X-ray structure of peptidyl-prolyl cis – trans isomerase A from Mycobacterium tuberculosis Lena M. Henriksson 1 , Patrik Johansson 1 , Torsten Unge 1 and Sherry L. Mowbray 2 1 Department of Cell and Molecular Biology, Uppsala University, Sweden; 2 Department of Molecular Biology, Swedish University of Agricultural Sciences, Uppsala, Sweden Peptidyl-prolyl cis–trans isomerases (EC 5.2.1.8) catalyse the interconversion of cis and trans peptide bonds and are therefore considered to be important for protein fold ing. They are also t hought to participate i n processes such as signalling, c ell surface recognition, chaperoning a nd heat- shock r esponse. Her e we report the soluble e xpression of recombinant Mycobacterium tuberculosis peptidyl-prolyl cis–trans isomerase PpiA in Escherichia coli, together with an investigation of its structure and biochemical properties. The protein w as shown t o be active in a spectrophotometric assay, with an estimated k cat /K m of 2.0 · 10 6 M )1 Æs )1 .The X-ray structure of PpiA was solved by molecular replace- ment, and r efined t o a resoluti on of 2.6 A ˚ with R and R free values of 21.3% and 22.9%, respectively. Comparisons to known structures show that the PpiA represents a slight variation o n t he peptidyl-prolyl cis–trans isomerase fold, previously not represented in the Protein Data Bank. Inspection of the active site suggests that specificity f or substrates and cyclosporin A will be similar to that found for most other enzymes of this structural family. Comparison to the sequence o f the second M. tuberculosis enzyme, P piB, suggests that binding of peptide substrates as well as cyclosporin A may differ in that case. Keywords: cyclophilin; peptidyl-prolyl cis-trans isomerase; PPIase; rotamase; Rv0009. According to the World H ealth Organization (http:// www.who.org), Mycobacterium tuberculosis, the causative pathogen of tuberculosis, currently infects one-third of the world’s population, and r esults in 2 million deaths each year. Due to the increased prevalence of drug-resistant and multidrug-resistant strains, a nd the lethal combination of tuberculosis and HIV, there is a great need for new therapies and drugs, as well a s better knowled ge of the bacteria’s basic biology. Cyclophilins, also known as rotamases or p eptidyl-prolyl cis–trans isomerases (Ppis), catalyse the cis –trans isomeri- zation of peptide bonds, preferring those preceding proline residues [2,3]. Ppis are found in many diverse organisms such as bacte ria, plants, and mammals, sometimes as single domain proteins and sometimes as components in a larger complex [ 4,5]. Multiple Ppis within a single organism are common. Their activity can accelerate protein folding both in vitro and in vivo; in some cases a chaperone function has been demonstrated to be independent of the catalytic action. Ppis also bind to and mediate the biological effects of the immunosuppressive agent cyclosporin A [6]. A complex of Ppi with cyclosporin A b inds to the protein phosphatase calcineurin, so inhibiting signal transduction in T cells [7]. As a result cyclosporin A is one of the most important drugs used for prevention of g raft rejection after transplant surgery [8]. Ppis a re also suggested t o take p art in other biological functions such as cell surface recognition [9] and heat-shock response [10]. M. tuberculosis has two distinct Ppi enzymes [11] (http:// genolist.pasteur.fr/TubercuList/). W e report here the clo- ning, expression, purification and X-ray structure of Rv0009, the putative P piA from this bacterium (MtPpiA) and demonstrate that it h as peptidyl-prolyl cis –trans isomerase activity. These results are discussed in the context of other sequence, structural and biochemical data. Experimental procedures Cloning, protein expression and purification The open reading frame encoding MtPpiA (Rv0009) was amplified by PCR from M. tuberculosis DNA strain H37Rv [11] using t he primers 5¢-ATGGCAGACTGTGATTC CGTGAC-3¢ (forward) and 5¢-CTAGGAGATGGTG ATCGACTCG-3¢ (reverse), and Taq DNA polymerase (Roche). An additional PCR was performed using the product from the first PCR a s template, and t he same reverse primer, but substituting the forward primer for 5¢-ATGGCCCATCATCATCATCATCATTCTGGTGC AGACTGTGATTCCGTGAC-3¢, in order to introduce an N-terminal His 6 tag. The PCR product was ligated into the Correspondence to S. Mowbray, Department of Molecular Biology, Swedish University of Agricultural Sciences, Uppsala Biomedical Center, Box 590, SE-751 24 Uppsala, Sweden. Fax: +46 18 53 69 71, Tel.: +46 18 471 49 90, E-mail: mowbray@xray.bmc.uu.se Abbreviations: Ppi, peptidyl-prolyl cis-trans isomerase; MtPpiA, PpiA from M. tuberculosis; PDB, Protein Data Bank. Enzyme: Peptidyl-prolyl cis–trans isomerases (EC 5.2.1.8). Note: Coordinates and structure factor data have been deposited at the PDB [Berman, H.M., Westbrook, J., Feng, Z., G illiland, G., Bhat, T.N., Weissig, H., Shindyalov, I.N. & Bourne, P.E. (2000) Nucleic Acids Res. 28, 235–242.] with entry code 1w74. (Received 7 July 2004, revised 24 August 2004, accepted 27 August 2004) Eur. J. Biochem. 271, 4107–4113 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04348.x pCRÒT7/CT-TOPOÒ vector using the pCR T7/CT TOPO TAÒ Express kit (Invitrogen), and then trans formed into E. coli TOP10F¢ cells (Invitrogen). Positive clones were selected on Luria agar plates containing 50 lgÆmL )1 ampi- cillin. Twelve colonies were picked and cultured for plasmid preparation using the QIAprepÒ Spin Miniprep kit proto- col (Qiagen). An analytical PCR was performed using the pCR T7/CT TOPO TAÒ Expression kit (Invitrogen), with the v5 (C-terminal) reverse primer, and the H is 6 forward primer. P lasmid (pCRT7::Rv0009) from one of the f our clones with the correct size insert was transformed i nto E. coli BL21-AI TM cells (Invitrogen). I n a test expression, cells were induced w ith 0.1 mg ÆmL )1 arabinose for 2 h at 37 °C. The a pparent molecular weight o f the expressed protein as deduced from SDS/PAGE was in agreement with the theoretical value, 20 kDa. The i solated gene encoding MtPpiA was further verified by DNA sequence ana- lysis (Uppsala Genome Center, Rudbeck Laboratory). On the p reparative scale, BL21-AI TM cells containing pCRT7::Rv0009 were grown in Luria broth, with 50 lgÆmL )1 ampicillin and 12 lgÆmL )1 tetracycline, at 37 °CtoD 550 ¼ 0.7–1.0. The culture was then transferred to 22 °C and induced with 0.001% (w/v) arabinose. Growth was continued for 2 h, after which the cells were harvested, washed w ith 1 · SSPE buffer (150 m M NaCl, 10 m M NaH 2 PO 4 pH 7.5, 1 m M EDTA), and stored at )20 °C. Thawed cells were treated with lysis buffer (50 m M NaH 2 PO 4 pH 8 .0, 300 m M NaCl, 10 m M imidazole, 4% glycerol) with 0.01 mgÆmL )1 RNase, 0.02 mgÆmL )1 DNase, and l ysed by using a Constant Cell Disruptio n System (Constant Systems Ltd) operated at 1.5 kbar. The cell lysate containing soluble Mt PpiA, was incubated for 30 min a t 4 °C with N i–NTA Agarose slurry (Qiagen) pre-equili- brated with native lysis buffer. The resin was washed with 10 column volumes l ysis buffer c ontaining 20 m M imidazole, and the protein eluted with four column volumes of the same buffer containing 250 m M imidazole. The protein was further purified on a size exclusion chromatography column (HiLoad TM 16/60 Superdex TM 75, Amersham Pharmacia Biotech), using a buffer containing 150 m M NaCl, and 20 m M Tris/HCl pH 7.5. Fractions containing MtPpiA were pooled and desalted using a PD10 column (Amersham Biosciences) with a solutio n of 10 m M 2-mercaptoethanol, and 20 m M Tris pH 7.5. The protein was concentrated to 29 mgÆmL )1 (based on the calculated absorbance o f 0.252 for a 1 mgÆmL )1 solution at 280 nm) using a Vivaspin concentrator (Vivascience) with a molecular cut-off of 10 kDa. The purification was monitored by SDS and native PAGE (PhastSystem TM , Amersham Biosciences). Assay The activity of MtPpiA was evaluated u sing a spectropho- tometric assay [ 12], in which the cisfitrans isomerization is measured using the chromogenic peptide N-succinyl- Ala-Ala-Pro-Phe-p-nitroanilide (Sigma). Peptide solution (7.8 m M ) was prepared the previous day in trifluorethanol with 0.45 M LiCl, in order to increase the fraction of the cis isomer [13]. Each assay included 910 lL0.1 M Tris/HCl pH 8.0 (maintained at 15 °C), 50 lL 600 l M a-chymotryp- sin, and 30 lLofMtPpiA (at 1.7 l M ,0.49l M or 0.33 l M ), which were mixed and preequilibrated in a cuvette at 1 5 °C for 2 min. The assay was initiated b y adding 10 lLof peptide solution resulting in a final c oncentration of 78 l M . The cisfitrans isomerization o f t he Ala-Pro bond (both spontaneous and enzyme-catalysed), coupled with the a-chymotryp sin cleavage of the trans peptide, was followed by the increase in absorbance at 390 nm at 15 °C(DUÒ 640 spectrophotometer, Beckman). Measurements were made every 0.5 s during 3 min. The final absorbance was estima- ted from e ach curve; the absorbance at each time point was subtracted from that value. A plot of the natural logarithm of these differences vs. time was linear for at least 10 s. The slope of this line was used to get an estimate of k obs ¼ (k cat / K m ) · [MtPpiA] for each experiment. A plot of k obs vs. [MtPpiA] gives a line with slope k cat /K m . Crystallization MtPpiA was cocrystallized, with a hexapeptide o f sequence HAGPIA [14] using vapour diffusion. The sitting drops contained 2 lL protein, with a final concentration of 1 m M of the p eptide dissolved in dimethyl sulfoxide, and 2 lL reservoir solution [30% (v/v) PEG-200, 5% (w/v) PEG 3000, 0.1 M MES/HCl pH 6 .0], at 22 °C. Needle-like crystals appeared within a few weeks. Crystallization conditions were optimized with the a id of seeding, and crystals grew in hanging drops to a size o f 0 .05 · 0.05 · 0.4 mm 3 over a period of 2–3 months. Prior to flash cooling, the crystals were p laced for 12 h in a d rop containing the reservoir solution plus 1 m M peptide, to favour peptide binding. Data collection, structure determination and refinement Data were collected under c ryo conditions at beam line ID14-2 at the European Synchrotron R adiation Facility (ESRF), Grenoble, equipped w ith an ADSC Q4 C CD detector (Area Detector Systems C orp.), k ¼ 0.933 A ˚ . Indexing and i ntegration of the d iffraction data were performed using MOSFLM [15], and the data were processed with SCALA [16] in space group P3 1 . The preliminary data set was 99.9% complete to 3.4 A ˚ resolution with an overall R meas of 0.13. The Matthews coefficient [17] suggested two or three molecules in the asymmetric unit; this value was predicted to be 3.1 with two molecules (60% solvent) and 2.1 with three molecules (40% solvent). Inspection of cumulative intensity distributions and other statistics indi- cated that no twinning was present. Exploiting the pseudo- translational symmetry observed in the native Patterson map, the structure was solved by means of molecular replacement using AMORE [18], with the human PpiA (Protein Data Bank (PDB) entry 1AWR [14], 37% sequence identity) as a search model. Two molecules were located in this way. The results of the molecular replacement solution were used, together with the MtPpiA sequence, to build the first model with the program SOD [19]. Initial refinement was performed using NCSREF and REFMAC 5[20]asimplemented in the CCP 4 program suite [21]. Rebuilding was carried out with the p rogram O [22]. A higher resolution data set was then collected at beam line I D14-1 ESRF, with an ADSC Q4R CCD detector (Area Detector Systems Corp.), k ¼ 0.934 A ˚ , e xtending to 2.3 A ˚ in two directions. How- ever, as observed for the earlier s et, t he diffraction was 4108 L. M. Henriksson et al. (Eur. J. Biochem. 271) Ó FEBS 2004 strongly anisotropic. Inspection of a number of criteria at various stages of the s olution and refinement (includ ing R free , figure of merit, map quality, etc.) s uggested that the 2.6 A ˚ cut-off was optimal. Data c ollection statistics for this set are shown in Table 1. The final rounds of refinement were carried out with REFMAC 5 using noncrys- tallographic symmetry restraints. Different weights were tested in the refinement, to find the best combination of R free and stereochemistry. Thirteen water molecu les were added after analysing the results from ARP/WARP water-building routines [23]. Final refinement statistics are shown in Table 1 . Coordinates and structure factor data have been deposited at the PDB with en try code 1w74. BLAST [24] was used for identifying similar sequences and structures. INDONESIA (http://xray.bmc.uu.se/dennis/ manual/) was used for ad ditional sequence and stru cture comparisons. Pictures were p repared using O , MOLRAY [25] and INDONESIA . Results and Discussion Enzyme properties The p rotein corresponding to MtPpiA with a His 6 tag attached to the N -terminus was e xpressed i n E. coli,and purified. It behaved as a homogeneou s monomer in size exclusion chromatography. Enzyme activity was shown using a spectrophotometric assay where the cisfitrans isomerization is m easured in a cou pled assay using the chromogenic peptide N-succinyl-Ala-Ala-Pro-Phe-p-nitro- anilide and a-chymotrypsin at 15 °C (Fig. 1). U nder t hese conditions, Mt PpiA has a k cat /K m of 2.0 · 10 6 M )1 Æs )1 ,and therefore shows s imilar activity to the Ppi from Brugia malayi,withak cat /K m of 7.9 · 10 6 M )1 Æs )1 [26], and to human PpiA, with a k cat /K m of 1.4 · 10 7 M )1 Æs )1 [27]. Ppis of this class process peptide substrates with quite b road specificity [5], and so the o bserved activity o f MtPpiA is likely to reflect that with physiologically relevant substrates. Overall structure TheX-raystructureofMtPpiA (182 residues, molecular mass 19.2 kDa) was solved by molecular replacement, using the structure of human P piA [14] a s a search model. A strong peak in the native Patterson map (Fig. 2) assisted in the location o f the two m olecules in the as ymmetric unit. Data collection and refinement statistics are shown in Table 1. Table 1. Data collection and refinement statistics. Values in parenthesis are for the highest resolution shell. Data collection statistics Cell axial lengths (A ˚ ) 65.3, 65.3, 102.5 Space group P3 1 Resolution range (A ˚ ) 32.62–2.60 (2.74–2.60) Number of reflections measured 80 277 Number of unique reflections 14 896 Average multiplicity 5.4 (5.4) Completeness (%) 99.6 (99.8) R meas 0.096 (0.590) <I>/<rI> 6.2 (1.3) Refinement statistics Resolution range (A ˚ ) 30.0–2.60 (2.67–2.60) Number of reflections used in working set 14,230 Number of reflections for R free calculation 756 R, R free (%) 21.3, 22.9 Number of nonhydrogen atoms 2575 Number of solvent waters 13 Mean B-factor, protein atoms (A ˚ 2 ) 61.8 Mean B-factor, solvent atoms (A ˚ 2 ) 51.9 Ramachandran plot outliers (%) a 3.4 rmsd from ideal bond length (A ˚ ) b 0.012 rmsd from ideal bond angle (°) b 1.4 a Calculated using a strict boundary Ramachandran plot [35]. b Using the parameters of Engh and Huber [36]. Fig. 1. Isomerization activity of MtPpiA. Activity of MtPpiA, at final enzyme concentrations of 50 n M (black l ine), 15 n M (dark g rey line), and 10 n M (grey line), measured in a coupled a ssay using the chromogenic peptide N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide and a-chymotrypsin, compared with th e spontaneous background rate of cisfitrans isomerization in the absence of MtPpiA (light grey line). The insert shows the linear relation between k obs ¼ (k cat /K m ) · [MtPpiA] and the protein concentration [MtPpiA]. Fig. 2. Pseudo translation. A large nonorigin peak was found in the native Patterso n map (contoured at 1.5 r with intervals of 0 .5 r, where r ¼ 0.32 eÆA ˚ )3 ), indicating the p ure translation between the two molecules in the asymmetric unit. Ó FEBS 2004 Structure of Mycobacterium tuberculosis PpiA (Eur. J. Biochem. 271) 4109 Residues 12–182 are present in the final m odel. It is unclear whether the absence of the residues from the extreme N-terminus is attributable to loss of this segment by proteolysis during the crystallization, or to disorder in that part of the s tructure. The main fold consists of an eight- stranded antiparallel b-barrel with one a-helix on each side (Fig. 3A). This structure is consistent with previous Ppi structures from the family, for example the human PpiA [14] and the Ppi from B. malayi [26]. Although the X-ray data were anisotropic, the use of noncrystallographic symmetry restraints resulted in strong density in virtually all areas of the structure (Fig. 3B). Some effects of the an isotropy are, however, apparent in the relatively high B-factors in the model (Table 1). Fig. 3. Structure of MtPpiA. (A) The overall structure of MtPpiA is illustrated i n a ribbon drawing. The chain is coloured beginning with blue at the N-terminus going through the rainbow to red at the C terminus. (B) Stereo view of the A molecule’s active site with refined |2F o -F c |mapcontouredat1r.For clarity, electron density for the protein alone is shown in this panel. (C) Stereo view of the active site showing only density attributable to partially occupied peptide. The expected site for binding the peptide HAGPIA was d eter- mined using a superposition of the human enzyme complex in PDB entry 1AWQ [14]. The superimposed peptide is shown in magenta. The |2F o -F c | map was then con- toured at 0.8 r in that region. Active site residues are labelled in black. 4110 L. M. Henriksson et al. (Eur. J. Biochem. 271) Ó FEBS 2004 Previous Ppi structures have shown that the active site is positioned at one side of the b-barrel [28]. Mt PpiA was crystallized in the p resence o f a hexa-peptide of sequence HAGPIA derived from the HIV c apsid p rotein s equence [14]. The expected site for binding the hexa-peptide is indicated using a superposition of the human enzyme complex ( Fig. 3C). Although electron d ensity con sistently appeared in this ar ea, it was not possible to place the substrate peptide in MtPpiA with confidence. Thus the affinity of MtPpiA for the HAGPIA peptide may be expected to be of the same order of magnitude as the concentration at which it was present in the crystallization solution (1 m M ). While K m values are not generally available f or Ppis, k cat has b een estimated for the human PpiA t o be 9000 s )1 with a different peptide [29]; taken together with an available k cat /K m estimated for that enzyme [30], a K m of  1m M is suggested. If t he k cat for Mt PpiA is similar, it too would have a K m in the millimolar range, and incomplete binding in the present crystallization experiment would not be surprising. Strong (similar to protein) density in both molecules also showed an unknown compound bound near the Ne of residue Lys50, and possibly stacking on the aromatic side chains of Tyr95 and Phe97. Among the reagents used during purification or crystallization, PEG appears to be the most likely candidate. However, as it is not near the active site, b inding of the unknown m olecule is not expected to have any impact o n activity. Comparison to other Ppi sequences and structures Proteins with sequences or str uctures similar to MtPpiA were found using BLAST ; some comparisons are s hown in Fig. 4. The t hree most similar structures, Mus musculus PpiC, B. malayi Ppi, and Ho mo s apiens PpiB were used, together with Mt PpiA, in a structure-base d sequence alignment. All three structures show an rmsd of approxi- mately 1.6 A ˚ from that of Mt PpiA, when the C a atoms are compared using a cut-off of 3.5 A ˚ (with  88% of the Cas matching). This is significantly larger than the r msd of 0.05 A ˚ observed when comparing the two NCS-restrained molecules of the Mt PpiA asymmetric unit. In the matched regions, the amino-acid sequence identity was  38%. The protein used for molecular replacement, H. sapiens PpiA shows a similar pattern in comparisons to MtPpiA. The structural alignment shows that MtPpiA represents a variant of Ppi not found among the structures in the PDB, with an extra insert, and a different N-terminal segment. A representative selection of Ppis that are expected to be similar to MtPpiA (60–90% sequence identity) is also shown in F ig. 4 . The catalytic arginine is completely conserved, and residues lining the active site are highly conserved, suggesting that substrate specificity will be similar in the two groups. Cyclosporin A binding by enzymes in the new group is also likely to resemble that of the human PpiA and PpiB and most others. In the binding site of the B. malayi Ppi,theequivalentofAla118is Fig. 4. Sequence alignment. The three structures most similar to MtPpiA were identified in a BLAST search and used for a structure-based sequence alignment with the program INDONESIA . The se sequences correspon d to the following entries in Gen Bank [34]: M. tuberculosis PpiA H37Rv (gi:15607151), Mus musculus PpiC (gi:1000033), B. malayi Ppi (gi:3212364), and H. sapiens PpiB (gi:1310882). A representative selection o f Ppis expected to be more similar to MtPpiA were further aligned with these, along with the protein used for molecular replacement (H. sapiens PpiA) and M. tuberculosis PpiB. These sequences are: M. leprae PpiA (gi:15826875), Corynebacterium diphtheriae PpiA (gi:38232667), Streptomyces avermitilis Ppi (gi:29830872), Thermobifida fusca Ppi (gi:23016930), H. sa piens PpiA (gi:2981743), and M. tuberculosis PpiB H37Rv (gi:15609719). Residues in the active site are indicated by ÔwÕ. Ó FEBS 2004 Structure of Mycobacterium tuberculosis PpiA (Eur. J. Biochem. 271) 4111 replaced by Lys, which has been suggested to account for its reduced binding of cyclosporin A [26]. The biological roles of MtPpiA have not yet been thoroughly investigated. Because it lacks an obvious signal sequence or m embrane-spanning segment, its location is presumably cytoplasmic. Its expression is decreased during iron depletion [31], suggesting that it is iron-regulated. It is upregulated slightly in an hspR and hrcA double deletion mutant, implying that it may be related to the heat shock response a nd possibly virulence [32]. The enzyme was not, however, f ound to be essential in transposon site hybrid- ization studies of M. tuberculosis [33]. Inspection of the M. tuberculosis PpiB sequence (Fig. 4) shows that it is different from the o ther Ppis. While the catalytic arginine is conserved, approximately one-third of the amino acids lining the active site are not. Thus its substrate specificity is probably d istinct from that of the other enzymes, including the human ones and MtPpiA; its sensitivity to cyclosporin A cannot be predicted. In addition, the sequence of this PpiB includes  140 residues preceding the catalytic domain. This region includes a membrane anchor that is most likely to position the active site on the extracellular surface. In this context, the fact that some Ppis have been reported to act as chaperones in protein folding is relevant [5]. Transposon site hybridization studies have also shown that this is an essential gene in M. tuberc ulosis [33]. Combined with the observed d ifferences from the human enzymes, these observations suggest t hat PpiB is worth further investigation as a potential drug target. Acknowledgements The authors thank Markus Dalin for help with cloning and i nitial crystallization experiments, Andrea Wilnerz on and Jimmy Lindbe rg for help with cry stall ization, and Jenny Berglund and Annette Roos for their aid in data collection. Financial s upport was received from the Swedish Research Council (VR), the Foundation for S trategic Research (SSF) and the European Commission programs SPINE (QLG2-CT-2002-00988) and X-TB (QLRT-2000-02018). References 1. Berman, H.M., Westbrook, J., Feng, Z., Gilliland, G., Bhat, T.N., Weissig,H.,Shindyalov,I.N.&Bourne,P.E.(2000)TheProtein Data Bank. Nucleic Acids Res. 28, 235–242. 2. Fischer, G., Wittmann-Liebold, B. , Lang, K., K iefhaber, T. & Schmid, F.X. (1989) Cyclophilin and peptidyl-prolyl cis-trans isomerase are probably identical proteins. Na ture 337, 476–478. 3. Takahashi, N., Hayano, T. & Suzuki, M. (1989) Peptidyl-prolyl cis-trans isomerase is the c yclosporin-A-binding protein cyclo- philin. Nature 33 7, 473–475. 4. Galat, A. & Met calfe, S.M. (1995) Peptidylproline cis/trans isomerases. Prog. Biophys. Mol. Biol. 63 , 67–118. 5. Schmid, F.X. (2001) Prolyl isomerases. Adv. Protein Chem. 59, 243–282. 6. Handschumacher, R.E., Harding, M.W., Rice, J., Drugge, R.J. & Speicher, D.W. ( 1984) Cyclophilin: a specific cytosolic binding protein for cyclosporin A. Science 226, 544–547. 7. Schreiber, S.L., Albers, M.W. & Brown, E .J. ( 1993) The cell- cycle, signal-transduction, and immunophilin ligand complexes. Acc. Chem. Res. 26, 412–420. 8. Borel, J.F. (1990) Pharmacology of cyclosporine (sandimmune). IV. Pharmacological properties in vivo. Pharmaco l. Rev. 41, 259– 371. 9. Anderson, S.K., Gallinger, S., Roder, J., Frey, J., Young, H.A. & Ortaldo, J.R. (1993) A cyclophilin-related protein involved in the function of natural k iller cells. Pro c. Natl Acad. Sci. USA 90, 542–546. 10. Sykes, K., Gething, M.J. & Sambrook, J . (1993) Proline i so- merases function during heat shock. Proc. Natl Acad. Sci. USA 90, 5853–5857. 11. Cole, S.T., Br osch, R., Parkhill, J., Garnier, T., Churcher, C., Harris, D., Gordon, S.V., Eiglmeier, K., Gas, S., Barry, C.E. III, Tekaia, F., Badcock, K., Basham, D., B rown, D., Chillingworth, T.,Connor,R.,Davies,R.,Devlin,K.,Feltwell,T.,Gentles,S., Hamlin, N., Holroyd, S., H ornsby, T., Jagels, K., Barrell, B.G. et al. (1998) Deciphering the biology of Mycobacterium tubercu- losis from the complete genome sequence. Nature 393, 537–544. 12. Fischer, G., B ang, H. & Mech, C. (1984) Determination of enzymatic catalysis for the cis-trans-isomerization of p eptide binding in proline-containing peptides. Biomed. Biochim. Acta 43, 1101–1111. 13. Ko fron, J.L., K uzmic, P., K ishore, V., Ge mmecke r, G., Fesik, S.W. & Rich, D.H. (1992) Lithium chloride perturbation of cis- trans peptide-bond equilibria – effect on conformational equilibria in cyclosporine-A and on time-dependent inhibition of cyclophilin. J. Am. Chem. Soc. 114, 2670–2675. 14. Vajdos,F.F.,Yoo,S.,Houseweart,M.,Sundquist,W.I.&Hill, C.P. (1997) Crystal structure of cyclophilin A complexed with a binding site peptide from the HIV-1 capsid protein. Protein Sci. 6, 2297–2307. 15. Leslie, A.G. (1999) Integration of m acromolecular diffraction data. Acta Crystallogr. D Biol Crystallogr. 55, 1696–1702. 16. Evans, P.R. (1993) Data reduction. In Proceedings of CCP4 Study Weekend on Data Collection and Processing, pp. 114–122. Dares- bury Laboratory, Warrington, UK. 17. Matthews, B.W. (1968) Solvent content of protein crystals. J. Mol. Biol. 33, 491–497. 18. Navaza, J. (1994) AMoRe – an automated package for molecular replacement. Acta Crystallogr. Sect. A50, 157–163. 19. Kleywegt, G.J., Zou, J.Y., Kjeldgaard, M. & Jones, T.A. (2001) Around O. In International Tables for Crystallography (Ross- mann, M.G. & Arnold, E., eds), Vol F, pp. 353–356. 20. Murshudov, G.N., Vagin, A.A. & Dodson, E.J. (1997) Refinement of macromolecular structures by the maximum-like- lihood method. Acta Crystallogr. Sect. D Biol. Crystallogr. 53, 240–255. 21. Collaborative Computing P roject Number 4 ( 1994) The CCP4 Suite: Programs for Protein Crystallography. Acta Crystallogr. D50, 760–763. 22. Jones, T.A., Zou, J.Y., Cowa n, S.W. & Kjeldgaard. ( 1991) Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A. 47, 110–119. 23. Lamzin,V.S.&Wilson,K.S.(1993)Automatedrefinementof protein models. Acta Crystallogr. Sect. D Biol. Crystallogr. 49, 129–147. 24. Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D.J. (1997) Gapped BLAST and PSI- BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402. 25. Harris, M. & Jones, T.A. (2001) Molray – a web interface between O and the POV-Ray ray tracer. Acta Crystallogr. Sect. D Biol. Crystallogr. 57, 1201–1203. 26. Taylor, P., Page, A.P., Kontopidis, G., Husi, H. & Walkinshaw, M.D. (1998) The X-ray structure of a divergent cyclophilin from the nematode parasite Brugia malayi. FEBS Lett. 425, 361–366. 27. Liu,J.,Albers,M.W.,Chen,C.M.,Schreiber,S.L.&Walsh,C.T. (1990) Cloning, expression, and purification of human cyclophilin in Escherichia coli and assessment of the catalytic role of cysteines 4112 L. M. Henriksson et al. (Eur. J. Biochem. 271) Ó FEBS 2004 by site-directed mutagenesis. Proc. Natl Acad. Sci. USA 87, 2304– 2308. 28.Fesik,S.W.,Gampe,R.T.Jr,Eaton,H.L.,Gemmecker,G., Olejniczak, E.T., Neri, P., Holzman, T.F., Egan, D.A., Edalji, R., Simmer, R. et al. (1991) NMR studies of [U-13C]cyclosporin A bound to cyclophilin: bound conformation and p ortions of cyclosporin involved in binding. Biochemistry 30, 6574–6583. 29.Eisenmesser,E.Z.,Bosco,D.A.,Akke,M.&Kern,D.(2002) Enzyme dynamics during catalysis. Science 295, 1520–1523. 30. Liu, J. & Walsh, C.T. ( 1990) Peptidyl-prolyl cis-trans-isomerase from Escherichia coli: a periplasmic homolog of cyclophilin that is not inhibited by cyclosporin A. Proc. Natl Acad. Sci. USA 87, 4028–4032. 31. Wong, D.K., Lee, B.Y., Horwitz,M.A.&Gibson,B.W.(1999) Identification of fur, aconitase, and other proteins expressed by Mycobacterium tuberculosis under conditions of low and high concentrations of iro n by c ombined two-dimensional gel elec tro- phoresis and mass spectrometry. Infect Immun. 67, 327–336. 32. Stewart, G.R., Wernisch, L., Stabler, R., Mangan, J.A., Hinds, J., Laing, K.G., Young, D.B. & Butcher, P.D. (2002) Dissection of the heat-shock response in Myc obacterium tuberculosis using mutants and microarrays. Microbiology 148, 3129–3138. 33. Sassetti, C.M., Boyd, D.H. & Rubin, E.J. (2003) Genes required for mycobacterial growth defined by high density mutagenesis. Mol. Microbiol. 48, 77–84. 34. Benson, D.A., Kars ch-Mizrachi, I., L ipman, D.J., O stell, J. & Wheeler, D.L. (2003) GenBank. Nucleic Acids Res. 31, 23–27. 35. Kleywegt, G.J. & Jones, T.A. (1996) Phi/psi-chology: Ram a- chandran revisited. Structure 4, 1395–1400. 36. Engh, R. & Huber, R. (1991) Accurate bond and angle parameters for X-ray p rotein s tructure refinement. Acta Crystallogr. A47, 392–400. Ó FEBS 2004 Structure of Mycobacterium tuberculosis PpiA (Eur. J. Biochem. 271) 4113 . purification and X-ray structure of Rv0009, the putative P piA from this bacterium (MtPpiA) and demonstrate that it h as peptidyl-prolyl cis –trans isomerase. primers 5¢-ATGGCAGACTGTGATTC CGTGAC-3¢ (forward) and 5¢-CTAGGAGATGGTG ATCGACTCG-3¢ (reverse), and Taq DNA polymerase (Roche). An additional PCR was performed