Mowbray2 1 Department of Cell and Molecular Biology, Uppsala University, Sweden;2Department of Molecular Biology, Swedish University of Agricultural Sciences, Uppsala, Sweden Peptidyl-pr
Trang 1X-ray structure of peptidyl-prolyl cis – trans isomerase A from
Mycobacterium tuberculosis
Lena M Henriksson1, Patrik Johansson1, Torsten Unge1and Sherry L Mowbray2
1
Department of Cell and Molecular Biology, Uppsala University, Sweden;2Department 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 folding
They are also thought to participate in processes such as
signalling, cell surface recognition, chaperoning and
heat-shock response Here we report the soluble expression 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 was shown to be active in a spectrophotometric
assay, with an estimated kcat/Kmof 2.0· 106M)1Æs)1 The
X-ray structure of PpiA was solved by molecular
replace-ment, and refined to a resolution of 2.6 A˚ with R and Rfree
values of 21.3% and 22.9%, respectively Comparisons to known structures show that the PpiA represents a slight variation on the peptidyl-prolyl cis–trans isomerase fold, previously not represented in the Protein Data Bank Inspection of the active site suggests that specificity for substrates and cyclosporin A will be similar to that found for most other enzymes of this structural family Comparison to the sequence of the second M tuberculosis enzyme, PpiB, 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 Health Organization (http://
www.who.org), Mycobacterium tuberculosis, the causative
pathogen of tuberculosis, currently infects one-third of the
world’s population, and results in 2 million deaths each
year Due to the increased prevalence of drug-resistant and
multidrug-resistant strains, and the lethal combination of
tuberculosis and HIV, there is a great need for new therapies
and drugs, as well as better knowledge of the bacteria’s
basic biology
Cyclophilins, also known as rotamases or peptidyl-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 bacteria, 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 vitroand 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 binds 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 graft rejection after transplant surgery [8] Ppis are also suggested to take part in other biological functions such as cell surface recognition [9] and heat-shock response [10]
M tuberculosishas two distinct Ppi enzymes [11] (http:// genolist.pasteur.fr/TubercuList/) We report here the clo-ning, expression, purification and X-ray structure of Rv0009, the putative PpiA from this bacterium (MtPpiA) and demonstrate that it has 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 the 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 as template, and the same reverse primer, but substituting the forward primer for 5¢-ATGGCCCATCATCATCATCATCATTCTGGTGC AGACTGTGATTCCGTGAC-3¢, in order to introduce an N-terminal His 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., Gilliland, 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)
Trang 2pCRT7/CT-TOPO vector using the pCR T7/CT TOPO
TA Express kit (Invitrogen), and then transformed 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 QIAprep Spin Miniprep kit
proto-col (Qiagen) An analytical PCR was performed using the
pCR T7/CT TOPO TA Expression kit (Invitrogen), with
the v5 (C-terminal) reverse primer, and the His6 forward
primer Plasmid (pCRT7::Rv0009) from one of the four
clones with the correct size insert was transformed into
E coliBL21-AITMcells (Invitrogen) In a test expression,
cells were induced with 0.1 mgÆmL)1arabinose for 2 h at
37C The apparent molecular weight of the expressed
protein as deduced from SDS/PAGE was in agreement with
the theoretical value, 20 kDa The isolated gene encoding
MtPpiA was further verified by DNA sequence
ana-lysis (Uppsala Genome Center, Rudbeck Laboratory)
On the preparative scale, BL21-AITM cells containing
pCRT7::Rv0009 were grown in Luria broth, with
50 lgÆmL)1 ampicillin and 12 lgÆmL)1 tetracycline, at
37C to D550¼ 0.7–1.0 The culture was then transferred
to 22C and induced with 0.001% (w/v) arabinose Growth
was continued for 2 h, after which the cells were harvested,
washed with 1· SSPE buffer (150 mM NaCl, 10 mM
NaH2PO4pH 7.5, 1 mMEDTA), and stored at)20 C
Thawed cells were treated with lysis buffer (50 mM
NaH2PO4 pH 8.0, 300 mM NaCl, 10 mM imidazole, 4%
glycerol) with 0.01 mgÆmL)1RNase, 0.02 mgÆmL)1DNase,
and lysed by using a Constant Cell Disruption System
(Constant Systems Ltd) operated at 1.5 kbar The cell lysate
containing soluble MtPpiA, was incubated for 30 min at
4C with Ni–NTA Agarose slurry (Qiagen)
pre-equili-brated with native lysis buffer The resin was washed with 10
column volumes lysis buffer containing 20 mM imidazole,
and the protein eluted with four column volumes of the
same buffer containing 250 mMimidazole The protein was
further purified on a size exclusion chromatography column
(HiLoadTM 16/60 SuperdexTM 75, Amersham Pharmacia
Biotech), using a buffer containing 150 mM NaCl, and
20 mM Tris/HCl pH 7.5 Fractions containing MtPpiA
were pooled and desalted using a PD10 column (Amersham
Biosciences) with a solution of 10 mM2-mercaptoethanol,
and 20 mM Tris pH 7.5 The protein was concentrated to
29 mgÆmL)1(based on the calculated absorbance of 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 (PhastSystemTM, Amersham Biosciences)
Assay
The activity of MtPpiA was evaluated using 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 mM) was prepared the previous day in trifluorethanol
with 0.45MLiCl, in order to increase the fraction of the cis
isomer [13] Each assay included 910 lL 0.1M Tris/HCl
pH 8.0 (maintained at 15C), 50 lL 600 lM
a-chymotryp-sin, and 30 lL of MtPpiA (at 1.7 lM, 0.49 lMor 0.33 lM),
which were mixed and preequilibrated in a cuvette at 15C
for 2 min The assay was initiated by adding 10 lL of peptide solution resulting in a final concentration of 78 lM The cisfitrans isomerization of the Ala-Pro bond (both spontaneous and enzyme-catalysed), coupled with the a-chymotrypsin cleavage of the trans peptide, was followed
by the increase in absorbance at 390 nm at 15C (DU 640 spectrophotometer, Beckman) Measurements were made every 0.5 s during 3 min The final absorbance was estima-ted from each 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 kobs¼ (kcat/
Km)· [MtPpiA] for each experiment A plot of kobs vs [MtPpiA] gives a line with slope kcat/Km
Crystallization MtPpiA was cocrystallized, with a hexapeptide of sequence HAGPIA [14] using vapour diffusion The sitting drops contained 2 lL protein, with a final concentration of 1 mM
of the peptide dissolved in dimethyl sulfoxide, and 2 lL reservoir solution [30% (v/v) PEG-200, 5% (w/v) PEG 3000, 0.1MMES/HCl pH 6.0], at 22C Needle-like crystals appeared within a few weeks Crystallization conditions were optimized with the aid of seeding, and crystals grew in hanging drops to a size of 0.05· 0.05· 0.4 mm3over a period of 2–3 months Prior to flash cooling, the crystals were placed for 12 h in a drop containing the reservoir solution plus 1 mM peptide, to favour peptide binding
Data collection, structure determination and refinement Data were collected under cryo conditions at beam line ID14-2 at the European Synchrotron Radiation Facility (ESRF), Grenoble, equipped with an ADSC Q4 CCD detector (Area Detector Systems Corp.), k¼ 0.933 A˚ Indexing and integration of the diffraction data were performed usingMOSFLM[15], and the data were processed withSCALA[16] in space group P31 The preliminary data set was 99.9% complete to 3.4 A˚ resolution with an overall
Rmeasof 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 programSOD[19] Initial refinement was performed usingNCSREFandREFMAC5 [20] as implemented
in theCCP4 program suite [21] Rebuilding was carried out with the programO[22] A higher resolution data set was then collected at beam line ID14-1 ESRF, with an ADSC Q4R CCD detector (Area Detector Systems Corp.),
k¼ 0.934 A˚, extending to 2.3 A˚ in two directions How-ever, as observed for the earlier set, the diffraction was
Trang 3strongly anisotropic Inspection of a number of criteria at
various stages of the solution and refinement (including
Rfree, figure of merit, map quality, etc.) suggested that
the 2.6 A˚ cut-off was optimal Data collection statistics
for this set are shown in Table 1 The final rounds of
refinement were carried out withREFMAC5 using
noncrys-tallographic symmetry restraints Different weights were
tested in the refinement, to find the best combination of Rfree
and stereochemistry Thirteen water molecules 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 entry code 1w74
BLAST [24] was used for identifying similar sequences
and structures.INDONESIA(http://xray.bmc.uu.se/dennis/
manual/) was used for additional sequence and structure
comparisons Pictures were prepared usingO,MOLRAY[25]
andINDONESIA
Results and Discussion
Enzyme properties
The protein corresponding to MtPpiA with a His6 tag
attached to the N-terminus was expressed in E coli, and
purified It behaved as a homogeneous monomer in size
exclusion chromatography Enzyme activity was shown
using a spectrophotometric assay where the cisfitrans
isomerization is measured in a coupled assay using the
chromogenic peptide
N-succinyl-Ala-Ala-Pro-Phe-p-nitro-anilide and a-chymotrypsin at 15C (Fig 1) Under these
conditions, MtPpiA has a kcat/Kmof 2.0· 106
M )1Æs)1, and therefore shows similar activity to the Ppi from Brugia malayi, with a kcat/Km of 7.9· 106M )1Æs)1 [26], and to human PpiA, with a kcat/Kmof 1.4· 107M )1Æs)1[27] Ppis
of this class process peptide substrates with quite broad specificity [5], and so the observed activity of MtPpiA is likely to reflect that with physiologically relevant substrates Overall structure
The X-ray structure of MtPpiA (182 residues, molecular mass 19.2 kDa) was solved by molecular replacement, using the structure of human PpiA [14] as a search model A strong peak in the native Patterson map (Fig 2) assisted in the location of the two molecules in the asymmetric 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 line), 15 n M (dark grey line), and 10 n M (grey line), measured in a coupled assay using the chromogenic peptide N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide and a-chymotrypsin, compared with the 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 Patterson map (contoured at 1.5 r with intervals of 0.5 r, where r ¼ 0.32 eÆA˚)3), indicating the pure translation between the two molecules in the asymmetric unit.
Trang 4Residues 12–182 are present in the final model 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 structure 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 anisotropy 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 in 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 | map contoured at 1 r 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 deter-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.
Trang 5Previous Ppi structures have shown that the active site is
positioned at one side of the b-barrel [28] MtPpiA was
crystallized in the presence of a hexa-peptide of sequence
HAGPIA derived from the HIV capsid protein sequence
[14] The expected site for binding the hexa-peptide is
indicated using a superposition of the human enzyme
complex (Fig 3C) Although electron density consistently
appeared in this area, 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 mM) While Km values are not generally
available for Ppis, kcathas been estimated for the human
PpiA to be 9000 s)1 with a different peptide [29]; taken
together with an available kcat/Kmestimated for that enzyme
[30], a Kmof 1 mMis suggested If the kcatfor MtPpiA is
similar, it too would have a Kmin 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, binding of the unknown molecule is not expected to
have any impact on activity
Comparison to other Ppi sequences and structures Proteins with sequences or structures similar to MtPpiA were found usingBLAST; some comparisons are shown in Fig 4 The three most similar structures, Mus musculus PpiC, B malayi Ppi, and Homo sapiens PpiB were used, together with MtPpiA, in a structure-based sequence alignment All three structures show an rmsd of approxi-mately 1.6 A˚ from that of MtPpiA, when the Ca atoms are compared using a cut-off of 3.5 A˚ (with 88% of the Cas matching) This is significantly larger than the rmsd of 0.05 A˚ observed when comparing the two NCS-restrained molecules of the MtPpiA 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 Fig 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, the equivalent of Ala118 is
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 These sequences correspond to the following entries in GenBank [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 of 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 sapiens PpiA (gi:2981743), and M tuberculosis PpiB H37Rv (gi:15609719).
Residues in the active site are indicated by w.
Trang 6replaced 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 membrane-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 and possibly virulence [32] The enzyme was not,
however, found 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 other 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 distinct 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 tuberculosis [33]
Combined with the observed differences from the human
enzymes, these observations suggest that PpiB is worth
further investigation as a potential drug target
Acknowledgements
The authors thank Markus Dalin for help with cloning and initial
crystallization experiments, Andrea Wilnerzon and Jimmy Lindberg for
help with crystallization, and Jenny Berglund and Annette Roos for
their aid in data collection Financial support was received from the
Swedish Research Council (VR), the Foundation for Strategic
Research (SSF) and the European Commission programs SPINE
(QLG2-CT-2002-00988) and X-TB (QLRT-2000-02018).
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