Energetic coupling between native-state prolyl isomerization and conformational protein folding

In folded proteins, prolyl peptide bonds are usually thought to be either trans or cis because only one of the isomers can be accommodated in the native folded protein. For the N-terminal domain of the gene-3 protein of the filamentous phage fd (N2 domain), Pro161 resides at the tip of a beta hairpin and was found to be cis in the crystal structure of this protein. Here we show that Pro161 exists in both the cis and the trans conformations in the folded form of the N2 domain. We investigated how conformational folding and prolyl isomerization are coupled in the unfolding and refolding of N2 domain. A combination of single-mixing and double-mixing unfolding and refolding experiments showed that, in unfolded N2 domain, 7% of the molecules contain a cis-Pro161 and 93% of the molecules contain a trans-Pro161. During refolding, the fraction of molecules with a cis-Pro161 increases to 85%. This implies that 10.3 kJ mol(-1) of the folding free energy was used to drive this 75-fold change in the Pro161 cis/trans equilibrium constant during folding. The stabilities of the forms with the cis and the trans isomers of Pro161 and their folding kinetics could be determined separately because their conformational folding is much faster than the prolyl isomerization reactions in the native and the unfolded proteins. The energetic coupling between conformational folding and Pro161 isomerization is already fully established in the transition state of folding, and the two isomeric forms are thus truly native forms. The folding kinetics are well described by a four-species box model, in which the N2 molecules with either isomer of Pro161 can fold to the native state and in which cis/trans isomerization occurs in both the unfolded and the folded proteins.     

Essential role of Pro 74 in stefin B amyloid-fibril formation: Dual action of cyclophilin A on the process

We report that Pro74 in human stefin B is critical for fibril formation and that proline isomerization plays an important role. The stefin B P74S mutant did not fibrillate over the time of observation at 25 °C, and it exhibited a prolonged lag phase at 30 °C and 37 °C. The peptidyl prolyl cis/trans isomerase cyclophilin A, when added to the wild-type protein, exerted two effects: it prolonged the lag phase and increased the yield and length of the fibrils. Addition of the inactive cyclophilin A R55A variant still resulted in a prolonged lag phase but did not mediate the increase of the final fibril yield. These results demonstrate that peptidyl prolyl cis/trans isomerism is rate-limiting in stefin B fibril formation.     

Cooperativity of a protein folding reaction probed at multiple chain positions by real-time 2D NMR spectroscopy

The refolding reaction of S54G/P55N ribonuclease T1 is a two-step process, where fast formation of a partly folded intermediate is followed by the slow reaction to the native state, limited by a trans --> cis isomerization of Pro39. The hydrodynamic radius of this kinetic folding intermediate was determined by real-time diffusion NMR spectroscopy. Its folding to the native state was monitored by a series of 128 very fast 2D (15)N-HMQC spectra, to observe the kinetics of 66 individual backbone amide probes. We find that the intermediate is as compact as the native protein with many native chemical shifts. All 66 analyzed amide probes follow the rate-limiting prolyl isomerization, which indicates that this cooperative refolding reaction is fully synchronized. The stability of the folding intermediate was determined from the protection factors of 45 amide protons derived from a competition between refolding and H/D exchange. The intermediate has already gained 40% of the Gibbs free energy of refolding with many protected amides in not-yet-native regions.     

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.     

Catalysis of proline-directed protein phosphorylation by peptidyl-prolyl cis/trans isomerases

Proline-directed protein phosphorylation was shown to depend on the capacity of the targeted Ser(Thr)-Pro bond to exhibit conformational polymorphism. The cis/trans isomer specificity underlying ERK2-catalyzed phosphate transfer leads to a complete discrimination of the cis Ser(Thr)-Pro conformer of oligopeptide substrates. We investigated in vitro the ERK2-catalyzed phosphorylation of Aspergillus oryzae RNase T1 containing two Ser-Pro bonds both of which share high stabilization energy in their respective native state conformation, the cis Ser54-Pro and the trans Ser72-Pro moiety. Despite trans isomer specificity of ERK2, a doubly phosphorylated RNase T1 was found as the final reaction product. Similarly, the RNase T1 S54G/P55N and RNase T1 P73V variants, which retain the prolyl bond conformations of the RNase T1-wt, were both monophosphorylated with a catalytic efficiency kcat/KM of 425 M(-1) s(-1) and 1228 M(-1) s(-1), respectively. However, initial phosphorylation rates did not depend linearly on the ERK2 concentration. The phosphorylation rate of the resulting plateau region at high ERK2 concentrations can be increased up to threefold for the RNase T1 P73V variant in the presence of the peptidyl-prolyl cis/trans isomerase Cyclophilin 18, indicating a conformational interconversion as the rate limiting step in the catalyzed phosphate group transfer. Using peptidyl-prolyl cis/trans isomerases with different substrate specificity, we identified a native state conformational equilibrium of the Ser54-Pro bond with the minor trans Ser54-Pro bond as the phosphorylation-sensitive moiety. This technique can therefore be used for a determination of the ratio and the interconversion rates of prolyl bond isomers in the native state of proteins.     

Parallel channels and rate-limiting steps in complex protein folding reactions: prolyl isomerization and the alpha subunit of Trp synthase,a TIM barrel protein

A kinetic folding mechanism for the alpha subunit of tryptophan synthase (alphaTS) from Escherichia coli, involving four parallel channels with multiple native, intermediate and unfolded forms, has recently been proposed. The hypothesis that cis/trans isomerization of several Xaa-Pro peptide bonds is the source of the multiple folding channels was tested by measuring the sensitivity of the three rate-limiting phases (tau(1), tau(2), tau(3)) to catalysis by cyclophilin, a peptidyl-prolyl isomerase. Although the absence of catalysis for the tau(1) (fast) phase leaves its assignment ambiguous, our previous mutational analysis demonstrated its connection to the unique cis peptide bond preceding proline 28. The acceleration of the tau(2) (medium) and tau(3) (slow) refolding phases by cyclophilin demonstrated that cis/trans prolyl isomerization is also the source of these phases. A collection of proline mutants, which covered all of the remaining 18 trans proline residues of alphaTS, was constructed to obtain specific assignments for these phases. Almost all of the mutant proteins retained the complex equilibrium and kinetic folding properties of wild-type alphaTS; only the P217A, P217G and P261A mutations caused significant changes in the equilibrium free energy surface. Both the P78A and P96A mutations selectively eliminated the tau(1) folding phase, while the P217M and P261A mutations eliminated the tau(2) and tau(3) folding phases, respectively. The redundant assignment of the tau(1) phase to Pro28, Pro78 and Pro96 may reflect their mutual interactions in non-random structure in the unfolded state. The non-native cis isomers for Pro217 and Pro261 may destabilize an autonomous C-terminal folding unit, thereby giving rise to kinetically distinct unfolded forms. The nature of the preceding amino acid, the solvent exposure, or the participation in specific elements of secondary structure in the native state, in general, are not determinative of the proline residues whose isomerization reactions can limit folding.     

Nuclear magnetic resonance characterization of the refolding intermediate of beta2-microglobulin trapped by non-native prolyl peptide bond

beta(2)-Microglobulin (beta2-m), a light chain of the major histocompatibility complex type I, is also found as a major component of amyloid fibrils formed in dialysis-related amyloidosis. Denaturation of beta2-m is considered to initiate the formation of fibrils. To clarify the mechanism of fibril formation, it is important to characterize the intermediate conformational states at the atomic level. Here, we investigated the refolding of beta2-m from the acid-unfolded state by heteronuclear magnetic resonance and circular dichroism spectroscopies. At low temperature, beta2-m refolded slowly, accumulating a rate-limiting intermediate with non-native chemical shift dispersions for several residues, but with compactness and secondary structures similar to those of the native protein. beta2-m has a cis proline residue at Pro32, located on the turn connecting the betaB and betaC strands. The slow refolding phase disappeared upon mutation of Pro32 to Val, indicating that Pro32 is responsible for the accumulation of the intermediate. The distribution of the perturbed residues in the intermediate suggests that the non-native prolyl peptide bond of Pro32 affects large areas of the molecule. A cis proline residue is common to various immunoglobulin domains involved in amyloidosis, implying that a non-native prolyl peptide bond that might occur under physiological conditions is related to the amyloidogenicity of these immunoglobulin domains.     

An examination of the steric effects of 5-tert-butylproline on the conformation of polyproline and the cooperative nature of type II to type I helical interconversion

The influence of steric effects on the helical geometry and the interconversion of type II to type I polyproline in water was examined by the synthesis and analysis of proline dimers and hexamers containing up to three (2S,5R)-5-tert-butylproline residues. In the dimers, the bulky 5-tert-butyl substituent was found to exert a significant influence on the local prolyl amide geometry such that the predominant trans-isomer in N-(acetyl)prolyl-prolinamide (1) was converted to 63% cis isomer in N-(acetyl)prolyl-5-tert-butylprolinamide (2) as measured by (1)H-nmr spectroscopy. Similarly, the presence of a 5-tert-butyl group on the C-terminal residue in the polyproline hexamer Ac-Pro(5)-t-BuPro-NH(2) (4) produced a local 5-tert-butylprolyl amide population containing 61% cis isomer in water. In spite of the presence of a local prolyl cis amide geometry, the downstream prolyl amides in 4 remained in the trans isomer as determined by NOESY spectroscopy. Conformational analysis by (13)C-nmr and CD spectroscopy indicated that Ac-Pro(6)-NH(2) (3) adopted the all-trans amide polyproline type II helix in water. As the amount of 5-tert-butylproline increased from one to three residues in hexamers 4-6, a gradual destabilization of the polyproline type II helical geometry was observed by CD spectroscopy in water; however, no spectrum was obtained, indicative of a complete conversion to a polyproline type I helix. The implications of these results are discussed with respect to the previously proposed theoretical mechanisms for the helical interconversion of polyproline, which has been suggested to occur by either a cooperative C- to N-terminal isomerization of the prolyl amide bonds or via a conformational intermediate composed of dispersed sequences of prolyl amide cis and trans isomers.     

Cyclophilin-promoted folding of mouse dihydrofolate reductase does not include the slow conversion of the late-folding intermediate to the active enzyme

Cyclophilins accelerate slow protein folding reactions in vitro by catalyzing the cis/trans isomerization of peptidyl-prolyl bonds. Cyclophilins were reported to be involved in a variety of cellular functions, including the promotion of protein folding by use of the substrate mouse dihydrofolate reductase (DHFR). The interaction of cyclophilin with DHFR has only been studied under limited conditions so far, not taking into account that native DHFR exists in equilibrium with a non-native late-folding intermediate. Here we report a systematic analysis of catalysis of DHFR folding by cyclophilins. The specific ligand methotrexate traps DHFR in its native state, permitting a specific analysis of the action of cyclophilin on both denatured DHFR with non-native prolyl bonds and denatured DHFR with all-native prolyl bonds. Cyclophilins from yeast and Neurospora crassa as well as the related prolyl isomerase b from Escherichia coli promote the folding of different forms of DHFR to the enzymatically active form, demonstrating the generality of cyclophilin-catalyzed folding of DHFR. The slow equilibrium between the late-folding intermediate and native DHFR suggests that prolyl isomerization may be required for this final phase of conversion to native DHFR. However, by reversible trapping of the intermediate, we analyze the slow interconversion between native and late-folding conformations in the backward and forward reactions and show a complete independence of cyclophilin. We conclude that cyclophilin catalyzes folding of DHFR, but surprisingly not in the last slow folding step.     

Conservation of cis prolyl bonds in proteins during evolution

In proteins and peptides, the vast majority of peptide bonds occurs in trans conformation, but a considerable fraction (about 5%) of X-Pro bonds adopts the cis conformation. Here we study the conservation of cis prolyl residues in evolutionary related proteins. We find that overall, in contrast to local, protein sequence similarity is a clear indicator for the conformation of prolyl residues. We observe that cis prolyl residues are more often conserved than trans prolyl residues, and both are more conserved than the surrounding amino acids, which show the same extent of conservation as the whole protein. The pattern of amino acid exchanges differs between cis and trans prolyl residues. Also, the cis prolyl bond is maintained in proteins with sequence identity as low as 20%. This finding emphasizes the importance of cis peptide bonds in protein structure and function.     

Support vector machines for prediction of peptidyl prolyl cis/trans isomerization.

A new method for peptidyl prolyl cis/trans isomerization prediction based on the theory of support vector machines (SVM) was introduced. The SVM represents a new approach to supervised pattern classification and has been successfully applied to a wide range of pattern recognition problems. In this study, six training datasets consisting of different length local sequence respectively were used. The polynomial kernel functions with different parameter d were chosen. The test for the independent testing dataset and the jackknife test were both carried out. When the local sequence length was 20-residue and the parameter d = 8, the SVM method archived the best performance with the correct rate for the cis and trans forms reaching 70.4 and 69.7% for the independent testing dataset, 76.7 and 76.6% for the jackknife test, respectively. Matthew's correlation coefficients for the jackknife test could reach about 0.5. The results obtained through this study indicated that the SVM method would become a powerful tool for predicting peptidyl prolyl cis/trans isomerization.     

Prolyl cis-trans isomerization as a molecular timer

Proline is unique in the realm of amino acids in its ability to adopt completely distinct cis and trans conformations, which allows it to act as a backbone switch that is controlled by prolyl cis-trans isomerization. This intrinsically slow interconversion can be catalyzed by the evolutionarily conserved group of peptidyl prolyl cis-trans isomerase enzymes. These enzymes include cyclophilins and FK506-binding proteins, which are well known for their isomerization-independent role as cellular targets for immunosuppressive drugs. The significance of enzyme-catalyzed prolyl cis-trans isomerization as an important regulatory mechanism in human physiology and pathology was not recognized until the discovery of the phosphorylation-specific prolyl isomerase Pin1. Recent studies indicate that both phosphorylation-dependent and phosphorylation-independent prolyl cis-trans isomerization can act as a novel molecular timer to help control the amplitude and duration of a cellular process, and prolyl cis-trans isomerization might be a new target for therapeutic interventions.     

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.     

Pin1-dependent prolyl isomerization regulates dephosphorylation of Cdc25C and tau proteins

The reversible protein phosphorylation on serine or threonine residues that precede proline (pSer/Thr-Pro) is a key signaling mechanism for the control of various cellular processes, including cell division. The pSer/Thr-Pro moiety in peptides exists in the two completely distinct cis and trans conformations whose conversion is catalyzed specifically by the essential prolyl isomerase Pin1. Previous results suggest that Pin1 might regulate the conformation and dephosphorylation of its substrates. However, it is not known whether phosphorylation-dependent prolyl isomerization occurs in a native protein and/or affects dephosphorylation of pSer/Thr-Pro motifs. Here we show that the major Pro-directed phosphatase PP2A is conformation-specific and effectively dephosphorylates only the trans pSer/Thr-Pro isomer. Furthermore, Pin1 catalyzes prolyl isomerization of specific pSer/Thr-Pro motifs both in Cdc25C and tau to facilitate their dephosphorylation by PP2A. Moreover, Pin1 and PP2A show reciprocal genetic interactions, and prolyl isomerase activity of Pin1 is essential for cell division in vivo. Thus, phosphorylation-specific prolyl isomerization catalyzed by Pin1 is a novel mechanism essential for regulating dephosphorylation of certain pSer/Thr-Pro motifs.     

Peptidyl-prolyl cis-trans isomerase activity as studied by dynamic proton NMR spectroscopy

Recently the identity of the peptidyl-prolyl cis-trans isomerase (PPIase), which accelerates the cis/trans isomerization of prolyl peptide bonds and cyclophilin, the binding protein for the immunosuppressive drug Cyclosporin A (CsA), was discovered. The PPIase catalysis toward the substrate Suc-Ala-Phe-Pro-Phe-pNA has been studied by 1H NMR spectroscopy. Using the bandshape analysis technique the rate of interconversion between the cis and trans isomers of the substrate could be measured in the presence of PPIase and under equilibrium conditions. The acceleration is inhibited by equimolar amounts of CsA. The results provide evidence that the PPIase catalysis is more complex than a simple exchange between two states.     

Bioactivity of folding intermediates studied by the recovery of enzymatic activity during refolding

The peptide bond preceding proline residues realizes a cis/trans conformational switch with high switching resistance in native proteins and folding intermediates. Therefore, individual isomers have the potential to differ in bioactivity. However, information about isomer-specific bioactivities is difficult to obtain because of the risk of affecting isomeric distribution by bioactivity assay components.Here we present an approach that allows for the measurement of the recovery of enzymatic activities of wild-type RNase T₁ and RNase T₁ variants during refolding under conditions where the population of enzyme-substrate or enzyme-product complexes is negligible. Recovery of enzymatic activity was continuously monitored within the visible range of the spectrum by addition of a fluorescence-labeled nucleotide substrate to the refolding sample. We found that a nonnative trans conformation at Pro39 renders the RNase T₁ almost completely inactive. A folding intermediate having a nonnative trans conformation at Pro55 shows about 46% of the enzymatic activity referred to the native state. Pro55, in contrast to the active site located Pro39, is situated in a solvent-exposed loop region remote from active-site residues. In both cases, peptidyl prolyl cis/trans isomerases accelerate the regain of nucleolytic activity. Our findings show that even if there is a considerable distance between the site of isomerization and the active site, conformational control of the bioactivity of proteins is likely to occur, and that the surface location of prolyl bonds suffices for the control of buried active sites mediated by peptidyl prolyl cis/trans isomerases.     

Role of phosphorylation in the conformation of tau peptides implicated in Alzheimer's disease

A series of peptides corresponding to isolated regions of Tau (tau) protein have been synthesized and their conformations determined by (1)H NMR spectroscopy. Immunodominant peptides corresponding to tau(224-240) and a bisphosphorylated derivative in which a single Thr and a single Ser are phosphorylated at positions 231 and 235 respectively, and which are recognized by an Alzheimer's disease-specific monoclonal antibody, were the main focus of the study. The nonphosphorylated peptide adopts essentially a random coil conformation in aqueous solution, but becomes slightly more ordered into beta-type structure as the hydrophobicity of the solvent is increased by adding up to 50% trifluoroethanol (TFE). Similar trends are observed for the bisphosphorylated peptide, with a somewhat stronger tendency to form an extended structure. There is tentative NMR evidence for a small population of species containing a turn at residues 229-231 in the phosphorylated peptide, and this is strongly supported by CD spectroscopy. A proposal that the selection of a bioactive conformation from a disordered solution ensemble may be an important step (in either tubulin binding or in the formation of PHF) is supported by kinetic data on Pro isomerization. A recent study showed that Thr231 phosphorylation affected the rate of prolyl isomerization and abolished tubulin binding. This binding was restored by the action of the prolyl isomerase Pin1. In the current study, we find evidence for the existence of both trans and cis forms of tau peptides in solution but no difference in the equilibrium distribution of cis-trans isomers upon phosphorylation. Increasing hydrophobicity decreases the prevalence of cis forms and increases the major trans conformation of each of the prolines present in these molecules. We also synthesized mutant peptides containing Tyr substitutions preceding the Pro residues and found that phosphorylation of Tyr appears to have an effect on the equilibrium ratio of cis-trans isomerization and decreases the cis content.     

Multiple parallel-pathway folding of proline-free Staphylococcal nuclease

When a protein exhibits complex kinetics of refolding, we often ascribe the complexity to slow isomerization events in the denatured protein, such as cis/trans isomerization of peptidyl prolyl bonds. Does the complex folding kinetics arise only from this well-known reason? Here, we have investigated the refolding of a proline-free variant of staphylococcal nuclease by stopped-flow, double-jump techniques, to examine the folding reactions without the slow prolyl isomerizations. As a result, the protein folds into the native state along at least two accessible parallel pathways, starting from a macroscopically single denatured-state ensemble. The presence of intermediates on the individual folding pathways has revealed the existence of multiple parallel pathways, and is characterized by multi-exponential folding kinetics with a lag phase. Therefore, a "single" amino acid sequence can fold along the multiple parallel pathways. This observation in staphylococcal nuclease suggests that the multiple folding may be more general than we have expected, because the multiple parallel-pathway folding cannot be excluded from proteins that show simpler kinetics.     

Effects of i and i+3 residue identity on cis-trans isomerism of the aromatic(i+1)-prolyl(i+2) amide bond: implications for type VI beta-turn formation

Cis-trans isomerization of amide bonds plays critical roles in protein molecular recognition, protein folding, protein misfolding, and disease. Aromatic-proline sequences are particularly prone to exhibit cis amide bonds. The roles of residues adjacent to a tyrosine-proline residue pair on cis-trans isomerism were examined. A short series of peptides XYPZ was synthesized and cis-trans isomerism was analyzed. Based on these initial studies, a series of peptides XYPN, X = all 20 canonical amino acids, was synthesized and analyzed by NMR for i residue effects on cis-trans isomerization. The following effects were observed: (a) aromatic residues immediately preceding Tyr-Pro disfavor cis amide bonds, with K(trans/cis)= 5.7-8.0, W > Y > F; (b) proline residues preceding Tyr-Pro lead to multiple species, exhibiting cis-trans isomerization of either or both X-Pro amide bonds; and (c) other residues exhibit similar values of K(trans/cis) (= 2.9-4.2), with Thr and protonated His exhibiting the highest fraction cis. beta-Branched and short polar residues were somewhat more favorable in stabilizing the cis conformation. Phosphorylation of serine at the i position modestly increases the stability of the cis conformer. In addition, the effect of the i+3 residue was examined in a limited series of peptides TYPZ. NMR data indicated that aromatic residues, Pro, Asn, Ala, and Val at the i+3 residue all favor cis amide bonds, with aromatic residues and Asn favoring more compact phi at Tyr(cis) and Ala and Pro favoring more extended phi at Tyr(cis). D-Alanine at the i+3 position particularly disfavors cis amide bonds.