Báo cáo khoa học: X-ray structure of peptidyl-prolyl cis–trans isomerase A from Mycobacterium tuberculosis potx

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:
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
( />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