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.     

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.     

Enzymatic catalysis of prolyl isomerization in an unfolding protein.

Prolyl isomerases are able to accelerate slow steps in protein refolding that are limited in rate by cis/trans isomerizations of Xaa-Pro peptide bonds. We show here that prolyl isomerizations in the course of protein unfolding are also well catalyzed. To demonstrate catalysis we use cytoplasmic prolyl isomerase from Escherichia coli as the enzyme and reduced and carboxymethylated ribonuclease T1 as the substrate. This form of ribonuclease T1 without disulfide bonds is nativelike folded only in the presence of moderate concentrations of NaCl. Unfolding can be induced by reducing the NaCl concentration at ambient temperature and in the absence of denaturants. Under these conditions prolyl isomerase retains its activity and it catalyzes prolyl cis/trans isomerization in the unfolding protein. Under identical conditions within the NaCl-induced transition unfolding and refolding are catalyzed with equal efficiency. The stability of the protein and thus the final distribution of unfolded and folded molecules attained at equilibrium is unchanged in the presence of prolyl isomerase. These results demonstrate that prolyl isomerase functions in protein folding as an enzyme and catalyzes prolyl isomerization in either direction.     

Combination of the human prolyl isomerase FKBP12 with unrelated chaperone domains leads to chimeric folding enzymes with high activity

Folding enzymes often use distinct domains for the binding of substrate proteins ("chaperone domains") and for the catalysis of slow folding reactions such as disulfide formation or prolyl isomerization. The human prolyl isomerase FKBP12 is a small single-domain protein without a chaperone domain. Its very low folding activity could previously be increased by inserting the chaperone domain from the homolog SlyD (sensitive-to-lysis protein D) of Escherichia coli. We now inserted three unrelated chaperone domains into human FKBP12: the apical domain of the chaperonin GroEL from E. coli, the chaperone domain of protein disulfide isomerase from yeast, or the chaperone domain of SurA from the periplasm of E. coli. All three conveyed FKBP12 with a high affinity for unfolded proteins and increased its folding activity. Substrate binding and release of the chimeric folding enzymes were found to be very fast. This allows rapid substrate transfer from the chaperone domain to the catalytic domain and ensures efficient rebinding of protein chains that were unable to complete folding. The advantage of having separate sites, first for generic protein binding and then for specific catalysis, explains why our construction of the artificial folding enzymes with foreign chaperone domains was successful.     

Binding analysis of a psychrotrophic FKBP22 to a folding intermediate of protein using surface plasmon resonance

SIB1 FKBP22 is a homodimer, with each subunit consisting of the C-terminal catalytic domain and N-terminal dimerization domain. This protein exhibits peptidyl prolyl cis-trans isomerase activity for both peptide and protein substrates. However, truncation of the N-terminal domain greatly reduces the activity only for a protein substrate. Using surface plasmon resonance, we showed that SIB1 FKBP22 loses the binding ability to a folding intermediate of protein upon truncation of the N-terminal domain but does not lose it upon truncation of the C-terminal domain. We propose that the binding site of SIB1 FKBP22 to a protein substrate of PPIase is located at the N-terminal domain.     

SlyD proteins from different species exhibit high prolyl isomerase and chaperone activities

SlyD is a putative folding helper protein from the Escherichia coli cytosol, which consists of an N-terminal prolyl isomerase domain of the FKBP type and a presumably unstructured C-terminal tail. We produced truncated versions without this tail (SlyD) for SlyD from E. coli, as well as for the SlyD orthologues from Yersinia pestis, Treponema pallidum, Pasteurella multocida, and Vibrio cholerae. They are monomeric in solution and unfold reversibly. All SlyD variants catalyze the proline-limited refolding of ribonuclease T1 with very high efficiencies, and the specificity constants (kcat/KM) are equal to approximately 10(6) M(-1) s(-1). These large values originate from the high affinities of the SlyD orthologues for unfolded RCM-T1, which are reflected in low KM values of approximately 1 microM. SlyD also exhibits pronounced chaperone properties. Permanently unfolded proteins bind with high affinity to SlyD and thus inhibit its prolyl isomerase activity. The unfolded protein chains do not need to contain proline residues to be recognized and bound by SlyD. The conservation of prolyl isomerase activity and chaperone properties within the SlyD family suggests that these proteins might act as true folding helpers in the bacterial cytosol. The SlyD proteins are also well suited for biotechnological applications. As fusion partners they facilitate the refolding and increase the solubility of aggregation-prone proteins such as the gp41 ectodomain fragment of HIV-1.     

Chemical shift assignments of the catalytic domain from the yeast proline isomerase Fpr4p

Yeast Fpr4p belongs to the FK506-binding protein (FKBP) class of peptidyl proline isomerases (PPIases), and has been implicated in regulating the cis-trans conversion of proline residues within histone tails. Here we report the (1)H, (13)C and (15)N chemical shift assignments for the bacterially expressed C-terminal PPIase catalytic domain of Fpr4p. Prediction of secondary structure reveals similarity to domains from other members of the FKBP proline isomerases, including yeast Fpr1p and the prototypic PPIase region from human FKBP12.     

Prolyl isomerases in a minimal cell. Catalysis of protein folding by trigger factor from Mycoplasma genitalium.

Peptidyl-prolyl cis/trans isomerases (PPIases) catalyze the isomerization of prolyl peptide bonds. Distinct families of this class of enzymes are involved in protein folding in vitro, whereas their significance in free living organisms is not known. Previously, we inspected the smallest known genome of a self-replicating organism and found that Mycoplasma genitalium is devoid of all known PPIases except the trigger factor. Despite the extensive sequence information becoming available, most genes remain hypothetical and enzyme activities in many species have not been assigned to an open reading frame. Therefore, we studied the PPIase activity in crude extracts of M. genitalium. We showed that this is solely attributed to a single enzyme activity, the trigger factor. Characterization of this enzyme revealed that its PPIase activity resides in a central 12-kDa domain. Only the complete trigger factor is able to cis/trans isomerize extended peptide substrates, while the PPIase domain alone can not. The N- and the C-terminal domains of the trigger factor seem to function in binding of proteins as substrates, as demonstrated by protein refolding experiments, in which the complete trigger factor catalyzed protein refolding towards a model protein 500-fold more efficiently than the isolated central PPIase domain. Protein modeling studies suggest that the PPIase domain can fold in a similar way as the PPIase domain of FK506 binding proteins (FKBPs), one class of PPIases, despite only very limited sequence homology. Differences at the active site explain why this enzyme is not inhibited by FK506 in contrast with FKBPs. Trigger factor expressed in Escherichia coli confirms its additional chaperone functions, as shown by its association with chaperones GroEL and GroES after induction of misfolding. In contrast, the isolated PPIase-domain lacks any association with chaperones from E. coli. In summary, trigger factor of M. genitalium is the single folding isomerase of this organism, which harbors an enzymatically active PPIase domain with structural homology to FKBPs. Its additional domains confer its ability to be an efficient catalyst of protein folding. The protein folding machinery is conserved and shows a dual function as a chaperone and a prolyl isomerase.     

Deletion of the C-terminal 138 amino acids of the wheat FKBP73 abrogates calmodulin binding,dimerization and male fertility in transgenic rice

Wheat FKBP73 (wFKBP73) belongs to the FK506-binding protein (FKBP) family which, in common with the cyclophilin and parvulin families, possesses peptidyl prolylcis-trans isomerase (PPIase) activity. Wheat FKBP73 has been shown to contain three FKBP12-like domains, a tetratricopeptide repeat (TPR) via which it binds heat shock protein 90 and a calmodulin-binding domain (CaMbd). In this study we investigated: (1) the contribution of the N-terminal and C-terminal moieties of wFKBP73 to its biological activity by over-expression of the prolyl isomerase domains in transgenic rice, and (2) the biochemical characteristics of the C-terminal moiety. The recombinant wFKBP73 was found to bind calmodulin via the CaMbd and to be present mainly as a dimer in solution. The dimerization was abrogated when 138 amino acids from the C-terminal half were deleted. Expression of the full-length FKBP73 produced fertile rice plants, whereas the expression of the peptidyl prolyl cis-trans isomerase domains in transgenic rice resulted in male-sterile plants. The male sterility was expressed at various stages of anther development with arrest of normal pollen development occurring after separation of the microspores from the tetrads. Although the direct cause of the dominant male sterility is not yet defined, we suggest that it is associated with a novel interaction of the prolyl isomerase domains with anther specific target proteins.     

The hsp70 chaperone DnaK is a secondary amide peptide bond cis-trans isomerase

Peptidyl prolyl cis-trans isomerases can enzymatically assist protein folding, but these enzymes exclusively target the peptide bond preceding proline residues. Here we report the identification of the Hsp70 chaperone DnaK as the first member of a novel enzyme class of secondary amide peptide bond cis-trans isomerases (APIases). APIases selectively accelerate the cis-trans isomerization of nonprolyl peptide bonds. Results from independent experiments support the APIase activity of DnaK: (i) exchange crosspeaks between the cis-trans conformers appear in 2D (1)H NMR exchange spectra of oligopeptides (ii) the rate constants for the cis-trans isomerization of various dipeptides increase and (iii) refolding of the RNase T1 P39A variant is catalyzed. The APIase activity shows both regio and stereo selectivity and is stimulated two-fold in the presence of the complete DnaK/GrpE/DnaJ/ATP refolding system. Moreover, known DnaK-binding oligopeptides simultaneously affect the APIase activity of DnaK and the refolding yield of denatured firefly luciferase in the presence of DnaK/GrpE/DnaJ/ATP. These results suggest a new role for the chaperone as a regioselective catalyst for bond rotation in polypeptides.     

Activation of colicin M by the FkpA prolyl cis-trans isomerase/chaperone

Colicin M (Cma) is specifically imported into the periplasm of Escherichia coli and kills the cells. Killing depends on the periplasmic peptidyl prolyl cis-trans isomerase/chaperone FkpA. To identify the Cma prolyl bonds targeted by FkpA, we replaced the 15 proline residues individually with alanine. Seven mutant proteins were fully active; Cma(P129A), Cma(P176A), and Cma(P260A) displayed 1%, and Cma(P107A) displayed 10% of the wild-type activity. Cma(P107A), Cma(P129A), and Cma(P260A), but not Cma(P176A), killed cells after entering the periplasm via osmotic shock, indicating that the former mutants were translocation-deficient; Cma(P129A) did not bind to the FhuA outer membrane receptor. The crystal structures of Cma and Cma(P176A) were identical, excluding inactivation of the activity domain located far from Pro-176. In a new peptidyl prolyl cis-trans isomerase assay, FkpA isomerized the Cma prolyl bond in peptide Phe-Pro-176 at a high rate, but Lys-Pro-107 and Leu-Pro-260 isomerized at only <10% of that rate. The four mutant proteins secreted into the periplasm via a fused signal sequence were toxic but much less than wild-type Cma. Wild-type and mutant Cma proteins secreted or translocated across the outer membrane by energy-coupled import or unspecific osmotic shock were only active in the presence of FkpA. We propose that Cma unfolds during transfer across the outer or cytoplasmic membrane and refolds to the active form in the periplasm assisted by FkpA. Weak refolding of Cma(P176A) would explain its low activity in all assays. Of the four proline residues identified as being important for Cma activity, Phe-Pro-176 is most likely targeted by FkpA.     

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.     

Cooperation of the prolyl isomerase and chaperone activities of the protein folding catalyst SlyD

The SlyD (sensitive to lysis D) protein of Escherichia coli is a folding enzyme with a chaperone domain and a prolyl isomerase domain of the FK506 binding protein type. Here we investigated how the two domains and their interplay are optimized for function in protein folding. Unfolded protein molecules initially form a highly dynamic complex with the chaperone domain of SlyD, and they are then transferred to the prolyl isomerase domain. The turnover number of the prolyl isomerase site is very high and guarantees that, after transfer, prolyl peptide bonds in substrate proteins are isomerized very rapidly. The Michaelis constant of catalyzed folding reflects the substrate affinity of the chaperone domain, and the turnover number is presumably determined by the rate of productive substrate transfer from the chaperone to the prolyl isomerase site and by the intrinsic propensity of the refolding protein chain to leave the active site with the native prolyl isomer. The efficiency of substrate transfer is high because dissociation from the chaperone site is very fast and because the two sites are close to each other. Protein molecules that left the prolyl isomerase site with an incorrect prolyl isomer can rapidly be re-bound by the chaperone domain because the association rate is very high as well.     

Osmolyte effects on kinetics of FKBP12 C22A folding coupled with prolyl isomerization

Unfolding and refolding kinetics of human FKBP12 C22A were monitored by fluorescence emission over a wide range of urea concentration in the presence and absence of protecting osmolytes glycerol, proline, sarcosine and trimethylamine-N-oxide (TMAO). Unfolding is well described by a mono-exponential process, while refolding required a minimum of two exponentials for an adequate fit throughout the urea concentration range considered. The bi-exponential behavior resulted from complex coupling between protein folding, and prolyl isomerization in the denatured state in which the urea-dependent rate constant for folding was greater than, equal to, and less than the rate constants for prolyl isomerization within the urea concentration range of zero to five molar. Amplitudes and the observed folding and unfolding rate constants were fitted to a reversible three-state model composed of two sequential steps involving the native state and a folding-competent denatured species thermodynamically linked to a folding-incompetent denatured species. Excellent agreement between thermodynamic parameters for FKBP12 C22A folding calculated from the kinetic parameters and those obtained directly from equilibrium denaturation assays provides strong support for the applicability of the mechanism, and provides evidence that FKBP12 C22A folding/unfolding is two-state, with prolyl isomer heterogeneity in the denatured ensemble. Despite the chemical diversity of the protecting osmolytes, they all exhibit the same kinetic behavior of increasing the rate constant of folding and decreasing the rate constant for unfolding. Osmolyte effects on folding/unfolding kinetics are readily explained in terms of principles established in understanding osmolyte effects on protein stability. These principles involve the osmophobic effect, which raises the Gibbs energy of the denatured state due to exposure of peptide backbone, thereby increasing the folding rate. This effect also plays a key role in decreasing the unfolding rate when, as is often the case, the activated complex exposes more backbone than is exposed in the native state.     

Suppression of EGFR autophosphorylation by FKBP12

FK506 binding proteins (FKBPs) represent a subfamily of peptidyl prolyl cis/trans isomerases that can control receptor-mediated intracellular signaling. The prototypic PPIase FKBP12 functionally interacts with EGFR. FKBP12 was shown to inhibit EGF-induced EGFR autophosphorylation with all internal phosphorylation sites equally affected. The inhibition of EGFR catalytic activity is conducted by targeting the EGFR kinase domain. The change of intracellular FKBP12 levels resulted in a change of EGFR autophosphorylation level. Collectively, our results demonstrate that FKBP12 forms an endogenous inhibitor of EGFR phosphorylation directly involved in the control of cellular EGFR activity.     

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.     

The Hsp90-binding peptidylprolyl isomerase FKBP52 potentiates glucocorticoid signaling in vivo

Hsp90 is required for the normal activity of steroid receptors, and in steroid receptor complexes it is typically bound to one of the immunophilin-related co-chaperones: the peptidylprolyl isomerases FKBP51, FKBP52 or CyP40, or the protein phosphatase PP5. The physiological roles of the immunophilins in regulating steroid receptor function have not been well defined, and so we examined in vivo the influences of immunophilins on hormone-dependent gene activation in the Saccharomyces cerevisiae model for glucocorticoid receptor (GR) function. FKBP52 selectively potentiates hormone-dependent reporter gene activation by as much as 20-fold at limiting hormone concentrations, and this potentiation is readily blocked by co-expression of the closely related FKBP51. The mechanism for potentiation is an increase in GR hormone-binding affinity that requires both the Hsp90-binding ability and the prolyl isomerase activity of FKBP52.     

Solution structure of the human parvulin-like peptidyl prolyl cis/trans isomerase, hPar14

The hPar14 protein is a peptidyl prolyl cis/trans isomerase and is a human parvulin homologue. The hPar14 protein shows about 30 % sequence identity with the other human parvulin homologue, hPin1. Here, the solution structure of hPar14 was determined by nuclear magnetic resonance spectroscopy. The N-terminal 35 residues preceding the peptidyl prolyl isomerase domain of hPar14 are unstructured, whereas hPin1 possesses the WW domain at its N terminus. The fold of residues 36-131 of hPar14, which comprises a four-stranded beta-sheet and three alpha-helices, is superimposable onto that of the peptidyl prolyl isomerase domain of hPin1. To investigate the interaction of hPar14 with a substrate, the backbone chemical-shift changes of hPar14 were monitored during titration with a tetra peptide. Met90, Val91, and Phe94 around the N terminus of alpha3 showed large chemical-shift changes. These residues form a hydrophobic patch on the molecular surface of hPar14. Two of these residues are conserved and have been shown to interact with the proline residue of the substrate in hPin1. On the other hand, hPar14 lacks the hPin1 positively charged residues (Lys63, Arg68, and Arg69), which determine the substrate specificity of hPin1 by interacting with phosphorylated Ser or Thr preceding the substrate Pro, and exhibits a different structure in the corresponding region. Therefore, the mechanism determining the substrate specificity seems to be different between hPar14 and hPin1.     

Evaluation of similarities in the cis/trans isomerase function of trigger factor and DnaK

Two functionally redundant enzymes, trigger factor and the hsp70 chaperone DnaK, have been found to assist de novo protein folding in E coli. Trigger factor is a peripheral peptidyl prolyl cis/trans isomerase (PPIase) of the large subunit of the ribosome. In contrast, DnaK displays two catalytic features: the secondary amide peptide bond cis/trans isomerase (APIase) function supplemented by the ATPase site. APIases accelerate the cis/trans isomerization of nonprolyl peptide bonds. Both enzymes have affinity for an unfolded polypeptide chain. The diminished low temperature cell viability in the presence of trigger factor variants with impaired PPlase activity indicates that the enhancement of folding rates plays a crucial role in protein folding in vivo. For the DnaK-mediated increase in the folding yield in vitro, the minimal model for APlase catalysis involves the catalyzed partitioning of a rapidly formed folding intermediate as could be inferred from the DnaK/DnaJ/GrpE/ATP-assisted refolding of GdmCl-denatured luciferase. Using three different peptide bond cis/trans isomerization assays in vitro, we could show that there is no overlapping substrate specificity of trigger factor and DnaK. We propose that only if trigger factor recruits supplementing molecules is it capable of exhibiting functional complementarity with DnaK in protein folding.     

Consequences of Domain Insertion on the Stability and Folding Mechanism of a Protein

SlyD, the sensitive-to-lysis protein from Escherichia coli, consists of two domains. They are not arranged successively along the protein chain, but one domain, the “insert-in-flap” (IF) domain, is inserted internally as a guest into a surface loop of the host domain, which is a prolyl isomerase of the FK506 binding protein (FKBP) type. We used SlyD as a model to elucidate how such a domain insertion affects the stability and folding mechanism of the host and the guest domain. For these studies, the two-domain protein was compared with a single-domain variant SlyDΔIF, SlyD* without the chaperone domain (residues 1-69 and 130-165) in which the IF domain was removed and replaced by a short loop, as present in human FKBP12. Equilibrium unfolding and folding kinetics followed an apparent two-state mechanism in the absence and in the presence of the IF domain. The inserted domain decreased, however, the stability of the host domain in the transition region and decelerated its refolding reaction by about 10-fold. This originates from the interruption of the chain connectivity by the IF domain and its inherent instability. To monitor folding processes in this domain selectively, a Trp residue was introduced as fluorescent probe. Kinetic double-mixing experiments revealed that, in intact SlyD, the IF domain folds and unfolds about 1000-fold more rapidly than the FKBP domain, and that it is strongly stabilized when linked with the folded FKBP domain. The unfolding limbs of the kinetic chevrons of SlyD show a strong downward curvature. This deviation from linearity is not caused by a transition-state movement, as often assumed, but by the accumulation of a silent unfolding intermediate at high denaturant concentrations. In this kinetic intermediate, the FKBP domain is still folded, whereas the IF domain is already unfolded.