Biochemical characterization of two Azotobacter vinelandii FKBPs and analysis of their interaction with the small subunit of carbamoyl phosphate synthetase

FK-506 binding proteins (FKBPs) belong to the peptidyl-prolyl cis/trans isomerase superfamily (PPIases, EC: 5.2.1.8) which catalyzes the interconversion of peptidyl-prolyl bonds while they can also act on polypeptides, as folding helper enzymes. Here, we biochemically characterize two recombinant FKBPs, AvfkbA1 and AvfkbA2, from the soil nitrogen-fixing bacterium Azotobacter vinelandii and show that both possess PPIase activity while AvfkbA2 possesses chaperone activity as well. Further, we demonstrate their physical interaction with AvcarA, the small subunit of carbamoyl phosphate synthetase. Using RT-qPCR, we show that AvfkbA1 and AvfkbA2 are co-expressed with AvcarA under the same growth conditions. A decrease in AvfkbA1 or AvfkbA2 PPIase activity, in the presence of AvcarA, further confirms each interaction. However, PPIase activity does not seem to be essential for these interactions since PPIase active site mutations of both FKBPs do not abolish the AvcarA binding. The P³⁵⁸ residue of AvcarA, possibly retaining a cis configuration, is critical only for the interaction with AvfkbA1. The presence of either of the two FKBPs did not influence the measured glutamine hydrolyzing activity of AvcarA. Taken together, these data indicate that although the two FKBPs have a common biological substrate they probably have differing physiological roles.     

The Role of SurA PPIase Domains in Preventing Aggregation of the Outer-Membrane Proteins tOmpA and OmpT

SurA is a conserved ATP-independent periplasmic chaperone involved in the biogenesis of outer-membrane proteins (OMPs). Escherichia coli SurA has a core domain and two peptidylprolyl isomerase (PPIase) domains, the role(s) of which remain unresolved. Here we show that while SurA homologues in early proteobacteria typically contain one or no PPIase domains, the presence of two PPIase domains is common in SurA in later proteobacteria, implying an evolutionary advantage for this domain architecture. Bioinformatics analysis of > 350,000 OMP sequences showed that their length, hydrophobicity and aggregation propensity are similar across the proteobacterial classes, ruling out a simple correlation between SurA domain architecture and these properties of OMP sequences. To investigate the role of the PPIase domains in SurA activity, we deleted one or both PPIase domains from E. coli SurA and investigated the ability of the resulting proteins to bind and prevent the aggregation of tOmpA (19 kDa) and OmpT (33 kDa). The results show that wild-type SurA inhibits the aggregation of both OMPs, as do the cytoplasmic OMP chaperones trigger factor and SecB. However, while the ability of SurA to bind and prevent tOmpA aggregation does not depend on its PPIase domains, deletion of even a single PPIase domain ablates the ability of SurA to prevent OmpT aggregation. The results demonstrate that the core domain of SurA endows its generic chaperone ability, while the presence of PPIase domains enhances its chaperone activity for specific OMPs, suggesting one reason for the conservation of multiple PPIase domains in SurA in proteobacteria.     

Functional interaction of Azotobacter vinelandii cytoplasmic cyclophilin with the biotin carboxylase subunit of acetyl-CoA carboxylase

Cyclophilins (E.C. 5.1.2.8) are protein chaperones with peptidyl-prolyl cis/trans isomerase activity (PPIase). In the present study, we demonstrate a physical interaction among AvppiB, encoding the cytoplasmic cyclophilin from the soil nitrogen-fixing bacterium Azotobacter vinelandii, and AvaccC, encoding the biotin carboxylase subunit of acetyl-CoA carboxylase, which catalyzes the committed step in long-chain fatty acid synthesis. A decrease in AvppiB PPIase activity, in the presence of AvaccC, further confirms the interaction. However, PPIase activity seems not to be essential for these interactions since a PPIase active site mutant of cyclophilin does not abolish the AvaccC binding. We further show that the presence of cyclophilin largely influences the measured ATP hydrolyzing activity of AvaccA in a way that is negatively regulated by the PPIase activity. Taken together, our data support a novel role for cyclophilin in regulating biotin carboxylase activity.     

The periplasmic chaperone SurA exploits two features characteristic of integral outer membrane proteins for selective substrate recognition

The Escherichia coli periplasmic chaperone and peptidyl-prolyl isomerase (PPIase) SurA facilitates the maturation of outer membrane porins. Although the PPIase activity exhibited by one of its two parvulin-like domains is dispensable for this function, the chaperone activity residing in the non-PPIase regions of SurA, a sizable N-terminal domain and a short C-terminal tail, is essential. Unlike most cytoplasmic chaperones SurA is selective for particular substrates and recognizes outer membrane porins synthesized in vitro much more efficiently than other proteins. Thus, SurA may be specialized for the maturation of outer membrane proteins. We have characterized the substrate specificity of SurA based on its natural, biologically relevant substrates by screening cellulose-bound peptide libraries representing outer membrane proteins. We show that two features are critical for peptide binding by SurA: specific patterns of aromatic residues and the orientation of their side chains, which are found more frequently in integral outer membrane proteins than in other proteins. For the first time this sufficiently explains the capability of SurA to discriminate between outer membrane protein and non-outer membrane protein folding intermediates. Furthermore, peptide binding by SurA requires neither an active PPIase domain nor the presence of proline, indicating that the observed substrate specificity relates to the chaperone function of SurA. Finally, we show that SurA is capable of associating with the outer membrane. Together, our data support a model in which SurA is specialized to interact with non-native periplasmic outer membrane protein folding intermediates and to assist in their maturation from early to late outer membrane-associated steps.     

Cyclophilins: Proteins in search of function

Cyclophilins constitute a subgroup of large family of proteins called immunophilins, which also include FKBPs and Parvulins. They are remarkably conserved in all genera, highlighting their pivotal role in important cellular processes. Most cyclophilins display PPIase enzymatic activity, multiplicity, diverse cellular locations and active role in protein folding which render them to be included in the class of diverse set of proteins called molecular chaperones. Due to their distinct PPIase function, besides protein disulfide isomerases and protein foldases, cyclophilins have been deemed necessary for in vivo chaperoning activity. Unlike other cellular chaperones, these proteins are specific in their respective targets. Not all cyclophilin proteins possess PPIase activity, indicating a loss of their PPIase activity during the course of evolution and gain of function independent of their PPIase activity. The PPIase function of cyclophilins is also compensated by their functional homologs, like FKBPs. Multiple cyclophilin members in plants like Arabidopsis and rice have been reported to be associated with diverse functions and regulatory pathways through their foldase, scaffolding, chaperoning or other unknown activities. Although many functions of plant cyclophilins were reported or suggested, the physiological relevance and molecular basis of stress-responsive expression of plant cyclophilins is still largely unknown. However, their wide distribution and ubiquitous nature signifies their fundamental importance in plant survival. Several of these members have also been directly linked to multiple stresses. This review attempts to deal with plant cyclophilins with respect to their role in stress response.     

Functional analysis of the two cyclophilin isoforms of <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis>

The nitrogen fixing Sinorhizobium meliloti possesses two genes, ppiA and ppiB, encoding two cyclophilin isoforms which belong to the superfamily of peptidyl prolyl cis/trans isomerases (PPIase, EC: 5.2.1.8). Here, we functionally characterize the two proteins and we demonstrate that both recombinant cyclophilins are able to isomerise the Suc-AAPF-pNA synthetic peptide but neither of them displays chaperone function in the citrate synthase thermal aggregation assay. Furthermore, we observe that the expression of both enzymes increases the viability of E. coli BL21 in the presence of abiotic stress conditions such as increased heat and salt concentration. Our results support and strengthen previous high-throughput studies implicating S. meliloti cyclophilins in various stress conditions.     

PPIase domain of trigger factor acts as auxiliary chaperone site to assist the folding of protein substrates bound to the crevice of trigger factor

Trigger factor (TF) is the first chaperone encountered by nascent chains in bacteria, which consists of two modules: peptidyl-prolyl-cis/trans-isomerase (PPIase) domain and a crevice built by both N- and C-terminal domains. While the crevice is suggested to provide a protective space over the peptide exit site of ribosome for nascent polypeptides to fold, it remains unclear whether PPIase domain is directly involved in assisting protein folding. Here, we introduced structural change into different regions of TF, and investigated their influence on the chaperone function of TF in assisting the folding of various substrate proteins, including oligomeric glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and monomeric carbonic anhydrase II (CA II) and lysozyme. Results showed that structural disturbances by site-specific mutations in the PPIase active site or by deletion of the PPIase domain from TF affected the chaperone activity of TF toward CA II and GAPDH but had no effect on TF-assisted lysozyme refolding, suggesting PPIase domain is involved in assisting the folding of substrates larger than lysozyme. Mutants with the structural disturbances in the crevice totally lost the chaperone activity toward all the substrates we used in this investigation. These results provide further evidence to confirm that the crevice is the major chaperone site of TF, and the hydrophobic pocket in PPIase domain acts as an auxiliary site to assist the folding of substrate proteins bound to the crevice in a substrate-dependent manner, which is beneficial for TF to provide appropriate assistance for protein folding by changing protective space and binding affinity.     

Functional analysis of the Hsp90-associated human peptidyl prolyl cis/trans isomerases FKBP51, FKBP52 and Cyp40

Large peptidyl-prolyl cis/trans isomerases (PPIases) are important components of the Hsp90 chaperone complex. In mammalian cells, either Cyp40, FKBP51 or FKBP52 is incorporated into these complexes. It has been suggested that members of this protein family exhibit both prolyl isomerase and chaperone activity. Here we define the structural and functional properties of the three mammalian large PPIases. We find that in all cases two PPIase monomers bind to an Hsp90 dimer. However, the affinities of the PPIases are different with FKBP52 exhibiting the strongest interaction and Cyp40 the weakest. Furthermore, in the mammalian system, in contrast to the yeast system, the catalytic activity of prolyl isomerization corresponds well to that of the respective small PPIases. Interestingly, Cyp40 and FKBP51 are the more potent chaperones. Thus, it seems that both the affinity for Hsp90 and the differences in their chaperone properties, which may reflect their interaction with the non-native protein in the Hsp90 complex, are critical for the selective incorporation of a specific large PPIase.     

Chaperone-like activity of peptidyl-prolyl cis-trans isomerase during creatine kinase refolding

Porcine kidney 18 kD peptidyl-prolyl cis-trans isomerase (PPIase) belongs to the cyclophilin family that is inhibited by the immunosuppressive drug cyclosporin A. The chaperone activity of PPIase was studied using inactive, active, and alkylated PPIase during rabbit muscle creatine kinase (CK) refolding. The results showed that low concentration inactive or active PPIase was able to improve the refolding yields, while high concentration PPIase decreased the CK reactivation yields. Aggregation was inhibited by inactive or active PPIase, and completely suppressed at 32 or 80 times the CK concentration (2.7 microM). However, alkylated PPIase was not able to prevent CK aggregation. In addition, the ability of inactive PPIase to affect CK reactivation and prevent CK aggregation was weaker than that of active PPIase. These results indicate that PPIase interacted with the early folding intermediates of CK, thus preventing their aggregation in a concentration-dependent manner. PPIase exhibited chaperone-like activity during CK refolding. The results also suggest that the isomerase activity of PPIase was independent of the chaperone activity, and that the proper molar ratio was important for the chaperone activity of PPIase. The cysteine residues of PPIase may be a peptide binding site, and may be an essential group for the chaperone function.     

All of the protein interactions that link steroid receptor.hsp90.immunophilin heterocomplexes to cytoplasmic dynein are common to plant and animal cells

Both plant and animal cells contain high molecular weight immunophilins that bind via tetratricopeptide repeat (TPR) domains to a TPR acceptor site on the ubiquitous and essential protein chaperone hsp90. These hsp90-binding immunophilins possess the signature peptidylprolyl isomerase (PPIase) domain, but no role for their PPIase activity in protein folding has been demonstrated. From the study of glucocorticoid receptor (GR).hsp90.immunophilin complexes in mammalian cells, there is considerable evidence that both hsp90 and the FK506-binding immunophilin FKBP52 play a role in receptor movement from the cytoplasm to the nucleus. The role of FKBP52 is to target the GR.hsp90 complex to the nucleus by binding via its PPIase domain to cytoplasmic dynein, the motor protein responsible for retrograde movement along microtubules. Here, we use rabbit cytoplasmic dynein as a surrogate for the plant homologue to show that two hsp90-binding immunophilins of wheat, wFKBP73 and wFKBP77, bind to dynein. Binding to dynein is blocked by competition with a purified FKBP52 fragment comprising its PPIase domain but is not affected by the immunosuppressant drug FK506, suggesting that the PPIase domain but not PPIase activity is involved in dynein binding. The hsp90/hsp70-based chaperone system of wheat germ lysate assembles complexes between mouse GR and wheat hsp90. These receptor heterocomplexes contain wheat FKBPs, and they bind rabbit cytoplasmic dynein in a PPIase domain-specific manner. Retention by plants of the entire heterocomplex assembly machinery for linking the GR to dynein implies a fundamental role for this process in the biology of the eukaryotic cell.     

PPIase activities and interaction partners of FK506-binding proteins in the wheat thylakoid

FK506-binding proteins (FKBPs) and cyclophilins, collectively called immunophilins, conserve peptidyl-prolyl cis/trans isomerase (PPIase) active sites, although many lack PPIase activity. The chloroplast thylakoid contains a large proportion of the plant immunophilin family, but their functions within this compartment are unclear. Some lumenal immunophilins are important for assembly of photosynthetic complexes, implicating them in the maintenance and turnover of the photosynthetic apparatus during acclimation processes. In this investigation into the functions of three FKBPs localized to the thylakoid of Triticum aestivum (wheat), we present the first evidence of PPIase activity in the thylakoid of a cereal plant, and also show that PPIase activity is not conserved in all lumenal FKBPs. Using yeast two-hybrid analysis we found that the PPIase-active FKBP13 interacts with the globular domain of the wheat Rieske protein, with potential impact on photosynthetic electron transfer. Specific interaction partners for PPIase-deficient FKBP16-1 and FKBP16-3 link these isoforms to photosystem assembly.     

Neurospora crassa FKBP22 is a novel ER chaperone and functionally cooperates with BiP

FK506 binding proteins (FKBPs) belong to the family of peptidyl prolyl cis-trans isomerases (PPIases) catalyzing the cis/trans isomerisation of Xaa-Pro bonds in oligopeptides and proteins. FKBPs are involved in folding, assembly and trafficking of proteins. However, only limited knowledge is available about the roles of FKBPs in the endoplasmic reticulum (ER) and their interaction with other proteins. Here we show the ER located Neurospora crassa FKBP22 to be a dimeric protein with PPIase and a novel chaperone activity. While the homodimerization of FKBP22 is mediated by its carboxy-terminal domain, the amino-terminal domain is a functional FKBP domain. The chaperone activity is mediated by the FKBP domain but is exhibited only by the full-length protein. We further demonstrate a direct interaction between FKBP22 and BiP, the major Hsp70 chaperone in the ER. The binding to BiP is mediated by the FKBP domain of FKBP22. Interestingly BiP enhances the chaperone activity of FKBP22. Both proteins form a stable complex with an unfolded substrate protein and thereby prevent its aggregation. These results suggest that BiP and FKBP22 form a folding helper complex with a high chaperoning capacity in the ER of Neurospora crassa.     

Escherichia coli Hsp31 functions as a holding chaperone that cooperates with the DnaK-DnaJ-GrpE system in the management of protein misfolding under severe stress conditions

Escherichia coli Hsp31 is a homodimeric protein that exhibits chaperone activity in vitro and is a representative member of a recently recognized family of heat shock proteins (Hsps). To gain insights on Hsp31 cellular function, we deleted the hchA gene from the MC4100 chromosome and combined the resulting null allele with lesions in other cytoplasmic chaperones. Although the hchA mutant only exhibited growth defects when cultivated at 48 degrees C, loss of Hsp31 had a strong deleterious effect on the ability of cells to survive and recover from transient exposure to 50 degrees C, and led to the enhanced aggregation of a subset of host proteins at this temperature. The absence of Hsp31 did not significantly affect the ability of the ClpB-DnaK-DnaJ-GrpE system to clear thermally aggregated proteins at 30 degrees C suggesting that Hsp31 does not possess disaggregase activity. Although it had no effect on the growth of groES30, Delta clpB or Delta ibpAB cells at high temperatures, the hchA deletion aggravated the temperature sensitive phenotype of dnaK756 and grpE280 mutants and led to increased aggregation in stressed dnaK756 cells. On the basis of biochemical, structural and genetic data, we propose that Hsp31 acts as a modified holding chaperone that captures early unfolding intermediates under prolonged conditions of severe stress and releases them when cells return to physiological conditions. This additional line of defence would complement the roles of DnaK-DnaJ-GrpE, ClpB and IbpB in the management of thermally induced cellular protein misfolding.     

Human and Escherichia coli cyclophilins: sensitivity to inhibition by the immunosuppressant cyclosporin A correlates with a specific tryptophan residue

The human T-cell protein cyclophilin shows high affinity for and is the proposed target of the major immunosuppressant drug cyclosporin A (CsA). Cyclophilin also has peptidyl prolyl cis-trans isomerase activity that is inhibited by CsA with an IC50 of 6 nM, while by contrast a homologous PPIase from Escherichia coli has been found to be much less sensitive to CsA, shown here to be 500-fold less potent at an IC50 of 3000 nM. This E. coli rotamase lacks the single highly conserved tryptophan residue of eukaryotic cyclophilins, and we show here that mutation of the natural F112 to W112 enhances E. coli rotamase susceptibility to CsA inhibition by 23-fold. Correspondingly, the human W121 mutations to F121 or A121 yield cyclophilins with 75- and 200-fold decreased sensitivity to CsA, while kcat/Km values of rotamase activity in a tetrapeptide assay drop only 2- and 13-fold, respectively. This complementary gain and loss of CsA sensitivity to mutation to or from tryptophan validate the indole side chain as a major determinant in immunosuppressant drug recognition and the separation of PPIase catalytic efficiency from CsA affinity.     

Transgenic mice overexpressing cyclophilin A are resistant to cyclosporin A-induced nephrotoxicity via peptidyl-prolyl cis-trans isomerase activity

Cyclosporin A (CsA) suppresses immune reaction by inhibiting calcineurin activity after forming complex with cyclophilins and is currently widely used as an immunosuppressive drug. Cyclophilin A (CypA) is the most abundantly and ubiquitously expressed family member of cyclophilins. We previously showed that CsA toxicity is mediated by ROS generation as well as by inhibition of peptidyl-prolyl cis-trans isomerase (PPIase) activity of CypA in CsA-treated myoblasts [FASEB J. 16 (2002) 1633]. Since CsA-induced nephrotoxicity is the most significant adverse effect in its clinical utilization, we here investigated the role of CsA inhibition of CypA PPIase activity in its nephrotoxicity using transgenic mouse models. Transgenic mice of either wild type (CypA/wt) or R55A PPIase mutant type (CypA/R55A), a dominant negative mutant of CypA PPIase activity, showed normal growth without any apparent abnormalities. However, CsA-induced nephrotoxicity was virtually suppressed in CypA/wt mice, but exacerbated in CypA/R55A mice, compared to that of littermates. Also, life expectancy was extended in CypA/wt mice and shortened in CypA/R55A mice during CsA administration. Besides, CsA-induced nephrotoxicity was inversely related to the levels of catalase expression and activity. In conclusion, our data provide in vivo evidence that supplement of CypA PPIase activity allows animal's resistance toward CsA-induced nephrotoxicity.     

In vivo and in vitro complementation study comparing the function of DnaK chaperone systems from halophilic lactic acid bacterium Tetragenococcus halophilus and Escherichia coli

In this study, we characterized the DnaK chaperone system from Tetragenococcus halophilus, a halophilic lactic acid bacterium. An in vivo complementation test showed that under heat stress conditions, T. halophilus DnaK did not rescue the growth of the Escherichia coli dnaK deletion mutant, whereas T. halophilus DnaJ and GrpE complemented the corresponding mutations of E. coli. Purified T. halophilus DnaK showed intrinsic weak ATPase activity and holding chaperone activity in vitro, but T. halophilus DnaK did not cooperate with the purified DnaJ and GrpE from either T. halophilus or E. coli in ATP hydrolysis or luciferase-refolding reactions under the conditions tested. E. coli DnaK, however, cross-reacted with those from both bacteria. This difference in the cooperation with DnaJ and GrpE appears to result in an inability of T. halophilus DnaK to replace the in vivo function of the DnaK chaperone of E. coli.     

Immunophilin AtFKBP13 sustains all peptidyl-prolyl isomerase activity in the thylakoid lumen from Arabidopsis thaliana deficient in AtCYP20-2

The physiological roles of immunophilins are unclear, but many possess peptidyl-prolyl isomerase (PPIase) activity, and they have been found in all organisms examined to date, implying that they are involved in fundamental, protein-folding processes. The chloroplast thylakoid lumen of the higher plant Arabidopsis thaliana contains up to 16 immunophilins (five cyclophilins and 11 FKBPs), but only two of them, AtCYP20-2 and AtFKBP13, have been found to be active PPIases, indicating that the other immunophilins in this cellular compartment may have lost their putative PPIase activities. To assess this possibility, we characterized two independent Arabidopsis knockout lines lacking AtCYP20-2 in enzymological and quantitative proteomic analyses. The PPIase activity in thylakoid lumen preparations of both mutants was equal to that of corresponding wild-type preparations, and comparative two-dimensional difference gel electrophoresis analyses of the lumenal proteins of the mutants and wild type showed that none of the potential PPIases was more abundant in the AtCYP20-2 deficient plants. Enzymatic analyses established that all PPIase activity in the mutant thylakoid lumen was attributable to AtFKBP13, and oxidative activation of this enzyme compensated for the lack of AtCYP20-2. Accordingly, sequence analyses of the potential catalytic domains of lumenal cyclophilins and FKBPs demonstrated that only AtCYP20-2 and AtFKBP13 possess all of the amino acid residues found to be essential for PPIase activity in earlier studies of human cyclophilin A and FKBP12. Thus, none of the immunophilins in the chloroplast thylakoid lumen of Arabidopsis except AtCYP20-2 and AtFKBP13 appear to possess prolyl isomerase activity toward peptide substrates.     

Structural and functional characterization of Escherichia coli peptidyl-prolyl cis-trans isomerases.

Peptidyl-prolyl cis-trans isomerases (PPIases), enzymes that catalyze the cis-trans isomerization of peptide bonds to which proline contributes the nitrogen, were purified from Escherichia coli. In this organism, at least two PPIases are present. Both the cationic (periplasmic) and anionic (cytoplasmic) PPIases are inhibited by cyclosporin A with a Ki of 25-50 microM, a concentration 1000-fold higher than that required for eukaryotic PPIases. Although isoelectric focusing indicates that the two enzymes differ in isoelectric point by at least 4.0 pH units, the specific activities of the enzymes toward the tetrapeptide substrate succinyl-Ala-Ala-Pro-Phe-methyl-coumarylamide are equivalent. The activity of both enzymes for a series of substituted succinyl-Ala-Xaa-Pro-Phe-para-nitroanilide tetrapeptides suggests that the structure and function of the active site of the prokaryotic proteins is similar to that of eukaryotic cyclophilins. Both enzymes are capable of catalyzing the refolding of thermally denatured type III collagen. Antibodies against the periplasmic PPIase do not recognize the cytoplasmic enzyme, indicating significant differences in epitopes between the two forms. Circular dichroism spectroscopy indicates that the secondary structure of the cationic protein consists of 17% alpha-helix, 34% beta-sheet, 17% turns, 33% random coil and is very similar to human cytosolic PPIase.