proteins

STRUCTURE O FUNCTION O BIOINFORMATICS

STRUCTURE NOTE

The crystal structure of Trypanosoma cruzi

arginine kinase
Pablo Fernandez,1 Ahmed Haouz,2 Claudio A. Pereira,3 Carlos Aguilar,4 and Pedro M. Alzari1*
1 Unité de Biochimie Structurale, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris, France
2 PF6, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris, France
3 Laboratorio de Biologı́a Molecular de Trypanosoma cruzi (LBMTC), Instituto de Investigaciones Médicas Alfredo Lanari

(IDIM, CONICET-UBA), Buenos Aires, Argentina

4 Laboratorio de Biologı́a Molecular Estructural, Facultad de Quı́mica, Bioquı́mica y Farmacia, Universidad Nacional de San Luis,

Av. Ejercito de los Andes Bloque I, 5700 San Luis, Argentina

Key words: Trypanosoma cruzi; arginine kinase; Chagas disease; phosphagen; phosphagen kinase; guanidino kinase.

INTRODUCTION conditions3 and seems to play a critical role as a regula-

Trypanosomes are protozoan organisms that cause im-
portant veterinary diseases and two of them are responsi-
ble for significant human diseases: Trypanosoma brucei is
the agent of sleeping sickness (African trypanosomiasis)
and T. cruzi causes Chagas disease in the American conti-
nent. Trypanosomes diverged early in evolution from
other eukaryotes and posses unique features in terms of
energetic metabolism. A peroxisome-like structure, the
glycosome, contains most of the glycolytic and pentose
phosphate pathway enzymes. In addition, the trypano-
some mitochondria present unusual structure and func-
tional properties that strongly depend on the different
environments encountered by parasites.1
Arginine kinase (AK, E.C. 2.7.3.3) is a phosphotrans-
ferase that catalyzes the interconversion between phos-
phoarginine and ATP. This enzyme is present in some
invertebrates, including the trypanosomatids, and repre-
sents an analogous system to the creatine kinases in ver-
tebrates. Phosphagens such as phosphoarginine and
phosphocreatine play a critical role as ‘‘energy storage’’
molecules because the high-energy phosphate can be rap-
idly transferred to ADP when the renewal of ATP is
needed.2 Phosphagens support bursts of cellular activity
until catabolic events such as glycogenolysis, glycolysis,
and oxidative phosphorylation are switched on. In T.
cruzi, AK overexpression was shown to increase the para-
site survival capability under pH and nutritional stress
tor of energetic reserves and cell growth. A similar situa-
tion occurs when a heterologous AK is expressed in
organisms lacking phosphagen kinases, such as yeast and
bacteria.4,5 More recently, it was observed that T. cruzi
epimastigotes treated with hydrogen peroxide showed a
significant increase in both AK expression and survival
capability during exposure, suggesting the participation
of AK in the parasite response to oxidative stress.6
Although the crystal structures of several vertebrate
creatine kinases are currently available, only a single AK
has been structurally characterized so far, that of Limulus
polyphemus (Horseshoe crab) AK.7 We have previously
characterized the molecular and biochemical characteri-
zation of the T. cruzi AK (TcAK)8,9 and we report here
the crystal structure of the ligand-free (open form) of the
enzyme at 1.9 Å resolution.
Grant sponsor: Institut Pasteur; Grant sponsor: CONICET; Grant number: PIP
5492; Grant sponsor: University of Buenos Aires; Grant number: UBACyT X073;
Grant sponsor: University of San Luis and Agencia Nacional de Promoción Cien-
tı́fica y Tecnológica; Grant number: FONCYT-PICT REDES 2003-00300.
Pablo Fernandez’s current address is Unité de Biologie des Interactions
Hôte-Parasite, Institut Pasteur, 25 rue du Dr. Roux, 75724 Paris, France
*Correspondence to: Pedro M. Alzari, Unité de Biochimie structurale, Institut Pasteur,
25 rue du Docteur Roux, 75724 Paris, France. E-mail: alzari@pasteur.fr
Received 16 February 2007; Accepted 2 April 2007
Published online 10 July 2007 in Wiley InterScience (www.interscience.wiley.com).
DOI: 10.1002/prot.21557

V
C 2007 WILEY-LISS, INC. PROTEINS 209
 P. Fernandez et al.

MATERIALS AND METHODS

Table I
Data Collection and Refinement Statistics
A fragment carrying the TcAK gene was obtained by
PCR and cloned into the pRSet A vector (Invitrogen) to
Beamline ID29 (ESRF)
obtain pRSetA-AK. Escherichia coli BL21(DE3)pLysS bac- Wavelength (
) 0.9756
teria transformed with pRSetA-AK were grown at 378C Data resolution (
)a 70–1.9 (2–1.9)
until late log phase in Luria-Bertani (LB) medium with Measured reflections 414,919
50 lg/mL ampicillin. Induction of expression was con- Unique reflections 44,532
Multiplicitya 9.3 (5.2)
ducted for 3 h at 308C after addition of 1 mM IPTG. Completeness (%)a 92.0 (67.6)
The bacterial pellet was resuspended in 50 mM Hepes Rsym (%)a,b 8.0 (41.3)
buffer, pH 7, 0.2M NaCl, in the presence of protease hI/ria 20.8 (2.7)
inhibitors, and sonicated. After lysate centrifugation, the Space group P3221
a ¼ b (
) 86.70
supernatant was applied to a Ni-column (Pharmacia) c (
) 138.82
using an FPLC system and eluted by an imidazole gradi- Resolution (
) 70–1.9
ent (0–0.5M). An additional gel filtration step (Superdex- Rcrystc [N8 refs] 0.194 [39,973]
75) allowed to separate the purified protein from aggre- Rfreec [N8 refs] 0.218 [2235]
Rms bonds (
) 0.018
gated material, as well as to remove imidazole and most Rms angles (degrees) 1.61
of the Niþ2-cations. The protein was subsequently con- Protein atoms 2695
centrated to 20 mg/mL and tested for biochemical activ- Water molecules 166
ity as described.8 a
Values in
P parentheses
P
apply
 to the

 high
P P resolution shell.
Crystals of TcAK were obtained by the hanging drop b
¼ hkl i
Ii ðhklÞ
IðhklÞ
Rsym P P


hkl i Ii ðhklÞ:
method, mixing 1.5 lL of protein solution and 1.5 lL of
c
R ¼ hkl jFðhÞobs
FðhÞcalcj hkl jFðhÞobsj. Rcryst and Rfree were calculated
from the working and test reflection sets, respectively.
the reservoir solution containing 2.5M ammonium sul-
fate, 0.1M Tris HCl, pH 7.5. Crystals grew within a week
at 188C to a maximum size of 0.2 mm 3 0.2 mm 3 0.3
mm. For freezing, crystals were transferred to a cryopro- eukaryotic creatine kinases, such as human type B or
tectant solution containing 2.5M ammonium sulfate and muscle creatine kinases (sequence identities 45–47%, rms
20% (v/v) glycerol. A native data set at 1.9 Å resolution deviations of 1.2–1.3 Å for 310–320 aligned residues).
was collected on beamline ID29 at the European Syn- The catalytic mechanism of phosphagen kinases has
chrotron Radiation Facility, and reduced using the pro- been thoroughly studied by classical methods. Despite
grams MOSFLM/SCALA from the CCP4 program pack- their different substrate specificities, these enzymes are
age10 (Table I). The 3D structure was determined by thought to share a common mechanism of direct, asso-
molecular replacement methods using the program ciative in-line g-phosphoryl transfer,16 and this notion is
AMoRe11 and the atomic coordinates of Limulus poly- reinforced by a significant conservation of AK amino
phemus arginine kinase (LpAK, PDB code 1M8012). acid sequences and overall structures. In particular, the
Crystallographic refinement was carried out with the pro- sequence and length of the ‘‘specificity loop’’ (residues
gram REFMAC13 using TLS matrices, alternated with 61–68), which has been postulated to mediate substrate
manual cycles of model reconstruction using COOT.14 specificity for different guanidine kinases,7,17 has a
Water molecules were added with programs Arp/Warp.15 nearly identical sequence in TcAK (LDSGIGVY) and
The parameters after the final refinement cycle are given LpAK (LDSGVGIY), and the loop also retains a similar
in Table I. Atomic coordinates and structure factors are conformation in the two structures. Two other well-con-
available from the PDB under accession code 2J1Q. served residues in guanidine kinases are Cys 271, which
plays an important role in substrate binding but is not
RESULTS AND DISCUSSION essential for catalysis,18 and Glu 225, postulated as one
of the two bases involved in the catalytic mechanism.19
The 3D structure of ligand-free TcAK was determined The corresponding structural regions are also well con-
by molecular replacement methods and refined at 1.9 Å served between the two structures of ligand-free AKs. On
resolution (Table I). Three regions of the protein (resi- the other hand, the second proposed catalytic base, Glu
dues 289–293, 310–320 and the two C-terminal residues, 314, belongs to a disordered loop in TcAK and is missing
356/7) are disordered in the crystal and were not mod- from the model of the open form of the enzyme.
elled. Also, a few solvent exposed side chains, mainly in The structure of TcAK corresponds to the open form
the loop 294–299, are missing in the model. The overall of the enzyme, in the absence of ligands. All our attempts
structure of TcAK [Fig. 1(A,B)] is very similar to that of to obtain crystals of TcAK in complex with substrates or
ligand-free Limulus polyphemus (Horseshoe crab) arginine analogs were unsuccessful. However, most residues that
kinase12 (LpAK, 72% sequence identity, rms deviation of were seen to undergo the largest changes in LpAK upon
0.9–1 Å for 334 aligned residues) and to those of several substrate binding16 are strictly conserved in TcAK,

210 PROTEINS DOI 10.1002/prot

 Structure of Trypanosomal arginine kinase
Figure 1
Overall structure of TcAK. A: Ribbon representation of TcAK, color-coded from
blue (N-terminus) to red (C-terminus). B: Molecular surface of TcAK, shown in
the same orientation as in (A) and color-coded according to surface charges. The
substrates (as observed in the LpAK complex, PDB code 1BG07) are shown in
stick representation. C: Superposition of the substrate-free (open form) of TcAK
(in grey) and the substrate-bound (closed form) of LpAK (in orange, with
substrates in cyan). Enzyme closure involves the rigid-body motion of three
subdomains (indicated with arrows) with respect to a fixed subdomain (in the
back). The substrate-induced structuring of the flexible loop 309–320 (in red)
completely shields the substrates from solvent access. [Color figure can be viewed
in the online issue, which is available at www.interscience.wiley.com.]
DOI 10.1002/prot
strongly suggesting that the induced-fit conformational
changes may be similar in both enzymes. Thus, the con-
served residues Ser122, His185 and His 284 (involved in
interactions with the nucleotide ring) and the positively
charged cluster Arg124, Arg126, Arg229 and Arg280
(which attract ADP/ATP to the active site) all occupy the
same conformational space in both ligand-free TcAK and
LpAK. Furthermore, the loop 310–320, which closes the
active site of LpAK when the enzyme forms a complex
with substrate analogs7 [Fig. 1(C)], is equally flexible in
both unliganded structures.
Significant conformational changes take place upon
substrate binding, as illustrated by the superposition of
ligand-free TcAK with the transition state analog complex
of LpAK (PDB code 1BG07), which shows a rms devia-
tion of 2.1 Å for 316 aligned residues. While AK may be
considered a two domain protein, the structural compari-
son of ligand-free and ligand-bound AKs strongly sug-
gests that the differences upon substrate binding can be
attributed to the motion of three rigid groups with
respect to a fourth, fixed, subdomain12 [Fig. 1(C)]. Since
these changes occur near the phosphoryl and nucleotide
acceptors, both ligands appear to be responsible for the
induction of the conformational changes that close the
active site. Indeed, such a mechanism could be useful to
prevent wasteful hydrolysis of ATP in the absence of
phosphagen.
The recently finished ‘‘Tritryp’’ genome projects (T.
cruzi, T. brucei, and Leishmania major) confirmed that
AK is present in the first two genomes but not in L.
major.20–22 T. cruzi has a single AK of 357 amino acids
length, whereas T. brucei has three isoforms of 356 (the
T. cruzi ortholog), 370, and 404 amino acids. Considering
the large-scale synteny between the trypanosomatid
genomes, the TcAK gene could have been acquired by
horizontal gene transfer, with a subsequent gene loss
event in Leishmania spp. Since AK is absent in mammals
but is well conserved in the human pathogens T. cruzi
and T. brucei (pairwise sequence identities of 82%
between T. brucei and T. cruzi AKs), the crystal structure
of the trypanosomal enzyme reported here could provide
a template for the rational development of new drugs
against African and American trypanosomiases.
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DOI 10.1002/prot