These studies showed that mutating Leu160 and Lys133 greatly reduced HGPRT activity, which confirms that these residues play an important Keywords crystal structure; enzymatic activity; H
Trang 1hypoxanthine-guanine phosphoribosyl transferase L160I
Qiang Chen1,2, Delin You1*, Yuhe Liang1,2, Xiaodong Su1,2, Xiaocheng Gu1, Ming Luo1,3
and Xiaofeng Zheng1,2
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
drug targets for the treatment of parasitic infections
[1,2] Hypoxanthine-guanine phosphoribosyltransferase
(HGPRT; EC 2.4.2.8) is a key enzyme in the salvage
pathway for purine nucleotide synthesis, and converts
the nucleobases hypoxanthine and guanine to IMP and GMP, respectively The active site lies in a cleft between the enzyme’s ‘core’ and ‘hood’ domains Some efforts have been made to identify inhibitors that tar-get the active site of HGPRT [3–6]
The crystal structure of wild-type HGPRT from Thermoanaerobacter tengcongensis was solved recently [7] In the present study, we sought to further our understanding of HGPRT’s chemical mechanism and
to identify key residues that contribute to its activity and regulation We constructed a series of point mutants, Leu160 and Lys133, and measured the enzyme activity These studies showed that mutating Leu160 and Lys133 greatly reduced HGPRT activity, which confirms that these residues play an important
Keywords
crystal structure; enzymatic activity; HGPRT;
mutant
Correspondence
X Zheng, College of Life Sciences, Peking
University, Beijing 100871, China
Fax: +86 10 6276 5913
Tel: +86 10 6275 5712
E-mail: xiaofengz@pku.edu.cn
*Present address
Shanghai Jiao Tong University, Shanghai,
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 subsimpor-tantially 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.
Trang 2role 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
Design of HGPRT mutants
Based on the crystal structure of wild-type HGPRT
that our laboratory solved previously [7], Lys133
inter-acts with the 6-oxo position on purines and is
pro-posed to be essential for substrate specificity Leu160 is
below the purine that, together with Ile103, stabilizes
purine binding by van der Waals interactions Both
Lys133 and Leu160 are relatively conserved among
dif-ferent HGPRTs from both eukaryotic and prokaryotic
organisms (Table 1), and the crystal structure confirms
that these residues are poised to function in catalysis
Therefore, in the present study, we chose to modify
HGPRT at Leu160 and Lys133 to better understand
how this enzyme has developed its specificity for
pur-ine nucleosides
Eleven mutants of HGPRT, L160I, L160V, L160T,
L160S, L160P, K133A, K133L, K133V, K133I, K133S
and K133T, were designed and crystallized and their
specific activities were compared (Table 2)
Protein purification and crystallization
All 11 HGPRT mutants were overexpressed and
iso-lated from the soluble fraction of Escherichia coli cells
The overexpressed protein represented approximately
30% of the total protein and purified to near
homoge-neity (data not shown) We obtained crystals of
mutants L160I, L160T, L160S, L160V and K133I in
addition to a His-tagged wild-type HGPRT (Fig 1)
Crystals of L160I diffracted to high resolution and
were subjected to structure determination Crystalliza-tion condiCrystalliza-tions of the other mutants are currently 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: C2221; 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 active site belonged to several N-terminal residues (RGSHM) of the neighboring molecule (Fig 3) Among these resides, four were from the His-tag (the full His-tag sequence is MGSSHHHHHHSSGLVPR-GSH), and one was the first residue Met of the L160I protein The N-terminal arginine occupied the active site whereas the upstream amino acids were exposed
to the solvent and could not be seen in the electron density map because of their disordered conforma-tions
Table 1 Comparison of HGPRT active site residues that are involved in substrate recognition.
Species
Above the purine
Below the purine
Interact with purine 6-oxo group
Near purine C2 group Cis-peptide
Proposed catalytic base
Coordinates with divalent metal ion
Trang 3Crystal 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 van der Waals contacts with Phe154, Val155, Ile103 and Ile160 to provide additional stabilization forces
A calcium ion was identified near the N-terminus connecting two tetramers The calcium ion was coordi-nated by the carboxyl side chains of Asp7 from one tetramer, Asp135 from a neighboring tetramer, and four water molecules, completing a perfect octahedral coordination sphere This interaction might promote the formation of the protein network
Enzyme activity The specific activities of the wild-type and mutant HGPRTs were measured and the results are summa-rized in Table 2
Leu160 and Lys133 mutants all showed decreased rates of activity with respect to wide-type HGPRT Using hypoxanthine as the substrate, activity of mutants L160S, K133V, K133I and L160I fell to 10%
to 19% compared to wild-type, whereas the activity of mutants L160T, L160P, K133A, K133L, K133S and K133T dropped to less than 5% When guanine was used as the substrate, the activity of L160T, K133A, K133T, K133V fell to 18%, 14%, 13.8% and 23.6%, respectively, compared to the wild-type, whereas the activity for L160S, L160P, K133L, K133I and K133S was less than 5% Based on all mutants, L160V showed the highest activity for both hypoxanthine and guanine, yet it was still substantially less than the wild-type HGPRT, especially when hypoxanthine was used
as substrate
1 Æmg
1 )
Wild-type (T7-tag) Wild-type (His-tag)
Trang 4In 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 L160I mutation is important in forming good crystal contacts The isoleucine probably provides an optimal environment for Arg-4, which only makes van der Waals interactions with the active site The cyclic pro-line may destroy the N-terminal peptide binding because no crystals were observed for L160P Among the six mutants at Lys133, only one, K133I, formed crystals which were very small (Fig 1), and this sug-gests that the hydrogen bond between Ser-2 and Lys133 is essential for the binding of the N-terminal peptide If the N-terminal peptide could not bind the active site properly, this peptide would become a dis-turbance to the ordered arrangement of protein mole-cules, thereby prohibiting crystallization
Although there was a four-fold excess of the product GMP to enzyme in the crystallization conditions, we could not detect any electron density that accounted for GMP in the L160I structure Instead, the N-termi-nal residues from a neighbor tetramer were found occupying the active site This observation suggests that, compared to the natural product GMP, the N-terminal peptide has stronger affinity for the active site of mutant L160I
The main goal of studying HGPRT function and structure is to design compounds that would be effec-tive inhibitors Almost all of the inhibitors available are analogues of the substrate or transition-state The main drawback of these compounds is that there is poor differential inhibition among various HGPRTs The reason maybe due to high structure similarity of
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).
Trang 5HGPRTs 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
this strategy In the present case, we propose that a
peptide that can bind to the groove between the core
domain and hood domain, and also block the active
site, could be an effective inhibitor One potential area
that may be exploited lies at Arg136 in T
tengcongen-sis HGPRT, which forms hydrogen bonds with the
N-terminal peptide After structural comparisons of
human HGPRT and all available parasitic HGPRTs
[11–15], we found that Tritrichomonas foetus,
Trypano-soma cruzi and Plasmodium falciparum HGPRTs
lacked a basic counterpart to Arg169 in human HGPRT Human HGPRT Arg169 corresponds to Arg136 in the L160I HGPRT variant from T tengcon-geensis Based on the crystal structure of mutant L160I, Arg136 forms hydrogen bonds with the N-ter-minal peptide using its side chain Such differences may provide the basis for designing inhibitors with preferential selectivity towards parasitic HGPRTs
Experimental procedures Protein preparation, crystallization and data collection
Wild-type HGPRT was cloned into a pET-15b vector (an N-terminal His-tag was used instead of a T7-tag reported in previous studies [7]) to facilitate protein purification and comparisons with mutants All mutants of T tengcongensis HGPRT, L160I (V, T, S and P) and K133A (L, V, I, S and T), were cloned into a pET-15b vector using the GeneEditor
in vitro site-directed mutagenesis system along with the wild-type vector as the template (Promega, Madison, WI, USA) All mutations were confirmed by plasmid DNA 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.
Trang 6protocols 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)1using Centricon
filter devices (Millipore, Billerica, MA, USA), set up in
16-well tissue-culture plates using the hanging-drop
vapor-diffusion method, and stored at 20C For initial screening,
the protein was equilibrated against Hampton’s Crystal
Screen and Crystal Screen 2 kits (Hampton Research,
River-side, CA, USA) Protein solution (1 lL) was mixed with
1 lL of well solution on a siliconized glass cover slip, which
was then sealed with high vacuum grease over a well
con-taining 0.4 mL of the respective crystallization solution To
fully saturate the enzyme binding site, GMP was added in a
4 : 1 molar ratio with respect to wild-type and mutant
HGPRTs Single crystals of the L160I mutant grew in 0.2 m
calcium chloride, 0.1 m Hepes (pH 7.5), 28% polyethylene
glycol 400 (Hampton Crystal Solution #14) within 2 weeks
X-ray diffraction data were collected by rotating the crystal
in 0.2 oscillations over a 180 wedge at k ¼ 1.5418 A˚, using
a Bruker SMART-6000 CCD detector (Bruker AXS GmbH,
Karlsruhe, Germany) Nitrogen gas was used to maintain
the crystal at 100 K and no cryoprotection was used Data
processing was performed with Bruker Proteum, as reported previously [8]
Structure determination and refinement The structure of the T tengcongensis HGPRT L160I mutant was determined by molecular replacement using the wild type HGPRT monomer (Protein Data Bank accession
no 1R3U) as the search model Rotation and translation searches were carried out using the software cns [17] to determine the position of one molecule in the asymmetric unit Initial rigid-body refinement was performed using data between 20.0 and 2.5 A˚ resolution resulting in a crystallo-graphic R-factor (Rcryst) 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 Rcrystand Rfreedropped
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 Ca2+in the active site Final Rcrystand Rfreevalues were 20.3% and 22.4%, respec-tively Data refinement statistics are summarized in Table 3
A
B
Fig 4 Crystal packing of HGPRT L160I
mutant (A) Each subunit of a tetramer (red)
links its N-terminus to the active site of a
neighboring tetramer subunit (green).
(B) Stereo diagram of the crystal packing
arrangement of the tetramers The unit cell
is outlined by a green box.
Trang 7Enzyme activity assay
To avoid complications due to the reverse reaction at longer
time points, all kinetic measurements were recorded using
ini-tial velocities of the forward reaction Data were collected
with an Ultrospec 2000 UV⁄ Visible Spectrophotometer
equipped with the kinetics program swift II (GE Healthcare,
Uppsala, Sweden) The formation of HGPRT-catalyzed IMP
or GMP was followed spectrophotometrically at 245 and
257 nm, respectively All kinetic measurements were carried
out in 100 mm Tris⁄ HCl buffer, pH 7.4, and 12 mm MgCl2
Given the temperature-dependent pH shift of Tris⁄ HCl
buffer (DpH¼)0.31 ⁄ 10 C), the buffer was readjusted to
pH 7.4 at the incubated temperatures Under these
conditions, the extinction coefficients between IMP and
5900 m)1Æcm)1, respectively [18] The assay was carried out
at 37C
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.
Data collection
Unit cell length (A ˚ ) a ¼ 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)
Subunits per asymmetric unit 1
Refinement
Number of protein atoms in
an asymmetric unit
1446 Number of water molecules
in an asymmetric unit
186
B-factor
a Rsym¼ S|I – <I>| ⁄ SI b Rcryst¼ S(||F o | ) |F c ||) ⁄ S|F o | c Rfreeis the
R-factor for a selected subset (approximately 10%) of the
reflec-tions that are not included in prior refinement calculareflec-tions.
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