Crystal structure of Thermoanaerobacter tengcongensis
hypoxanthine-guanine phosphoribosyl transferase L160I
mutant ) insights into inhibitor design
Qiang Chen
1,2
, Delin You
1
*, Yuhe Liang
1,2
, Xiaodong Su
1,2
, Xiaocheng Gu
1
, Ming Luo
1,3
and Xiaofeng Zheng
1,2
1 National Laboratory of Protein Engineering and Plant Genetic Engineering, Peking University, Beijing, China
2 Department of Biochemistry and Molecular Biology, College of Life Sciences, Peking University, Beijing, China
3 Department of Microbiology, University of Alabama at Birmingham, AL, USA
Parasites cause a wide variety of human and animal
diseases. These infections are routinely treated
using therapeutics such as chemotherapy. A common
approach for developing drug treatments against para-
sites is to target the biochemical and physiological
differences between a pathogen and host. In living sys-
tems, including humans, purine nucleotides are synthe-
sized using a de novo pathway and salvage pathway.
Most, if not all, protozoan parasites lack the de novo
pathway for synthesizing purine nucleotides. For this
reason, enzymes in the salvage pathway are potential

China
(Received 23 April 2007, revised 17 June
2007, accepted 2 July 2007)
doi:10.1111/j.1742-4658.2007.05970.x
Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is a potential
target for structure-based inhibitor design for the treatment of parasitic dis-
eases. We created point mutants of Thermoanaerobacter tengcongensis
HGPRT and tested their activities to identify side chains that were impor-
tant for function. Mutating residues Leu160 and Lys133 substantially
diminished the activity of HGPRT, confirming their importance in cataly-
sis. All 11 HGPRT mutants were subject to crystallization screening. The
crystal structure of one mutant, L160I, was determined at 1.7 A
˚
resolution.
Surprisingly, the active site is occupied by a peptide from the N-terminus
of a neighboring tetramer. These crystal contacts suggest an alternate strat-
egy for structure-based inhibitor design.
Abbreviation
HGPRT, hypoxanthine-guanine phosphoribosyltransferase.
4408 FEBS Journal 274 (2007) 4408–4415 ª 2007 The Authors Journal compilation ª 2007 FEBS
role in catalysis. The crystal structure of the L160I
mutant of T. tengcongensis HGPRT was determined at
1.7 A
˚
resolution. Unexpectedly, the enzyme active site
was occupied by the N-terminus of a neighboring pep-
tide. This interaction suggests an alternative and
potentially useful strategy for designing inhibitors
against HGPRT.
Results

being optimized to improve diffraction resolution and
quality.
Structure of the mutant L160I
The overall structure of the L160I mutant is in excel-
lent agreement with wild-type HGPRT (calculations
using the peptide backbone reveals an rmsd of 0.5 A
˚
between the two structures) [7]. The active site is in a
cleft between two domains: the core and hood. One of
the striking differences between the two structures is at
the N-terminus. Although the wild-type HGPRT has a
disordered N-terminus, the mutant L160I has an
extended loop (Fig. 2). The tetramer formation
observed for the L160I mutant is similar to that of the
wild-type HGPRT reported previously [7], although
the crystals belong to different space groups (wild-type:
C222
1
; L160I: I222). The four subunits of mutant
L160I tetramer are related by two orthorhombic two-
fold axes, whereas two subunits of wild-type HGPRT
in the asymmetric unit are related by a noncrystallo-
graphic two-fold axis and two asymmetric units
formed the tetramer through a crystallographic two-
fold axis.
We initially thought that the electron density in the
active site was GMP because the crystallization condi-
tions included four-fold excess GMP versus the pro-
tein [8]. However, after structure refinement, it was
clear that the electron density surrounding the

Homo sapiens F186 I135, L192 K165 V187, D193 L67, K68 D137 E133, D134
Toxoplasma gondii W199 I148, Y205 K178 I200, D206 L78, K79 D150 E146, D147
Tritrichomonas foetus Y156 I104, F162 K134 V157, D163 L46, T47 D106 E102, D103
Trypanosoma cruzi F164 I113, L170 K143 V165, D171 L51, K52 D115 E111, D112
Plasmodium falciparum F197 I146, L203 K176 V198, D204 L76, K77 D148 E144, D145
Q. Chen et al. Crystal structure of HGPRT L160I
FEBS Journal 274 (2007) 4408–4415 ª 2007 The Authors Journal compilation ª 2007 FEBS 4409
Crystal packing
L160I mutants exist as a tetramer in both solution and
crystals. The N-terminus of each subunit of the tetra-
mer forms crystal contacts with the active site of a
subunit of the neighboring tetramer. As a result, one
tetramer links to four other tetramers via the active
site and the recombinant N-terminal, which allows the
proteins to form an ordered network in the crystal lat-
tice (Fig. 4). This is likely the major reason why the
crystal could diffract to high resolution (> 1.7 A
˚
).
Several hydrogen bonds are present between the
N-terminus and active site. Ser-2 (residues in the recom-
binant His-tag are denoted with a minus sign to distin-
guish them from residues in the native protein) has a
backbone oxygen that hydrogen bonds with the side
chains of Lys133 and Arg136. Gly-3 backbone oxygen
interacts with Val155 backbone nitrogen and Lys153
backbone oxygen. Met1 backbone nitrogen interacts
with the Asp152 side chain. Several ordered water mole-
cules were present in the interaction between the
N-terminus and the active site. In addition, Arg-4 makes

)1
Æmg
)1
) of HGPRT wild-type and mutants. Reactions were carried out in 100 mM Tris ⁄ HCl buffer, pH 7.4, and 12 mM MgCl
2
at 37 °C. Data are
reported as the mean ± SD of triplicate measurements.
Substrate
Wild-type
(T7-tag)
Wild-type
(His-tag) L160I L160V L160T L160S L160P K133A K133L K133V K133I K133S K133T
Hypoxanthine 21.0 ± 0.66 18.4 ± 0.82 3.5 ± 0.14 8.5 ± 0.35 0.7 ± 0.03 1.8 ± 0.08 0.03 ± 0.01 0.7 ± 0.03 1.2 ± 0.05 3.3 ± 0.14 2.2 ± 0.09 0.2 ± 0.09 0.5 ± 0.03
Guanine 10.5 ± 0.45 14.4 ± 0.70 2.6 ± 0.13 10.3 ± 0.52 4.3 ± 0.22 0.9 ± 0.04 0.07 ± 0.02 0.2 ± 0.01 0.5 ± 0.03 3.4 ± 0.16 0.01 ± 0.01 0.09 ± 0.02 0.2 ± 0.02
Crystal structure of HGPRT L160I Q. Chen et al.
4410 FEBS Journal 274 (2007) 4408–4415 ª 2007 The Authors Journal compilation ª 2007 FEBS
Discussion
In the wild-type T. tengcongensis HGPRT structure,
Leu160, together with Ile103, stabilizes purine binding
by van der Waals interactions, and Lys133 is proposed
to be essential for substrate specificity [7]. Point
mutants of Leu160 and Lys133 had much weaker
activity compared to the wild-type, which confirms that
Leu160 and Lys133 are two key residues in catalysis.
Based on the crystal packing, the interaction
between the N-terminus and active site is essential for
the formation of well-ordered crystals. Qualitatively,
crystals of His-tagged wild-type HGPRT, mutant
L160V, L160T and L160S appear to be in good shape;
however, they diffracted weakly implying that the

His-tag WT L160I L160T
L160S L160V K133I
Fig. 1. Photographs of wild-type and mutant
HGPRT crystals.
N-terminal loop
II
III
I
IV
Fig. 2. Ribbon representation of T. tengcongensis HGPRT L160I
mutant subunit. The core domain contains a central five-stranded
parallel b-sheet flanked by three a-helices. The hood domain con-
sists of a small antiparallel b-sheet and two small 3–10 helices. The
four loops that make up the active site are labeled (I–IV).
Q. Chen et al. Crystal structure of HGPRT L160I
FEBS Journal 274 (2007) 4408–4415 ª 2007 The Authors Journal compilation ª 2007 FEBS 4411
HGPRTs from different species (especially the active
site) even though there is only moderate sequence
homology. The key residues in the active site are
highly similar among HGPRTs (Table 1). Thus, in
addition to targeting the substrate and cofactor bind-
ing sites, we propose another strategy to design inhibi-
tors that target unique regions surrounding the active
site. A compound that specifically binds such regions
in parasitic HGPRTs and that can also block the
active site may be a good approach for tackling
the differential inhibition problem. A similar strategy
was suggested previously [9,10], and the interaction
between the N-terminal residues and the active site in
the mutant L160I structure supports the feasibility of

sequencing. Detailed protein overexpression and purification
A
B
Fig. 3. Close-up view showing the interac-
tions between the active site of T. teng-
congensis HGPRT L160I mutant with the
N-terminus of a neighboring molecule. (A)
Stereoview of an Fo–Fc omit electron den-
sity map for the N-terminal loop. The elec-
tron density map was contoured at 3r.
(B) Overview depicting two subunits
involved in protein–protein interactions
across subunits. One subunit is shown in
ribbons whereas its neighboring subunit is
rendered in Van der Waals surface. The
N-terminal loop is shown in CPK representa-
tion and C atoms are colored cyan to distin-
guish them from the other subunit.
Crystal structure of HGPRT L160I Q. Chen et al.
4412 FEBS Journal 274 (2007) 4408–4415 ª 2007 The Authors Journal compilation ª 2007 FEBS
protocols are reported elsewhere [8,16]. Briefly, overexpres-
sion of the protein was carried out in E. coli BL21(DE3) ⁄
pLysS cells. A two-step purification procedure involving a
nickel chelating column followed by a Superdex-75 size
exclusion gel-filtration was used to obtain near homogenous
protein. For crystallization trials, the purified protein was
concentrated to approximately 10 mgÆmL
)1
using Centricon
filter devices (Millipore, Billerica, MA, USA), set up in

between 20.0 and 2.5 A
˚
resolution resulting in a crystallo-
graphic R-factor (R
cryst
) of 36.7%. Manual substitution for
the L160I modification and model fitting were performed
using the software o [18]. Multiple rounds of conjugate gra-
dient minimization, simulated annealing and individual
B-factor refinement were performed. R
cryst
and R
free
dropped
to 28.4% and 34.2%, respectively. 3Fo)2Fc and Fo–Fc elec-
tron density maps were calculated using the refined model
phases. Data were collected to 1.70 A
˚
resolution, which
allowed for calcium ions and water molecules to be incorpo-
rated into the model during the latter stages of refinement.
Electron density maps showed one Ca
2+
in the active site.
Final R
cryst
and R
free
values were 20.3% and 22.4%, respec-
tively. Data refinement statistics are summarized in Table 3.

Æcm
)1
, respectively [18]. The assay was carried out
at 37 °C.
Acknowledgements
We would like to thank Dr Rieko Yajima for critical
reading of the manuscript and Professor Yicheng
Dong for helpful discussions. We thank Quan Yu for
help with protein gel-filtration analysis. This work was
supported by the National Science Foundation of
China (No. 30328006).
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Table 3. Data collection and refinement statistics. Values in paren-
theses refer to the highest resolution shell.
Characteristic HGPRT mutant L160I
Data collection
Temperature (°K) 100
Space group I222
Unit cell length (A
˚
)a¼ 52.21, b ¼ 88.36, c ¼ 93.03
Unit cell angle (°) a ¼ b ¼ c ¼ 90
Resolution range (A
˚
) 20.0–1.70 (1.79–1.70)
Completeness (%) 97.4 (87.3)
R
sym
(%)
a
3.79 (10.76)
I ⁄ r (I) 14.4 (3.9)
Redundancy 3.53 (1.72)
Unique reflections 23 486
Subunits per asymmetric unit 1
Solvent content (%) 45.2
Refinement
Number of protein atoms in
an asymmetric unit
1446
Number of water molecules

c
R
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tions that are not included in prior refinement calculations.
Crystal structure of HGPRT L160I Q. Chen et al.
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