Prolyl isomerases show low sequence specificity toward the residue following the proline

Prolyl isomerases catalyze the cis/trans isomerization of peptide bonds preceding proline. Previously, we had determined the specificity toward the residue before the proline for cyclophilin-, FKBP-, and parvulin-type prolyl isomerases by using proline-containing oligopeptides and refolding proteins as model substrates. Here, we report the specificities of members of these three prolyl isomerase families for the residue following the proline, again in short peptide and in refolding protein chains. Human cyclophilin 18 and parvulin 10 from Escherichia coli show high activity, but low specificity, with respect to the residue following the proline. Human FKBP12 prefers hydrophobic residues at this position in the peptide assays and shows a very low activity in the protein folding assays. This activity was strongly improved, and the sequence specificity was virtually eliminated after the insertion of a chaperone domain into the prolyl isomerase domain of human FKBP12.     

Insertion of a chaperone domain converts FKBP12 into a powerful catalyst of protein folding

The catalytic activity of human FKBP12 as a prolyl isomerase is high towards short peptides, but very low in proline-limited protein folding reactions. In contrast, the SlyD proteins, which are members of the FKBP family, are highly active as folding enzymes. They contain an extra "insert-in-flap" or IF domain near the prolyl isomerase active site. The excision of this domain did not affect the prolyl isomerase activity of SlyD from Escherichia coli towards short peptide substrates but abolished its catalytic activity in proline-limited protein folding reactions. The reciprocal insertion of the IF domain of SlyD into human FKBP12 increased its folding activity 200-fold and generated a folding catalyst that is more active than SlyD itself. The IF domain binds to refolding protein chains and thus functions as a chaperone module. A prolyl isomerase catalytic site and a separate chaperone site with an adapted affinity for refolding protein chains are the key elements for a productive coupling between the catalysis of prolyl isomerization and conformational folding in the enzymatic mechanisms of SlyD and other prolyl isomerases, such as trigger factor and FkpA.     

TrbB from conjugative plasmid F is a structurally distinct disulfide isomerase that requires DsbD for redox state maintenance

TrbB, a periplasmic protein encoded by the conjugative plasmid F, has a predicted thioredoxin-like fold and possesses a C-X-X-C redox active site motif. TrbB may function in the conjugative process by serving as a disulfide bond isomerase, facilitating proper folding of a subset of F-plasmid-encoded proteins in the periplasm. Previous studies have demonstrated that a ΔtrbB F plasmid in Escherichia coli lacking DsbC(E.coli), its native disulfide bond isomerase, experiences a 10-fold decrease in mating efficiency but have not provided direct evidence for disulfide bond isomerase activity. Here we demonstrate that trbB can partially restore transfer of a variant of the distantly related R27 plasmid when both chromosomal and plasmid genes encoding disulfide bond isomerases have been disrupted. In addition, we show that TrbB displays both disulfide bond isomerase and reductase activities on substrates not involved in the conjugative process. Unlike canonical members of the disulfide bond isomerase family, secondary structure predictions suggest that TrbB lacks both an N-terminal dimerization domain and an α-helical domain found in other disulfide bond isomerases. Phylogenetic analyses support the conclusion that TrbB belongs to a unique family of plasmid-based disulfide isomerases. Interestingly, although TrbB diverges structurally from other disulfide bond isomerases, we show that like those isomerases, TrbB relies on DsbD from E. coli for maintenance of its C-X-X-C redox active site motif.     

ERp57 and PDI: multifunctional protein disulfide isomerases with similar domain architectures but differing substrate-partner associations.

Secretory proteins become folded and acquire stabilizing disulfide bonds in the endoplasmic reticulum (ER). Correct disulfide bond formation is a key step in ER quality control (ERQC). Proteins with incorrect disulfide bonds are recognized by the quality control machinery and are retrotranslocated into the cytosol where they are degraded by the proteasome. The mammalian ER contains 17 disulfide isomerases and at least one of them, ERp57, works in conjunction with the ER lectin-like chaperones calnexin and calreticulin. The targeting of ERp57 to calnexin-calreticulin is mediated by its noncatalytic b' domain, and analogous domains in other disulfide isomerases likely determine their substrate and partner preferences. This review discusses some explanations for the multiplicity of disulfide isomerases and highlights structural differences in the b' domains of PDI and ERp57 as an example of how noncatalytic domains define specialized roles in oxidative folding.     

Structure of the Non-Catalytic Domain of the Protein Disulfide Isomerase-Related Protein (PDIR) Reveals Function in Protein Binding

Protein disulfide isomerases comprise a large family of enzymes responsible for catalyzing the proper oxidation and folding of newly synthesized proteins in the endoplasmic reticulum (ER). Protein disulfide isomerase-related (PDIR) protein (also known as PDIA5) is a specialized member that participates in the folding of α₁-antitrypsin and N-linked glycoproteins. Here, the crystal structure of the non-catalytic domain of PDIR was determined to 1.5 Å resolution. The structure adopts a thioredoxin-like fold stabilized by a structural disulfide bridge with a positively charged binding surface for interactions with the ER chaperones, calreticulin and ERp72. Crystal contacts between molecules potentially mimic the interactions of PDIR with misfolded substrate proteins. The results suggest that the non-catalytic domain of PDIR plays a key role in the recognition of protein partners and substrates.     

Cyclophilin 20 is involved in mitochondrial protein folding in cooperation with molecular chaperones Hsp70 and Hsp60.

We studied the role of mitochondrial cyclophilin 20 (CyP20), a peptidyl-prolyl cis-trans isomerase, in preprotein translocation across the mitochondrial membranes and protein folding inside the organelle. The inhibitory drug cyclosporin A did not impair membrane translocation of preproteins, but it delayed the folding of an imported protein in wild-type mitochondria. Similarly, Neurospora crassa mitochondria lacking CyP20 efficiently imported preproteins into the matrix, but folding of an imported protein was significantly delayed, indicating that CyP20 is involved in protein folding in the matrix. The slow folding in the mutant mitochondria was not inhibited by cyclosporin A. Folding intermediates of precursor molecules reversibly accumulated at the molecular chaperones Hsp70 and Hsp60 in the matrix. We conclude that CyP20 is a component of the mitochondrial protein folding machinery and that it cooperates with Hsp70 and Hsp60. It is speculated that peptidyl-prolyl cis-trans isomerases in other cellular compartments may similarly promote protein folding in cooperation with chaperone proteins.     

The cyclophilin homolog NinaA functions as a chaperone, forming a stable complex in vivo with its protein target rhodopsin.

In Drosophila, biogenesis of the major rhodopsin, Rh1, is dependent on the presence of a photoreceptor cell-specific cyclophilin, NinaA. In ninaA mutants, Rh1 is retained within the endoplasmic reticulum and rhodopsin levels are reduced > 100-fold. Cyclophilins have been shown to be peptidyl-prolyl cis-trans isomerases and have been implicated in catalyzing protein folding. We have generated transgenic animals expressing different functional rhodopsins containing a histidine tag. We isolated these molecules from wild-type and ninaA mutant retinas, and have demonstrated that in vivo NinaA forms a specific stable protein complex with its target Rh1. We also expressed ninaA under an inducible promoter and showed that NinaA is required quantitatively for Rh1 biogenesis. These results provide the first evidence for a biologically relevant physical interaction between a cyclophilin and its cellular target, and suggest that the normal cellular role of this class of cyclophilins is to function as chaperones, possibly escorting their protein substrates through the secretory pathway.     

Central functions of the lumenal and peripheral thylakoid proteome of Arabidopsis determined by experimentation and genome-wide prediction

Experimental proteome analysis was combined with a genome-wide prediction screen to characterize the protein content of the thylakoid lumen of Arabidopsis chloroplasts. Soluble thylakoid proteins were separated by two-dimensional electrophoresis and identified by mass spectrometry. The identities of 81 proteins were established, and N termini were sequenced to validate localization prediction. Gene annotation of the identified proteins was corrected by experimental data, and an interesting case of alternative splicing was discovered. Expression of a surprising number of paralogs was detected. Expression of five isomerases of different classes suggests strong (un)folding activity in the thylakoid lumen. These isomerases possibly are connected to a network of peripheral and lumenal proteins involved in antioxidative response, including peroxiredoxins, m-type thioredoxins, and a lumenal ascorbate peroxidase. Characteristics of the experimentally identified lumenal proteins and their orthologs were used for a genome-wide prediction of the lumenal proteome. Lumenal proteins with a typical twin-arginine translocation motif were predicted with good accuracy and sensitivity and included additional isomerases and proteases. Thus, prime functions of the lumenal proteome include assistance in the folding and proteolysis of thylakoid proteins as well as protection against oxidative stress. Many of the predicted lumenal proteins must be present at concentrations at least 10,000-fold lower than proteins of the photosynthetic apparatus.     

Periplasmic peptidyl prolyl cis-trans isomerases are not essential for viability, but SurA is required for pilus biogenesis in Escherichia coli

In Escherichia coli, FkpA, PpiA, PpiD, and SurA are the four known periplasmic cis-trans prolyl isomerases. These isomerases facilitate proper protein folding by increasing the rate of transition of proline residues between the cis and trans states. Genetic inactivation of all four periplasmic isomerases resulted in a viable strain that exhibited a decreased growth rate and increased susceptibility to certain antibiotics. Levels of the outer membrane proteins LamB and OmpA in the quadruple mutant were indistinguishable from those in the surA single mutant. In addition, expression of P and type 1 pili (adhesive organelles produced by uropathogenic strains of E. coli and assembled by the chaperone/usher pathway) were severely diminished in the absence of the four periplasmic isomerases. Maturation of the usher was significantly impaired in the outer membranes of strains devoid of all four periplasmic isomerases, resulting in a defect in pilus assembly. Moreover, this defect in pilus assembly and usher stability could be attributed to the absence of SurA. The data presented here suggest that the four periplasmic isomerases are not essential for growth under laboratory conditions but may have significant roles in survival in environmental and pathogenic niches, as indicated by the effect on pilus production.     

Retarded protein folding of the human Z-type α1-antitrypsin variant is suppressed by Cpr2p

The human Z-type α₁-antitrypsin variant has a strong tendency to accumulate folding intermediates due to extremely slow protein folding within the endoplasmic reticulum (ER) of hepatocytes. Human α₁-antitrypsin has 17 peptidyl-prolyl bonds per molecule; thus, the effect of peptidyl-prolyl isomerases on Z-type α₁-antitrypsin protein folding was analyzed in this study. The protein level of Cpr2p, a yeast ER peptidyl-prolyl isomerase, increased more than two-fold in Z-type α₁-antitrypsin-expressing yeast cells compared to that in wild-type α₁-antitrypsin-expressing cells. When CPR2 was deleted from the yeast genome, the cytotoxicity of Z-type α₁-antitrypsin increased significantly. The interaction between Z-type α₁-antitrypsin and Cpr2p was confirmed by co-immunoprecipitation. In vitro folding assays showed that Cpr2p facilitated Z-type α₁-antitrypsin folding into the native state. Furthermore, Cpr2p overexpression significantly increased the extracellular secretion of Z-type α₁-antitrypsin. Our results indicate that ER peptidyl-prolyl isomerases may rescue Z-type α₁-antitrypsin molecules from retarded folding and eventually relieve clinical symptoms caused by this pathological α₁-antitrypsin.     

SurA assists the folding of Escherichia coli outer membrane proteins.

Many proteins require enzymatic assistance in order to achieve a functional conformation. One rate-limiting step in protein folding is the cis-trans isomerization of prolyl residues, a reaction catalyzed by prolyl isomerases. SurA, a periplasmic protein of Escherichia coli, has sequence similarity with the prolyl isomerase parvulin. We tested whether SurA was involved in folding periplasmic and outer membrane proteins by using trypsin sensitivity as an assay for protein conformation. We determined that the efficient folding of three outer membrane proteins (OmpA, OmpF, and LamB) requires SurA in vivo, while the folding of four periplasmic proteins was independent of SurA. We conclude that SurA assists in the folding of certain secreted proteins.     

Interaction of the periplasmic peptidylprolyl cis-trans isomerase SurA with model peptides. The N-terminal region of SurA id essential and sufficient for peptide binding

One of the rate-limiting steps in protein folding has been shown to be the cis-trans isomerization of proline residues, which is catalyzed by a range of peptidylprolyl cis-trans isomerases. To characterize the interaction between model peptides and the periplasmic peptidylprolyl cis-trans isomerase SurA from E. coli, we employed a chemical cross-linking strategy that has been used previously to elucidate the interaction of substrates with other folding catalysts. The interaction between purified SurA and model peptides was significant in that it showed saturation and was abolished by denaturation of SurA; however the interaction was independent of the presence of proline residues in the model peptides. From results obtained by limited proteolysis we conclude that an N-terminal fragment of SurA, comprising 150 amino acids that do not contain the active sites involved in the peptidylprolyl cis-trans isomerization, is essential for the binding of peptides by SurA. This was confirmed by probing the interaction of the model peptide with the recombinant N-terminal fragment, expressed in Escherichia coli. Hence we propose that, similar to protein disulfide isomerase and other folding catalysts, SurA exhibits a modular architecture composed of a substrate binding domain and distinct catalytically active domains.     

An interaction map of endoplasmic reticulum chaperones and foldases

Chaperones and foldases in the endoplasmic reticulum (ER) ensure correct protein folding. Extensive protein-protein interaction maps have defined the organization and function of many cellular complexes, but ER complexes are under-represented. Consequently, chaperone and foldase networks in the ER are largely uncharacterized. Using complementary ER-specific methods, we have mapped interactions between ER-lumenal chaperones and foldases and describe their organization in multiprotein complexes. We identify new functional chaperone modules, including interactions between protein-disulfide isomerases and peptidyl-prolyl cis-trans-isomerases. We have examined in detail a novel ERp72-cyclophilin B complex that enhances the rate of folding of immunoglobulin G. Deletion analysis and NMR reveal a conserved surface of cyclophilin B that interacts with polyacidic stretches of ERp72 and GRp94. Mutagenesis within this highly charged surface region abrogates interactions with its chaperone partners and reveals a new mechanism of ER protein-protein interaction. This ability of cyclophilin B to interact with different partners using the same molecular surface suggests that ER-chaperone/foldase partnerships may switch depending on the needs of different substrates, illustrating the flexibility of multichaperone complexes of the ER folding machinery.     

The periplasmic peptidyl prolyl cis-trans isomerases PpiD and SurA have partially overlapping substrate specificities

One of the rate-limiting steps in protein folding has been shown to be the cis-trans isomerization of proline residues, catalysed by a range of peptidyl prolyl cis-trans isomerases (PPIases). In the periplasmic space of Escherichia coli and other Gram-negative bacteria, two PPIases, SurA and PpiD, have been identified, which show high sequence similarity to the catalytic domain of the small PPIase parvulin. This observation raises a question regarding the biological significance of two apparently similar enzymes present in the same cellular compartment: do they interact with different substrates or do they catalyse different reactions? The substrate-binding motif of PpiD has not been characterized so far, and no biochemical data were available on how this folding catalyst recognizes and interacts with substrates. To characterize the interaction between model peptides and the periplasmic PPIase PpiD from E. coli, we employed a chemical crosslinking strategy that has been used previously to elucidate the interaction of substrates with SurA. We found that PpiD interacted with a range of model peptides independently of whether they contained proline residues or not. We further demonstrate here that PpiD and SurA interact with similar model peptides, and therefore must have partially overlapping substrate specificities. However, the binding motif of PpiD appears to be less specific than that of SurA, indicating that the two PPIases might interact with different substrates. We therefore propose that, although PpiD and SurA have partially overlapping substrate specificities, they fulfil different functions in the cell.     

Oxidative protein folding in the plant endoplasmic reticulum

For most of the proteins synthesized in the endoplasmic reticulum (ER), disulfide bond formation accompanies protein folding in a process called oxidative folding. Oxidative folding is catalyzed by a number of enzymes, including the family of protein disulfide isomerases (PDIs), as well as other proteins that supply oxidizing equivalents to PDI family proteins, like ER oxidoreductin 1 (Ero1). Oxidative protein folding in the ER is a basic vital function, and understanding its molecular mechanism is critical for the application of plants as protein production tools. Here, I review the recent research and progress related to the enzymes involved in oxidative folding in the plant ER. Firstly, nine groups of plant PDI family proteins are introduced. Next, the enzymatic properties of plant Ero1 are described. Finally, the cooperative folding by multiple PDI family proteins and Ero1 is described.     

De novo design and evolution of artificial disulfide isomerase enzymes analogous to the bacterial DsbC

The Escherichia coli disulfide isomerase, DsbC is a V-shaped homodimer with each monomer comprising a dimerization region that forms part of a putative peptide-binding pocket and a thioredoxin catalytic domain. Disulfide isomerases from prokaryotes and eukaryotes exhibit little sequence homology but display very similar structural organization with two thioredoxin domains facing each other on top of the dimerization/peptide-binding region. To aid the understanding of the mechanistic significance of thioredoxin domain dimerization and of the peptide-binding cleft of DsbC, we constructed a series of protein chimeras comprising unrelated protein dimerization domains fused to thioredoxin superfamily enzymes. Chimeras consisting of the dimerization domain and the alpha-helical linker of the bacterial proline cis/trans isomerase FkpA and the periplasmic oxidase DsbA gave rise to enzymes that catalyzed the folding of multidisulfide substrate proteins in vivo with comparable efficiency to E. coli DsbC. In addition, expression of FkpA-DsbAs conferred modest resistance to CuCl2, a phenotype that depends on disulfide bond isomerization. Selection for resistance to elevated CuCl2 concentrations led to the isolation of FkpA-DsbA mutants containing a single amino acid substitution that changed the active site of the DsbA domain from CPHC into CPYC, increasing the similarity to the DsbC active site (CGYC). Unlike DsbC, which is resistant to oxidation by DsbB-DsbA and does not normally catalyze disulfide bond formation under physiological conditions, the FkpA-DsbA chimeras functioned both as oxidases and isomerases. The engineering of these efficient artificial isomerases delineates the key features of catalysis of disulfide bond isomerization and enhances our understanding of its evolution.     

The prolyl isomerase domain of PpiD from Escherichia coli shows a parvulin fold but is devoid of catalytic activity

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 parvulin‐like 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.     

Catalysis of protein folding by cyclophilins from different species.

Cyclophilins are a class of ubiquitous proteins with yet unknown function. They were originally discovered as the major binding proteins for the immunosuppressant cyclosporin A. The only known catalytic function of these proteins in vitro is the cis/trans isomerization of Xaa-Pro bonds in oligopeptides. This became clear after the discovery that bovine cyclophilin is identical with porcine prolyl isomerase. This enzyme accelerates slow, proline-limited steps in the refolding of several proteins. Here we demonstrate that the cyclophilins from man, pig, Neurospora crassa, Saccharomyces cerevisiae, and Escherichia coli are all active as prolyl isomerases and as catalysts of protein folding. This evolutionary conservation suggests that catalysis of prolyl peptide bond isomerization may be an important function of the cyclophilins. It could be related with de novo protein folding or be involved in regulatory processes. Catalysis of folding is very efficient in the presence of the high cellular concentrations of prolyl isomerase.     

Novel protein-disulfide isomerases from the early-diverging protist Giardia lamblia.

Protein-disulfide isomerase is essential for formation and reshuffling of disulfide bonds during nascent protein folding in the endoplasmic reticulum. The two thioredoxin-like active sites catalyze a variety of thiol-disulfide exchange reactions. We have characterized three novel protein-disulfide isomerases from the primitive eukaryote Giardia lamblia. Unlike other protein-disulfide isomerases, the giardial enzymes have only one active site. The active-site sequence motif in the giardial proteins (CGHC) is characteristic of eukaryotic protein-disulfide isomerases, and not other members of the thioredoxin superfamily that have one active site, such as thioredoxin and Dsb proteins from Gram-negative bacteria. The three giardial proteins have very different amino acid sequences and molecular masses (26, 50, and 13 kDa). All three enzymes were capable of rearranging disulfide bonds, and giardial protein-disulfide isomerase-2 also displayed oxidant and reductant activities. Surprisingly, the three giardial proteins also had Ca(2+)-dependent transglutaminase activity. This is the first report of protein-disulfide isomerases with a single active site that have diverse roles in protein cross-linking. This study may provide clues to the evolution of key functions of the endoplasmic reticulum in eukaryotic cells, protein disulfide formation, and isomerization.     

Generation of a Highly Active Folding Enzyme by Combining a Parvulin-Type Prolyl Isomerase from SurA with an Unrelated Chaperone Domain

Parvulins are small prolyl isomerases and serve as catalytic domains of folding enzymes. SurA (survival protein A) from the periplasm of Escherichia coli consists of an inactive (Par1) and an active (Par2) parvulin domain as well as a chaperone domain. In the absence of the chaperone domain, the folding activity of Par2 is virtually abolished. We created a chimeric protein by inserting the chaperone domain of SlyD, an unrelated folding enzyme from the FKBP family, into a loop of the isolated Par2 domain of SurA. This increased its folding activity 450-fold to a value higher than the activity of SurA, in which Par2 is linked with its natural chaperone domain. In the presence of both the natural and the foreign chaperone domain, the folding activity of Par2 was 1500-fold increased. Related and unrelated chaperone domains thus are similarly efficient in enhancing the folding activity of the prolyl isomerase Par2. A sequence analysis of various chaperone domains suggests that clusters of exposed methionine residues in mobile chain regions might be important for a generic interaction with unfolded protein chains. This binding is highly dynamic to allow frequent transfer of folding protein chains between chaperone and catalytic domains.