase 2A (PP2A), protein phosphatase 2B (PP2B, calcineurin, CaN), and protein phosphatase 2C (PP2C), has proven to be successful (1). Okadaic acid and microcystin are strong inhibitors of PP1 and PP2A, but they exhibit poor inhibition of the Ca 2ϩ /calmodulinregulated CaN and PP2C. It was difficult to identify CaN function in cell signaling until the membrane-penetrable cyclopeptide cyclosporin A (CsA) and the peptidomacrolide FK506 were found to inhibit CaN specifically under certain conditions (2). Currently, most reports about CaN involvement in cellular processes are based on CsA and FK506 susceptibility of the appropriate bioassays. CaN inhibition by these drugs is characterized by their prior binding to the 18-kDa cyclophilin (Cyp18) and 12-kDa FK506-binding protein (FKBP12), respectively, indicating a gain-of-function mechanism (3). These mammalian prototypes of two different families of the enzyme class of peptidyl-prolyl-cis/trans-isomerases (EC 5.2.1.8; PPIases) function as molecular matchmakers because the drugs cannot bind to CaN on their own. The prototypic P...