High enzymatic activity and chaperone function are mechanistically related features of the dimeric E. coli peptidyl-prolyl-isomerase FkpA

Ramm, Kathrin; Plückthun, Andreas

doi:10.1006/jmbi.2001.4747

Cited by 73 publications

(90 citation statements)

References 49 publications

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“…TF is different, because both activities involve the same substrate binding pocket in the central PPIase domain. A similar finding was only observed for the periplasmic chaperone and PPIase FkpA from E. coli (15).…”

supporting

confidence: 82%

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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

Journal of Biological Chemistry

105

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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...

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supporting

confidence: 82%

“…Two findings support this hypothesis. (i) Members of the FKBP family show PPIase-independent chaperone activity (15). (ii) There is no ribosome-associated PPIase known in eukaryotic cells.…”

Section: Discussionmentioning

confidence: 99%

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

Journal of Biological Chemistry

105

View full text Add to dashboard Cite

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“…Mutants show attenuated virulence and reduced adhesion, and antibodies raised against the Trypanosoma enzyme reduce infectivity. The same family of enzymes has been reported as having chaperone activity facilitating preferentially maturation of outer membrane proteins and thus, proline racemases may play a dual role (71). Although these enzymes do not harbor any signal/anchoring motifs, they have been reported to be present on the surface and hence immuno-accessible (80).…”

Section: Resultsmentioning

confidence: 96%

Genome-Based Bioinformatic Selection of ChromosomalBacillus anthracisPutative Vaccine Candidates Coupled with Proteomic Identification of Surface-Associated Antigens

et al. 2003

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“…[2][3][4] Several of these proteins, in particular FkpA, SurA, and PpiD, combine prolyl isomerase domains with chaperone domains. [5][6][7][8] The SurA protein participates in the maturation of outer membrane proteins. It consists of an N-terminal chaperone domain that is followed by two prolyl isomerase domains of the parvulin type.…”

Section: Introductionmentioning

confidence: 99%

The prolyl isomerase domain of PpiD fromEscherichia colishows a parvulin fold but is devoid of catalytic activity

et al. 2009

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PpiD is a periplasmic folding helper protein of Escherichia coli. It consists of an N-terminal helix that anchors PpiD in the inner membrane near the SecYEG translocon, followed by three periplasmic domains. The second domain (residues 264-357) shows homology to parvulinlike prolyl isomerases. This domain is a well folded, stable protein and follows a simple two-state folding mechanism. In its solution structure, as determined by NMR spectroscopy, it resembles most closely the first parvulin domain of the SurA protein, which resides in the periplasm of E. coli as well. A previously reported prolyl isomerase activity of PpiD could not be reproduced when using improved protease-free peptide assays or assays with refolding proteins as substrates. The parvulin domain of PpiD interacts, however, with a proline-containing tetrapeptide, and the binding site, as identified by NMR resonance shift analysis, colocalized with the catalytic sites of other parvulins. In its structure, the parvulin domain of PpiD resembles most closely the inactive first parvulin domain of SurA, which is part of the chaperone unit of this protein and presumably involved in substrate recognition.Keywords: parvulin; Pin1; prolyl isomerase; SurA; protein maturation; periplasm; SecYEG Abbreviations: PpiD, periplasmic folding helper protein from Escherichia coli; PpiD*, parvulin-like domain of PpiD (residues 264-357); PPIase, peptidyl-prolyl cis/trans isomerase; CD, circular dichroism; T M , midpoint of a thermal unfolding transition; DH D , vań t Hoff enthalpy of denaturation at T M ; DG D , Gibbs free energy of denaturation; m, cooperativity value of a denaturant-induced equilibrium unfolding transition; Ds, time constant of a folding reaction; k u , microscopic rate constant and m u (¼ qlnk u /q[urea]), kinetic m-value of unfolding; k f , m f , microscopic rate constant and kinetic m-value of refolding; NOESY, nuclear Overhauser enhancement and exchange spectroscopy; HSQC, hetero single quantum coherence; hNOE, 15 N hetero nuclear NOE; HX, hydrogen exchange; NH, amide proton; RCM-T1, reduced and carboxymethylated form of the S54G/P55N double mutant of ribonuclease T 1 ; N2, N2 domain of the gene 3 protein of phage fd.Additional Supporting Information may be found in the online version of this article.

show abstract

High enzymatic activity and chaperone function are mechanistically related features of the dimeric E. coli peptidyl-prolyl-isomerase FkpA

Cited by 73 publications

References 49 publications

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

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

Genome-Based Bioinformatic Selection of ChromosomalBacillus anthracisPutative Vaccine Candidates Coupled with Proteomic Identification of Surface-Associated Antigens

The prolyl isomerase domain of PpiD fromEscherichia colishows a parvulin fold but is devoid of catalytic activity

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