Solution structure of the parvulin-type PPIase domain of Staphylococcus aureus PrsA – Implications for the catalytic mechanism of parvulins
© Heikkinen et al; licensee BioMed Central Ltd. 2009
Received: 04 December 2008
Accepted: 24 March 2009
Published: 24 March 2009
Staphylococcus aureus is a Gram-positive pathogenic bacterium causing many kinds of infections from mild respiratory tract infections to life-threatening states as sepsis. Recent emergence of S. aureus strains resistant to numerous antibiotics has created a need for new antimicrobial agents and novel drug targets. S. aureus PrsA is a membrane associated extra-cytoplasmic lipoprotein which contains a parvulin-type peptidyl-prolyl cis-trans isomerase domain. PrsA is known to act as an essential folding factor for secreted proteins in Gram-positive bacteria and thus it is a potential target for antimicrobial drugs against S. aureus.
We have solved a high-resolution solution structure of the parvulin-type peptidyl-prolyl cis-trans isomerase domain of S. aureus PrsA (PrsA-PPIase). The results of substrate peptide titrations pinpoint the active site and demonstrate the substrate preference of the enzyme. With detailed NMR spectroscopic investigation of the orientation and tautomeric state of the active site histidines we are able to give further insight into the structure of the catalytic site. NMR relaxation analysis gives information on the dynamic behaviour of PrsA-PPIase.
Detailed structural description of the S. aureus PrsA-PPIase lays the foundation for structure-based design of enzyme inhibitors. The structure resembles hPin1-type parvulins both structurally and regarding substrate preference. Even though a wealth of structural data is available on parvulins, the catalytic mechanism has yet to be resolved. The structure of S. aureus PrsA-PPIase and our findings on the role of the conserved active site histidines help in designing further experiments to solve the detailed catalytic mechanism.
Staphylococcus aureus is a Gram-positive bacterium causing many kinds of infections from mild respiratory tract infections to life-threatening states as sepsis. It produces many toxins and has a remarkable ability to acquire resistance to antimicrobial drugs. Many S. aureus strains have acquired resistance to commonly used antibiotics and some strains are becoming multi-resistant. Methicillin-resistant strain of Staphylococcus aureus (MRSA) is the principal cause of severe nosocomial infections which can be fatal to compromised patients. Whole genome sequencing of two MRSA strains in 2001 was regarded as a way to find targets for novel antibiotics against infections caused by MRSA .
PrsA protein is found ubiquitously in Gram-positive bacteria, including S. aureus [Swiss-Prot:P60747], but not in Gram-negative ones [2, 3]. By sequence homology PrsA contains a parvulin-type peptidyl-prolyl cis-trans isomerase (PPIase) domain and flanking N- and C-terminal domains. PPIases are enzymes that catalyze cis-trans-isomerization of the peptide bonds preceding proline residues . Biological role of PPIases is to act as chaperones or foldases in protein folding and remodelling. FK506 binding proteins (FKBPs), cyclophilins and parvulins form the three classes of PPIases each having their own fold, substrate specificity and catalytic mechanism.
PrsA is localized at the space between plasma membrane and cell wall and it is bound to the cell membrane through a lipid-anchor attached to the N-terminal cysteine residue [2, 3]. It has been shown to have a role as folding catalyst of secreted proteins. In bacteria, secreted proteins include enzymes important for formation of the cell wall and toxins. Due to importance of the catalyzed reaction in protein folding PrsA is a potential target for novel antimicrobial drugs. PrsA has been previously shown to be an essential protein for viability of B. subtilis .
Parvulin-type PPIases are ~100 residues long globular protein domains folding into a four-stranded antiparallel β-sheet core surrounded by four α-helices (βα3βαβ2 parvulin-fold) . Parvulin-type PPIases have been found both in bacteria and in eukaryotes. At present there are structures of 7 different parvulins available in the Protein Data Bank: human Pin1 (e.g. [PDB:1PIN, 1NMV and 1NMW]) [5, 6] and Par14 [PDB:1EQ3], Pin1At from Arabidopsis thaliana [PDB:1J6Y], Par10 [PDB:1JNS]  and SurA [PDB:1M5Y] from Escherichia coli, Ess1 from Candida albicans [PDB:1YW5] and PrsA-PPIase from Bacillus subtilis [PDB:1ZK6]. Also several other parvulin-type PPIases are known, e.g. Par27 from Bordetella pertussis , but their structures are still to be solved. The subtypes of parvulins differ in length and composition of the S1-H1 loop. In hPin1-type parvulins the loop has a high number of positively charged residues and this is thought to induce the preference for substrates having a negatively charged residue, preferably a phosphorylated serine/threonine, before the processed proline . In Par14-type parvulins this loop is missing and in SurA PPIase domain I the S1-H1 loop contains mainly hydrophobic residues [7, 10].
PrsA of S. aureus shows 24% amino acid sequence conservation to PrsA protein from Bacillus subtilis [Swiss-Prot:P24327]. The PPIase domain is the most conserved area of the sequence (42% of the residues conserved). Sequence comparison of B. subtilis and S. aureus PrsA-PPIases shows that they differ in length and nature of the S1-H1 loop. S. aureus PrsA-PPIase contains a long loop rich of lysine residues whereas in B. subtilis PrsA the loop is very short. This suggests that the structure and the substrate specificity of S. aureus PrsA-PPIase would rather resemble hPin1-type parvulins than B. subtilis PrsA-PPIase.
Since PrsA is known to be an essential protein for other gram-positive bacteria  it is a potential target for antimicrobial drugs against S. aureus infections. Exact knowledge of the structure and catalysis mechanism of PrsA-PPIase is a prerequisite for successful design of efficient and selective enzyme inhibitors to be used as antibacterial agents against Gram-positive bacteria. We have studied structure and function of the parvulin-type PrsA-PPIase from S. aureus using NMR spectroscopy.
Protease-coupled PPIase assay
Structure statistics of PrsA-PPIase
Total distance restraints
Short-range |i - j| ≤ 1
Medium-range, 1 < |i - j| < 5
Long-range, |i - j| ≥ 5
Restraints per residue
Maximum NOE restraint violation (Å)
Number of NOE violations > 0.10 Å
3 ± 2
Average restraint violation energy (kcal/mol ± SD)
9.55 ± 0.86
Average AMBER energy (kcal/mol ± SD)
-3259.69 ± 8.55
RMS deviations from ideal covalent geometry
Bond lengths (Å ± SD)
0.0096 ± 0.0001
Bond angles (° ± SD)
1.93 ± 0.02
Atomic coordinate RMSD (Å ± SD) for residues 140–243 and (140–152, 160–243)
0.55 ± 0.18 (0.31 ± 0.05)
1.07 ± 0.20 (0.80 ± 0.06)
Ramachandran map regions (%)
Residues in most favoured regions
Additionally allowed regions
Generously allowed regions
Dynamics and exchange
In this study we have investigated the structure and function of the parvulin-type PPIase domain of PrsA protein from S. aureus. NMR spectroscopic structure determination of PrsA-PPIase yielded a high-quality structure which enabled investigation of the catalytic site in detail. Solution structure of PrsA-PPIase shows close structural similarity to hPin1-type parvulins but also some important differences in constitution of the active site. The original hypothesis on the catalysis mechanism of the parvulin-type PPIases is based on the crystal structure of hPin1 . However, the recent studies of hPin1 [21–23] have provided new insight into the functional status of the active site residues and thus have brought the original catalysis mechanism into question. The solution structure of S. aureus PrsA-PPIase supports these findings but also brings out some new aspects into the debate.
The results of protease-coupled PPIase assay indeed confirm that PrsA-PPIase functions as a prolyl-isomerase. The most efficient catalysis was observed with Suc-AEPF-p NA peptide. The substrate preference of S. aureus PrsA-PPIase resembles that of hPin1 which was somewhat expected based on common S1-H1 loop rich of positively charged residues. Binding of multivalent anions to the S1-H1 loop of PrsA-PPIase was also confirmed by NMR titrations with sodium sulphate (data not shown). Clear chemical shift perturbations resembling the ones Bayer et al. observed with hPin1  were detected at the S1-H1 loop. Paradoxically, the protease-coupled PPIase assay showed practically no prolyl-isomerase activity towards Suc-A(pS/pT)PF-p NA peptides.
The active site of PrsA-PPIase was mapped by NMR titrations with parvulin substrate peptides. Our results conform well with the previous studies with other parvulins [5, 12]. Largest chemical shift changes occurred at H3 helix, S2 strand and at S2-H4 and S3-S4 loops which face the active site and contain the residues thought to participate in the catalysis mechanism (Figure 6). Based on the NMR titrations the dissociation constant for all tested peptides was in millimolar regime and most of the spectral changes were practically the same with all the three peptides. During the Suc-AEPF-p NA peptide titration, but not with the other peptides, we observed consistent chemical shift perturbations at the S1-H1 loop. Backbone amide titration data demonstrates involvement of the S1-H1 loop in substrate binding when the substrate contains a negatively charged glutamate residue before the processed proline.
Referring to previously published NMR titration data, Bailey et al. concluded recently that parvulin active site histidines are not involved in substrate binding . It should be noted, however, that NMR chemical shift perturbation studies are commonly done using only backbone N-H correlations (i.e. using 1H-15N-HSQC spectrum). Participation of the active site histidines (H146 and H239) in the substrate binding is not easily observed in 1H-15N-HSQC-based NMR titrations since backbone amides reside quite far from the peptide binding site. Involvement of the histidine side chains in substrate binding is however clearly evidenced by the chemical shift perturbations in the 1H-13C-HSQC spectrum of the aromatic residues (see Figure 3).
The original parvulin catalysis mechanism presented by Ranganathan et al.  has been disproved [21–23]. Comparison of the active site structure of S. aureus PrsA-PPIase and other parvulin PPIases questions the residues proposed to be responsible for the catalysis in parvulins . The cysteine residue C113 of hPin1 was originally claimed to act as a nucleophile starting the catalysis (Figure 10b). This residue is replaced in S. aureus and in B. subtilis PrsAs by an aspartate (D194 and D154, respectively) which is also a potential nucleophile. In fact, Behrsin et al. have proved that the Pin1 C113D mutant remains functional . Forthcoming steps of the original catalysis mechanism  include participation of a serine residue (S154 of hPin1) acting as a proton donor. In S. aureus PrsA-PPIase this residue is replaced by phenylalanine (F236) which is not capable of carrying out protonation/deprotonation steps. The same situation is also faced with other parvulin PPIases e.g. E. coli Par10  and hPar14 . In some parvulin PPIases this residue is replaced by valine and for example in B. subtilis PrsA by tyrosine . Evidently the original catalysis mechanism proposed based on the crystal structure of hPin1 cannot be a universal route of the reaction for all parvulins. A closer inspection of the active site of S. aureus PrsA-PPIase reveals a somewhat symmetric assembly of aspartate and serine residues on both sides of the histidine pair (Figure 10a). Similar set of residues is also found in B. subtilis PrsA . Mutation studies of B. subtilis PrsA have shown that D154A substitution (corresponding to D194 in S. aureus PrsA) destroys only half of the catalytic activity of PrsA . Obviously some other residue can perform the role of the nucleophilic residue when it is inactivated by mutation. The symmetrical assembly of aspartates and serines and the charge relay system through the active site histidines would imply a protonation/deprotonation step as part of the catalytic mechanism. The charge relay system (Figure 10c) could facilitate deprotonation of the aspartates which would enhance their nucleophilic character. In light of the diverse structural and functional data on parvulin PPIases one inevitably raises a question whether all parvulin PPIases even share the same catalysis mechanism.
The solution structure of PrsA-PPIase from S. aureus enables detailed study of its function and target based design of inhibitors. Highly conserved protein sequences are also found in other Staphylococcus subspecies. Exact biological role and importance of PrsA are still unclear although it is known to act as a foldase of secreted proteins (e.g. bacterial toxins)  and it is shown to be essential for B. subtilis . Natural substrates of S. aureus PrsA-PPIase are not known at present, but the enzyme may prefer substrates where a negatively charged residue precedes the processed proline. The structure of the catalytic site of S. aureus PrsA-PPIase conflicts with the original hypothetical catalysis mechanism of parvulin PPIases. Recent studies also recognize the deficiencies of the parvulin catalysis mechanism [21–23]. The orientation and the tautomeric state of the active site histidine residues of S. aureus PrsA-PPIase suggest that the catalytic mechanism includes a protonation/deprotonation step facilitated by a charge relay system through the active site histidine pair. On the other hand, the hydrogen bonding between the active site histidines might merely serve as a structural stabilisation mechanism of the enzyme fold. Apparently the catalysis mechanism of parvulin-type PPIases still needs some clarifications. Existing structural data on parvulins can be used to design further experiments, e.g. site-directed mutagenesis, to decipher the detailed catalysis mechanism.
Protein expression and purification
The PPIase domain (residues 140–245) was expressed as glutathione S-transferase (GST) fusion. The protein was overexpressed in E. coli BL21 strain containing the pGEX-2T expression vector (GE Healthcare). For enzymatic studies, the cells were grown and harvested as described earlier . For NMR samples, the cells were grown in M9 medium containing either 15NH4Cl as the sole nitrogen source or [13C6]-D-glucose/15NH4Cl as the sole carbon and nitrogen sources, respectively. The expression of protein was induced by addition of 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at A600 of 0.8. The cells were grown 4 additional hours and harvested. For both enzymatic and NMR studies the cells were broken by French Press and centrifuged. The supernatant was applied to a glutathione-Sepharose FF (GE Healthcare) column, and washed with phosphate-buffered saline (PBS). The precission protease was added to the column and incubated 4 h at +5°C to release the protein. The cleaved protein was eluted with PBS, and the fractions containing the protein were concentrated with Vivaspin 2 (Sartorius Stedim Biotech). For NMR samples, the buffer was changed to 20 mM Bis-Tris pH 6.8, and D2O was added to the final concentration of 8% (v/v). The final protein concentration was ~1 mM.
Protease-coupled PPIase assay
Prolyl isomerase activity of PrsA-PPIase was determined with α-chymotrypsin-coupled PPIase assay as described by Fischer et al. . The catalytic activity was tested with synthetic succinyl-AXPF-p-nitroanilide (Suc-AXPF-p NA) peptides where X is alanine (A), lysine (K), asparagine (N), glutamic acid (E), phosphoserine (pS) or phosphothreonine (pT). The peptides with A, K and E were purchased from Bachem (Bubendorf, Switzerland). Suc-ANPF-p NA was synthesized by Ale Närvänen in University of Kuopio, Finland. The phosphorylated peptides were purchased from EZBiolab Inc. (Westfield, IN). p-Nitroanilide was cleaved off by α-chymotrypsin and the increase of released p-nitroanilide was monitored in absorbance at 390 nm. Cyclophilin from calf thymus (Sigma-Aldrich) was used as a positive control.
NMR spectroscopy for the structure determination was performed on Varian INOVA 600 MHz and 800 MHz spectrometers with 5 mm inverse z-gradient triple-resonance probe heads at 25°C. The acquisition and processing were conducted with VNMR 6.1C software (Varian Inc., Palo Alto, CA). A conventional set of three-dimensional triple resonance experiments i.e. iHNCA , HN(CO)CA, HNCACB, HN(CO)CACB, HNCO, HN(CA)CO [28, 29] was recorded for sequential backbone assignment. The aliphatic side chain resonances were assigned using three-dimensional HCCH-COSY and HCCH-TOCSY experiments with the help of CC(CO)NH and HCC(CO)HN experiments . (Hβ)Cβ(CγCδ)Hδ, (Hβ)Cβ(CγCδCε)Hε  experiments and 13C-edited three-dimensional HSQC-NOESY spectrum were used in assignment of aromatic side chain resonances. Sparky 3.110 program  was used to analyze the NMR spectra.
The distance restraints for structure calculation were extracted from signal intensities of 15N- and 13C-edited three-dimensional HSQC-NOESY spectra. Automated NOESY signal assignment and structure calculation was conducted with CYANA 2.1 software . In addition to NOE derived distance restraints, 146 φ and ψ dihedral angle constraints (average of the TALOS database hits used in the prediction ± 2 SD) were generated from chemical shift data with TALOS program (version 2003.027.13.05) . After torsion angle dynamics run 40 structures were chosen from 400 calculated structures based on lowest target function value. These 40 structures were refined with molecular dynamics using Born implicit solvent model in AMBER 8.0 . The final ensemble of 25 structures was chosen based on lowest AMBER energy and restraint violation energy. Quality of the final structures was analyzed with PROCHECK-NMR  and WHAT_CHECK  programs. Tautomeric state of the active site histidines H146 and H239 was determined using JCN intensity modulated constant time 1H-13C-HSQC spectrum  both in presence and in absence of the substrate peptide Suc-AEPF-p NA. Molecule visualization programs MOLMOL  and PyMOL  were used in preparation of the figures representing the protein structure.
The Suc-AXPF-p NA tetrapeptides, where X = A, K or E, were tested for binding to PrsA-PPIase. The 1H-15N-HSQC-based titration experiments were conducted with 0.3 mM 15N-labeled PrsA-PPIase samples adding the unlabeled peptide as concentrated solution in sample buffer. The 1H-15N-HSQC spectrum was recorded after each peptide addition. Large excess of peptide was used at the last titration point to obtain high proportion of ligand-bound form of the protein. Total chemical shift change of the backbone amide signals at the titration end-point was calculated with the equation Δδ = [(0.17* ΔδN)2+(ΔδH)2]1/2. Determination of 15N relaxation rates, heteronuclear NOEs, amide proton exchange rates and tautomeric state of the histidine side chains in presence of Suc-AEPF-p NA peptide substrate were done at the titration end point (20-fold excess of peptide to protein).
15N R1 and R2 relaxation rates of the backbone amide groups were measured using three-dimensional relaxation rate-resolved 1H-15N-HSQC spectra [35, 36]. Inverse Laplace transform was applied to the relaxation dimension enabling extraction of the relaxation rate constants simply by peak picking. Heteronuclear NOEs of the backbone amide nitrogens were determined with conventional methods . The data for the analysis of protein dynamics was recorded with a Bruker DRX 500 MHz spectrometer equipped with a 5 mm z-gradient inverse broadband probehead from 0.3 mM 15N-labeled PrsA-PPIase sample. Generalized order parameters for each backbone amide were extracted from the relaxation data using Modelfree 4.1 program [38, 39] with FASTModelfree interface . Proton exchange rates between backbone amides and water were measured through exchange rate-resolved 1H-15N-HSQC spectrum . Relaxation and exchange rates as well as heteronuclear NOEs were determined both in absence and in presence of substrate peptide Suc-AEPF-p NA.
The resonance assignments of S. aureus PrsA-PPIase and the distance constraints used in structure calculation have been deposited in BioMagResBank under accession number 15628. The atomic coordinates of S. aureus PrsA-PPIase structure ensemble have been deposited in Protein Data Bank under accession code 2JZV.
This work was supported by The Finnish Academy (grant no. 123318 to IK), Sigrid Juselius Foundation (to PP & IK), and Helsinki University's Research Funds (to PP). We thank Tuula Lunden, Ph.D., for preparation of chromosomal DNA from S. aureus and Anne Hakonen for excellent technical assistance.
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