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 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 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 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 conditions 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: 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 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 Thermoanaerobacter tengcongensis F154 I103, L160 K133 V155, D161 L44, K45 D105 E101, D102 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 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. Table 2. The specific activity (lmolÆmin )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 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 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 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. 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 16-well tissue-culture plates using the hanging-drop vapor- diffusion method, and stored at 20 °C. 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 (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. 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. Q. Chen et al. Crystal structure of HGPRT L160I FEBS Journal 274 (2007) 4408–4415 ª 2007 The Authors Journal compilation ª 2007 FEBS 4413 Enzyme 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 MgCl 2 . Given the temperature-dependent pH shift of Tris ⁄ HCl buffer (DpH ¼ )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 hypoxanthine, GMP and guanine were 1900 and 5900 m )1 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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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 in an asymmetric unit 186 R cryst (%) b 20.4 R free (%) c 22.6 B-factor Average 18.0 Protein 16.4 Water 23.0 a R sym ¼ S|I – <I>| ⁄SI. b R cryst ¼ S(||F o | ) |F c ||) ⁄S|F o |. c R free is the R-factor for a selected subset (approximately 10%) of the reflec- tions that are not included in prior refinement calculations. Crystal structure of HGPRT L160I Q. 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Acta Crystallogr A 47, 110–119. Q. Chen et al. Crystal structure of HGPRT L160I FEBS Journal 274 (2007) 4408–4415 ª 2007 The Authors Journal compilation ª 2007 FEBS 4415 . Crystal structure of Thermoanaerobacter tengcongensis hypoxanthine-guanine phosphoribosyl transferase L160I mutant ) insights into inhibitor design Qiang. (199 9) Crystal structure of Toxoplasma gondii hypoxanthine-guanine phosphoribosyltransferase with XMP, pyrophosphate, and two Mg(2 +) ions bound: insights into