Structural and functional studies of the human phosphoribosyltransferase domain containing protein ˚ Martin Welin1,*, Louise Egeblad2,*, Andreas Johansson1, Pal Stenmark1, , Liya Wang2, ´ Susanne Flodin1, Tomas Nyman1, Lionel Tresaugues1, Tetyana Kotenyova1, Ida Johansson1, Staffan Eriksson2, Hans Eklund3 and Par Nordlund1 ă Structural Genomics Consortium, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, Uppsala, Sweden Department of Molecular Biology, Biomedical Center, Swedish University of Agricultural Sciences, Uppsala, Sweden Keywords characterization; crystal structure; homolog; HPRT; phosphoribosyltransferase; PRTFDC1 Correspondence P Nordlund or S Eriksson, Structural Genomics Consortium, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-17177 Stockholm, Sweden; Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, Box 575, SE-75123 Uppsala, Sweden Fax: +46 524 868 50; +46 18 55 0762 Tel: +46 524 868 60; +46 18 471 4187 E-mail: par.nordlund@ki.se; staffan.eriksson@afb.slu.se *These authors contributed equally to this work  Present address Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, Sweden Database Structural data are available in the Protein Data Bank under the accession number 2JBH Human hypoxanthine-guanine phosphoribosyltransferase (HPRT) (EC 2.4.2.8) catalyzes the conversion of hypoxanthine and guanine to their respective nucleoside monophosphates Human HPRT deficiency as a result of genetic mutations is linked to both Lesch–Nyhan disease and gout In the present study, we have characterized phosphoribosyltransferase domain containing protein (PRTFDC1), a human HPRT homolog of unknown ˚ function The PRTFDC1 structure has been determined at 1.7 A resolution with bound GMP The overall structure and GMP binding mode are very similar to that observed for HPRT Using a thermal-melt assay, a nucleotide metabolome library was screened against PRTFDC1 and revealed that hypoxanthine and guanine specifically interacted with the enzyme It was subsequently confirmed that PRTFDC1 could convert these two bases into their corresponding nucleoside monophosphate However, the catalytic efficiency (kcat ⁄ Km) of PRTFDC1 towards hypoxanthine and guanine was only 0.26% and 0.09%, respectively, of that of HPRT This low activity could be explained by the fact that PRTFDC1 has a Gly in the position of the proposed catalytic Asp of HPRT In PRTFDC1, a water molecule at the position of the aspartic acid side chain position in HPRT might be responsible for the low activity observed by acting as a weak base The data obtained in the present study indicate that PRTFDC1 does not have a direct catalytic role in the nucleotide salvage pathway Structured digital abstract l MINT-7996314: PRTFDC1 (uniprotkb:Q9NRG1) and PRTFDC1 (uniprotkb:Q9NRG1) bind (MI:0407) by x-ray crystallography (MI:0114) (Received 15 November 2009, revised 18 August 2010, accepted 30 September 2010) doi:10.1111/j.1742-4658.2010.07897.x Abbreviations DSLS, differential static light scattering; HPRT, human hypoxanthineguanine phosphoribosyltransferase; ImmGP, immucillinGP; PRPP, a-D-5-phosphoribosyl 1-pyrophosphate; PRTFDC1, phosphoribosyltransferase domain containing protein 1; TCEP, tris(2-carboxyethyl)phosphine 4920 FEBS Journal 277 (2010) 4920–4930 ª 2010 The Authors Journal compilation ª 2010 FEBS M Welin et al Studies of the human PRTFDC1 Introduction Human hypoxanthine guanine phosphoribosyltransferase (HPRT) (EC 2.4.2.8) is an important enzyme in the salvage pathway of purine nucleotides It catalyzes the transfer of a hypoxanthine or guanine base to a-d-5-phosphoribosyl 1-pyrophosphate (PRPP), producing IMP or GMP and pyrophosphate Several of the identified mutations leading to disease are spread over the entire protein, and are not just restricted to the active site [1,2] Among the metabolic consequences of having HPRT deficiency are an overproduction of uric acid and elevated levels of PRPP [3] Patients having mutations resulting in partial HPRT deficiency often suffer from gouty arthritis However, more severe deficiencies lead to Lesch–Nyhan syndrome, a disease with symptoms of self mutilation and mental retardation [4] The underlying mechanisms of Lesch– Nyhan syndrome are still not well understood, and the absence of HPRT gives rise to a complex altered gene expression profile [5] Tissue culture, HPRT knockout mice and neonatal dopamine lesion models have been used to elucidate the biochemical and physiological processes taking place in these diseases [6] Furthermore, protozoan parasites have no de novo purine nucleotide synthesis and must rely solely on salvage enzymes, which therefore makes HPRT a potential antiparasital drug target [7] A homolog with 65% identity to HPRT, phosphoribosyltransferase domain containing protein (PRTFDC1) is present in the human genome A recent study identified this homologue as a gene potentially involved in the development of ovarian cancer In a genome-wide screening for differently methylated promoter islands, PRTFDC1 was transcribed in ovarian cancer cell lines with unmethylated DNA but not in cancer cell lines with methylated DNA [8] A similar study demonstrated that restoring PRTFDC1 in oral squamous cell carcinomas cells lacking its expression inhibited cell growth [9] HPRT is well characterized using both biochemical and structural methods, whereas PRTFDC1 is poorly characterized Sequence analysis of a wide range of species indicates that HPRT have eleven completely conserved amino acids, whereas PRTFDC1 genes not show full conservation of these eleven residues [10,11] Because one of the differences in PRTFDC1 is Gly145, which corresponds to the proposed catalytic residue Asp137 in HPRT [12], it was suggested that the PRTFDC1 proteins have lost their phosphoribosyltransferase activity [10] The structural characterization of numerous complexes of the human HPRT and several bacterial and protozoan HPRTs have been undertaken [13–17] The structure of human HPRT can be divided into two domains: a core domain and a hood domain [14,18] The HPRTs have a flexible loop, referred to as loop II, that covers the active site in the substrate bound forms [13,14,17,19] Human HPRT has been shown to be a dimer or a tetramer in solution depending on ionic strength [20] In the present study, we report the structural and functional characterization of the human HPRT homolog PRTFDC1 To shed light on the potential function of PRTFDC1, the protein was screened against a nucleotide metabolome library containing 81 potential substrates or regulatory ligands for enzymes in the human nucleotide metabolism These were selected as substrates, intermediates and products of other enzymes in the human nucleotide metabolism The library was screened against PRTFDC1 using differential static light scattering (DSLS) thermal-melt assay PRPP, IMP and GMP were the top hits and therefore were identified as potential substrates, products or regulatory ligands for PRTFDC1 The catalytic efficiency and substrate specificity of PRTFDC1 was further characterized using a radiochemical assay with tritium-labeled bases as substrates, whereas the structural basis for substrate recognition and activity was revealed by solving the structure of PRTFDC1 in com˚ plex with the product GMP at 1.7 A resolution Results Overall structure The structure of full-length human PRTFDC1 was ˚ determined at 1.7 A resolution with two subunits in the asymmetric unit Most of the polypeptide chains could be traced in the electron density, with only a few residues at the N-terminus and two short flexible loops left unmodeled The 3D structures of HPRTs have been divided into a core and a hood domain [14] The core domain of PRTFDC1 contains a six-stranded twisted parallel b-sheet surrounded by three a-helices b4 in the core domain extends into a b-ribbon with b5, stabilizing loop II The hood domain is mainly built up by residues from the C-terminus and consists of a two-stranded anti-parallel b-sheet composed of b2 and b9 and an a-helix from the C-terminus (Fig 1A) The two subunits in the asymmetric unit, together with two symmetry-related subunits, form a plausible tetramer similar to the tetramer in HPRT (Fig 1B) However, the gel filtration profile reveals a dimeric protein (data FEBS Journal 277 (2010) 4920–4930 ª 2010 The Authors Journal compilation ª 2010 FEBS 4921 Studies of the human PRTFDC1 A M Welin et al B C Fig The structure of PRTFDC1 (A) monomer and (B) tetramer generated using symmetry-related molecules Interactions with GMP (C) GMP, phosphate and calcium ions are shown as sticks and spheres colored pink and black, respectively not shown) The addition of PRPP has been observed to induce tetramerization for HPRTs [21,22] and the use of 10 mm PRPP in crystallization set-up for PRTFDC1 could explain the different oligomeric states for PRTFDC1 in solution and crystal structure Nucleotide binding In the initial electron density maps, a clear difference density was found in the active site corresponding to a nucleoside monophosphate, despite no nucleotide being added to crystallization solutions The ligand bound was interpreted as GMP because the N2 of the base makes a hydrogen bond to a main chain carbonyl Binding of a xanthine base from XMP in the same position, would lead to the loss of a hydrogen bond and a less favorable interaction To investigate this further, the protein was heated until precipitated, and the remaining solute was scanned using a spectrophotometer The UV-absorption spectrum of the solute from the precipitated protein displayed a similar UV-absorption spectrum as a GMP solution, suggesting that GMP was bound to the protein A similar GMP bound enzyme was observed in a xanthine phosphoribosyltransferase family member in Bacillus subtil4922 is [23] A likely explanation for bound GMP is that it originates from expression in Escherichia coli cells In the structure the base of bound GMP is stacked between Val143 and Phe194 N1, N2 and O6 of the nucleotide form hydrogen bonds to main chain atoms, whereas the side chain of Lys173 interacts with both O6 and N7 (Fig 1C) The 2¢OH of the ribose is hydrogen bonded to the main chain and the side chain of Asp142 (Fig 1C) Both hydroxyl groups of the ribose are involved in coordinating a putative Ca2+ ion This Ca2+ is likely to have been exchanged with the active site Mg2+ ion normally used by this family as a result of the high concentration of Ca2+ present in the crystallization buffer The geometry is consistent with a Ca2+ ion coordinated by Glu141, Asp142, 2¢ and 3¢OH of the ribose and three water molecules with ˚ coordination distances of approximately 2.4 A The phosphate of the GMP is hydrogen bonded to main chain and side chain atoms of amino acids 145–149 (Fig 1C) A second Ca2+ ion is coordinated by the side chain of Asp201, a phosphate ion and five water molecules The phosphate involved in this interaction is bound by main chain interactions with Lys76 and Gly77 and the side chain of Arg207 The phosphate has some FEBS Journal 277 (2010) 4920–4930 ª 2010 The Authors Journal compilation ª 2010 FEBS M Welin et al Studies of the human PRTFDC1 additional density, suggesting that it may be a degradation product of PRPP from the crystallization solution ing 81 compounds of substrates, products and regulators of other enzymes in the human nucleotide metabolism (Table S1) The library can then be seen to constitute the specific metabolome of the nucleotide metabolism, which would be the most likely source for a substrate or a regulator of PRTFDC1 The compounds producing the largest increase in calculated DTagg (i.e the difference in midpoint of the aggregation process measured by DSLS) were PRPP, GMP and IMP Large increase in thermal shift is an indication of protein-compound binding The high thermal shifts for GMP, and IMP indicated that PRTFDC1 could have similar activity as HPRT or, alternatively, be regulated by these nucleotides Therefore, PRTFDC1 was rescreened against nucleobases hypoxanthine, guanine, cytidine, uracil, adenine and xanthine (X), as well as their respective nucleoside monophosphates Although IMP and GMP produced large shifts, adding only the bases to the enzyme did not produce any thermal shifts However, in the presence of 50 lm PRPP thermal shifts were observed for hypoxanthine and guanine (Table 1) These results imply that PRPP is required for the nucleobases to bind For comparison the DSLS thermal melt assay was run for HPRT but, unfortunately HPRT gave uninterruptable aggregation temperature profiles Comparison with HPRTs Superposition of PRTFDC1 and human HPRT results ˚ in a rmsd of 1.0–1.7 A for approximately 200 Ca atoms depending on which HPRT complex is compared (Protein Data Bank code: 1Z7G, 1D6N, 1BZY, 1HMP) The overall structures are very similar with the exception of loop II, which, in some HPRT complexes with transition state analogs, closes over the active site In the B subunit of PRTFDC1, this loop interacts with a symmetry-related molecule and thus the whole loop is visible This open conformation of the loop has also been observed in Giardia lamblia guanine phosphoribosyltransferase where the conformation is also stabilized by crystal contacts [24] The bound phosphate ion superposes well with one of the phosphates of pyrophosphate in the transition state analog complex of human HPRT with immucillinGP (ImmGP) and pyrophosphate bound (Fig 2A) [17] The conserved Lys76 in PRTFDC1 is in a cis-peptide conformation, which also has been observed in the human HPRT in complex with the transition state analog, ImmGP [17] Steady-state kinetic analysis Screening of nucleotide metabolome library to identify PRTFDC1 substrates To examine whether PRTFDC1 possessed any phosphoribosyltransferase activity, a radiochemical method was used with tritium-labeled bases and PRPP as substrates Reaction products were separated from substrate and quantified by using the DE-81 filter paper To investigate the potential function of PRTFDC1 a DSLS thermal-melt assay was used to screen for interactions with a nucleotide metabolome library contain- B A S103 G145/D137 Y112 D201/193 L75/67 G145/D137 E141/133 K173/165 R207/199 G77/69 D142/134 G197/189 Fig Superposition of PRTFDC1 with HPRT-ImmGP (Protein Data Bank code: 1BZY) (A) Residues in the PRPP binding motif, nucleotides, pyrophosphate and phosphate are shown as sticks in brown and pink, respectively Metals are shown with same coloring scheme (B) Superposition of PRTFDC1 with HPRT-GMP (Protein Data Bank code: 1HMP) and HPRT-ImmGP (Protein Data Bank code: 1BZY) the structures are colored brown, white and pink The water molecule in proximity to the Asp137 is colored in blue FEBS Journal 277 (2010) 4920–4930 ª 2010 The Authors Journal compilation ª 2010 FEBS 4923 Studies of the human PRTFDC1 M Welin et al Table DSLS thermal-melt assay The results are based on two independent experiments each containing the samples in triplicate and given as the mean ± SD Ligand DTagg (°C) 500 lMa DTagg (°C) 10 mMb PRPP IMP GMP CMP UMP AMP XMP 5.8 5.1 4.5 )0.2 )0.2 0.0 0.6 12.7 7.8 10.5 0.2 0.5 1.4 3.5 Ligand DTagg (°C) 500 lMc PRPP Hypoxanthine ⁄ PRPP Guanine ⁄ PRPP Cytosine ⁄ PRPP Uracil ⁄ PRPP Adenine ⁄ PRPP Xanthine ⁄ PRPP 4.7 2.9 )0.1 0.2 )0.2 0.0 ± ± ± ± ± ± ± 0.1 0.7 0.4 0.4 0.4 0.4 0.6 ± ± ± ± ± ± ± 0.8 0.1 0.2 0.2 0.6 0.5 0.4 DTagg (°C) 50 lMd 0.4 ± 0.3 ± ± ± ± ± ± 1.2 0.4 0.1 0.1 0.1 0.1 a Condition; [ligand]: 500 lM in 20 mM Hepes, 300 mM NaCl, mM MgCl2 DTagg calculated using control Tagg; 53.0 ± 0.7 °C b Condition; [ligand]: 10 mM in 20 mM Hepes, 300 mM NaCl, 10 mM MgCl2 DTagg calculated using control Tagg; 52.5 ± 0.6 °C c [PRPP]: 50 lM; [base]: 500 lM in 20 mM Hepes, 300 mM NaCl, 10 mM MgCl2, 1.25% dimethylsulfoxide DTagg calculated using control Tagg; 52.7 ± 0.4 °C d [PRPP]: 50 lM in 20 mM Hepes, 300 mM NaCl, 10 mM MgCl2, 1.25% dimethylsulfoxide technique taking the advantage of charge difference between substrate and product Phosphoribosyltransferase activity was detected with hypoxanthine and guanine and there was no detectable activity with adenine Subsequently, the kinetic constants were determined Human HPRT was characterized in parallel to compare their catalytic efficiency The Km (hypoxanthine) value for PRTFDC1 was 23.3 ± 6.8 lm, and the Vmax value was 0.340 ± 0.037 lmolỈmin)1Ỉmg)1, whereas the values for HPRT were 3.9 ± 1.5 lm and 23.3 ± 2.0 lmolỈ min)1Ỉmg)1, respectively (Fig 3A,C and Table 2) With hypoxanthine as a substrate, the Km value is approximately six-fold higher for PRTFDC1, and Vmax is approximately 70-fold lower compared to those of HPRT Thus, the kcat ⁄ Km value with PRTFDC1 is only 0.26% of that of HPRT The Km (guanine) value for PRTFDC1 was 36.1 ± 14.3 lm, and the Vmax was value 2.9 ± 0.7 lmolỈmin)1Ỉmg)1, whereas the values for HPRT were 9.9 ± 0.2 lm and 899 ± 117 lmolỈmin)1Ỉ mg)1, respectively (Fig 3B,D and Table 2) This indicates that, with guanine as a substrate, the Km value is approximately four-fold higher, and the Vmax value is approximately 310-fold lower compared to HPRT Therefore, the catalytic efficiency (kcat ⁄ Km value) of PRTFDC1 is 0.09% compared to HPRT 4924 Discussion Because PRTFDC1 was annotated as a protein with unknown function, the use of a DSLS thermal-melt assay proved to be a good initial step for elucidating which compounds could stabilize the protein The increased DTagg for IMP, GMP, hypoxanthine ⁄ PRPP and guanine ⁄ PRPP indicated a similar binding profile as for HPRT The DSLS results also suggest a sequential mechanism where PRPP binds first, inducing a conformation change, then allowing the purine base to bind in accordance with the mechanism of human HPRT [19,25] This conclusion can be made based on the lack of thermal shifts when only the bases were added, whereas the addition of PRPP led to a significant thermal shift Kinetic studies of PRTFDC1 showed that this enzyme had similar substrate specificity as HPRT, with guanine as the best substrate However, the catalytic efficiency (kcat ⁄ Km) of PRTFDC1 is much lower than that of HPRT The differences in catalytic efficiency could be explained by the difference in amino acid residues involved in catalysis between PRTFDC1 and HPRT In the structure, residues interacting with PRPP in PRTFDC1 are slightly different than those in HPRT (Fig 4), with the most striking difference being a Gly substitution in the position of the HPRT Asp137 [18] Mutations of Asp137in human HPRT to Asn resulted in a 290-fold and 18-fold decrease in kcat values for nucleotide formation with guanine and hypoxanthine bases, respectively, indicating that Asp137 functions as a catalytic base [12] However, a tight binding of N7 of the purine base and Asp137 was observed in HPRT in complex with ImmGP, indicating a role in transition state stabilization during catalysis [17] A series of mutations were made of the invariant Asp in HPRT from Trypanosoma cruzi The D137G mutation in this enzyme, corresponding to the situation observed in PRTFDC1, was shown to have some residual activity for the forward reaction [26] When the HPRT in complex with GMP and ImmGP are superposed with the PRTFDC1 structure, a water molecule is found at approximately the same distance from N7 of the guanine base as is the oxygen of the aspartic acid side chain in the HPRT complex (Fig 2B) It is possible that this water molecule could provide some catalytic power by acting as a weak base in the reaction Both Km and Vmax values for HPRT with hypoxanthine determined in the present study are in agreement with values reported in the literature [45] However, the Vmax for HPRT with guanine as substrate using the DE-81 filter assay was 899 lmolỈmin)1Ỉmg)1, which is 20-fold higher than reported previously [45] This FEBS Journal 277 (2010) 4920–4930 ª 2010 The Authors Journal compilation ª 2010 FEBS M Welin et al Studies of the human PRTFDC1 B A PRTFDC1 25 HPRT 20 1000 0.2 800 600 400 0.1 200 –50 50 10 10 50 100150 200 250 S 200 –50 250 50 1200 100 150 [Hx] (µM) 2.0 PRTFDC1 50 100 150 200 250 S HPRT D C 0.0 12 15 S/V V (µmol·min–1·mg–1) 0.3 S/V V (µmol·min–1·mg–1) 0.4 100 150 [Hx] (µM) 200 250 50 1.0 40 30 20 0.5 10 800 600 0.10 0.08 400 S/V V (µmol·min–1·mg–1) 60 S/V Fig Characterization of PRTFDC1 and HPRT with hypoxanthine and guanine Michaelis–Menten and Hanes–Woolf plots illustrate the kinetic pattern The experiments were repeated three to four times (A) PRTFDC1 and hypoxanthine (B) HPRT and hypoxanthine (C) PRTFDC1 and guanine (D) HPRT and guanine V (µmol·min–1·mg–1) 1000 1.5 200 0 20 40 60 80 [G] (µM) 100 120 0.04 0.02 –40 –20 20 40 60 80 100 120 S 0.0 0.06 –10 10 20 30 40 50 60 S 10 20 30 40 [G] (µM) 50 60 Table Km and Vmax values for Hx and G in the presence of mM PRPP, determined using the DE-81 filter paper assay and tritium-labeled substrates Experiments have been repeated three to four times and the data are given as the mean ± SD kcat was calculated using Mw (HPRT) = 27132 Da and Mw (PRTFDC1) = 28226 Da The kcat ⁄ Km for HPRT was set to 100% as a reference for both substrates, and kcat ⁄ Km for PRTFDC1, shown in parenthesis, was calculated in relation to this Km (lM) Hypoxanthine PRTFDC1 HPRT Guanine PRTFDC1 HPRT Vmax (lmolỈmin)1Ỉmg)1) kcat (s)1) kcat ⁄ Km (s)1ỈM)1) 23.3 ± 6.8 3.9 ± 1.5 0.340 ± 0.037 23.3 ± 2.0 0.16 ± 0.02 10.5 ± 0.9 7.4 · 103 ± 1.9 · 103 (0.26%) 2.9 · 106 ± 1.0 · 106 (100%) 36.1 ± 14.3 9.9 ± 0.2 2.9 ± 0.7 899 ± 117 1.36 ± 0.34 406 ± 53 3.9 · 104 ± 9.3 · 103 (0.09%) 4.5 · 107 ± 1.0 · 107 (100%) discrepancy might be explained by the different methods used We used a radiochemical method in which the amount of products was quantified directly, and therefore it is a more accurate and sensitive method compared to the spectrophotometric methods Because the enzymatic activity of PRTFDC1 towards hypoxanthine and guanine is only 0.26% and 0.09%, respectively, of the activity of HPRT, the role of the PRTFDC1 in purine metabolism remains unclear and it is uncertain whether it has the capacity to compensate for a deficiency or partial deficiency in HPRT Knowledge of the expression pattern of PRTFDC1 in healthy individuals and patients with an impaired HPRT gene has not yet provided clues for whether PRTFDC1 over-expression in individuals carrying mutations in HPRT might lead to a milder disease phenotype The concentration of hypoxanthine available for salvage in human tissues is 8.2 ± 1.3 lm [28,29] compared to the Km for PRTFDC1, which is 23 lm, indicated that PRTFDC1 is not turning over this substrate to a larger extent Indeed, these data indicate that PRTFDC1 does not have a direct catalytic role in the nucleotide salvage pathway When PRTFDC1 has been shown to interact with HPRT FEBS Journal 277 (2010) 4920–4930 ª 2010 The Authors Journal compilation ª 2010 FEBS 4925 Studies of the human PRTFDC1 Loop II M Welin et al PRPP motif [29], one possibility is that it participates in forming heterooligomers containing subunits of both PRTFDC1 and HPRT, thereby providing an additional means of regulating the activity of HPRT Recently, Suzuki et al [9] showed that PRTFDC1 expression often was silenced in oral squamous-cell carcinoma lines By reintroducing PRTFDC1 expression in silenced oral squamous-cell carcinoma cells, growth was reduced This indicates that PRTFDC1 might have an important regulatory role, although the molecular basis for this activity remains to be elucidated The elucidation of the structure of PRTFDC1 and the definition of its ligand binding specificity from a large metabolome library provides an initial start point for defining the molecular function of PRTFDC1 The availability of a high resolution structure may also assist efforts aiming to develop chemical probes that could be used to pinpoint the function of PRTFDC1 using chemical biology strategies Experimental procedures Cloning, expression and purification The PRTFDC1 and HPRT gene was obtained from National Institute of Health Mammalian Gene Collection 4926 Fig Sequence alignment of PRTFDC1 (accession number: NP_064585), HPRT (accession number: AAA36012), Plasmodium falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase (PfHGXPRT) (accession number: NP_700595), TzHPRT (accession number: XP_816917) and GlGPRT (accession number: XP_779753) Loop II and the PRPP motif are shown in the alignment Secondary structure for PRTFDC1 is shown above the sequence alignment, g-310-helix Black and white boxes refer to identical and similar residues, respectively (accession numbers: BC008662 and BC000578) The sequences encoding residues 1–225 (PRTFDC1) and 1–218 (HPRT) were amplified by PCR and inserted into pNIC28Bsa4 vector by ligation independent cloning Constructs included an N-terminal tag containing a 6-His sequence (MHHHHHHSSGVDLGTENLYFQSM) The pNIC-Bsa4 vector containing the insert was transformed into E coli BL21(DE3) strain and stored at )80 °C for further use One hundred and fifty milliliters of LB supplemented with 50 lgỈmL)1 kanamycin was inoculated and grown at 37 °C overnight; 40 mL of this culture were used to inoculate L of TB supplemented with 50 lgỈmL)1 kanamycin and approximately 50 lL of Breox antifoam (Cognis Performance Chemical UK Ltd) in glass flasks using the large scale expression system Cells were grown at 37 °C until D600 of 1.2 was reached followed by down-tempering to 18 °C for h in water bath Expression was induced by the addition of isopropyl thio-b-d-glactoside with a concentration of 0.5 mm and growth was allowed to continue overnight Cells were harvested by centrifugation at 3500 g for 20 and frozen at )80 °C Before purification, the cell pellet was re-suspended in 50 mm Hepes, 500 mm NaCl, 10% glycerol, 10 mm imidazole and 0.5 mm tris(2-carboxyethyl)phosphine (TCEP) supplemented with one tablet per 50 mL of Complete EDTA-free protease inhibitor tablet (Santa Cruz Biotechnlogy, Santa Cruz, CA, USA) and lL per 50 mL of benzonase Cells were disrupted by high FEBS Journal 277 (2010) 4920–4930 ª 2010 The Authors Journal compilation ª 2010 FEBS M Welin et al Studies of the human PRTFDC1 pressure homogenization run three times at 10 000 p.s.i and samples were centrifuged for 40 at 50 228 g The soluble fraction was filtered through 0.2 lm lters and subă jected to further purication on an AKTAprime system Table Data collection and refinement statistics Values in parentheses refer to the highest resolution shell data (GE Healthcare) Columns used were HiTrap Chelating mL and HiLoadÔ 16 ⁄ 60 Superdex 200 Prep Grade (GE Healthcare) The mL HiTrap chelating HP column was equilibrated with IMAC buffer [50 mm Hepes (pH 7.5), 10 mm imidazole, 500 mm NaCl, 10% glycerol, 0.5 mm TCEP], washed with IMAC buffer followed by IMAC buffer [50 mm Hepes (pH 7.5), 30 mm imidazole, 500 mm NaCl, 10% glycerol, 0.5 mm TCEP] and eluated with [50 mm Hepes (pH 7.5), 500 mm imidazole, 500 mm NaCl, 10% glycerol, 0.5 mm TCEP] Additional purification was conducted on a Superdex 200 column using gel-filtration buffer [20 mm Hepes (pH 7.5), 300 mm NaCl, 10% glycerol, mm TCEP] The purity of the protein was estimated on SDS ⁄ PAGE The protein concentration was measured using Bradford reagent [27] A similar protocol was used for the expression and purification of HPRT, with the exception that sonication was used instead of high pressure homogenization, the ă purication was conducted on an AKTAxpress system (GE Healthcare) European Synchrotron Radiation Facility beam line ˚ Wavelength (A) Space group ˚ Unit cell parameters (A) Crystallization, data collection and structure determination Crystals were initially obtained from the JCSG screen [28] [#D10, 100 mm sodium cacodylate (pH 6.5), 200 mm calcium acetate, 40% PEG 300] co-crystallized with 10 mm PRPP and 10 mm MgCl2 at room temperature Crystallization experiments were set up using the Phoenix crystallization robot (Art Robbins Instrument, Sunnyvale, CA, USA) In the optimized condition with 100 mm sodium cacodylate (pH 6.1), 200 mm calcium acetate and 34% PEG 300, using a protein concentration of 20.5 mgỈmL)1, 10 mm PRPP and 10 mm MgCl2 3D crystals grew in a few days using hanging drop vapor diffusion A crystal was transferred into a cryosolution containing 100 mm sodium cacodylate (pH 6.1), 200 mm calcium acetate and 40% PEG 300 before being flash frozen in liquid nitrogen The human PRTFDC1 crystals belong to space group P321 and have a solvent content of 56.4% The asymmetric unit contained two subunits Data were collected at ID29 of the European Synchrotron Radiation Facility (Grenoble, ˚ France) using a wavelength of 1.04 A The data were integrated using mosflm [29] and scaled using scala from the ccp4 software suit [30] The structure was solved with the molecular replacement software molrep [31] using coordinates from a monomer of HPRT (Protein Data Bank code: 1HMP) After simulated annealing using cns [32], most of the structure could be manually rebuilt using coot [33] Refinement was carried out using refmac5 [34] Data collection and refinement statistics are shown in Table PRTFDC1-GMP Content of the asymmetric unit ˚ Resolution (A) Completeness (%) Rmeas (%) Redundancy Number of unique reflections Refinement Rwork (%)a Rfree (%)b Rmsd ˚ Bond length (A) Bond angle (o) ˚ Mean B-value (A2) Ramachandran plot (%)c Favored regions Additionally allowed regions ID29 1.04 P321 a = b = 139.2 c = 52.1 Two subunits 1.7 (1.70–1.79) 100 (100) 10.0 (48.2) 19.4 (4.2) 10.8 (11.0) 63939 18.2 21.5 0.012 1.4 20.2 98.04 1.96 Rwork is defined as R||Fobs| ) |Fcalc|| ⁄ R|Fobs|, where Fobs and Fcalc are observed and calculated structure-factor amplitudes, respectively b Rfree is the R factor for the test set (5% of the data) c According to MOLPROBITY [35] a [35] Residues 7–225 and 9–225 could be modeled for subunits A and B, respectively A covalent modification on Cys82 was observed likely as the result of a reaction with cacodylate creating S-(dimethylarsenic)cysteine Library files and coordinates for GMP and S-(dimethylarsenic)cysteine were obtained from the HIC-UP ligand database [36] and generated at the prodrg server [37] All figures were condtructed using pymol [38] and the alignments were performed using clustalw [39] and ESPript [40] Superpositions used in the text and figures were made using the ssm superposition algorithm in coot [33,41] The coordinates and structure factors are published in the Protein Data Bank under accession number 2JBH Characterization Using DSLS thermal denaturation [42–43], PRTFDC1 was screened against a nucleotide metabolome library consisting of 81 nucleoside, (deoxy)nucleotide(mono-, di-, tri-phosphate), and various other metabolites of both the purine and pyrimidine pathways (Table S1) The samples were run twice in duplicate on a StarGazer-384 (Harbinger Biotechnology and Engineering Corporation, Markham, Ontario, Canada) Assay conditions were: 500 lm compound, 0.2 mgỈmL)1 (7.1 lm) protein, 20 mm Hepes (pH 7.5), FEBS Journal 277 (2010) 4920–4930 ª 2010 The Authors Journal compilation ª 2010 FEBS 4927 Studies of the human PRTFDC1 M Welin et al 300 mm NaCl, and mm MgCl2 Following initial screening, a subset consisting of IMP, GMP, XMP, AMP, CMP and PRPP were screened at 500 lm and 10 mm, with mm and 10 mm MgCl2 respectively [20 mm Hepes (pH 7.5), 300 mm NaCl] A second subset was screened against 50 lm PRPP in combination with 500 lm nucleobase (hypoxanthine, guanine, adenine, xanthine, cytosine, uracil) in 20 mm Hepes, 300 mm NaCl, mm MgCl2 and 1.25% dimethylsulfoxide All StarGazer-384 screening was conducted using 384 well optical clear-bottom plates (#242764; Nunc, Rochester, NY, USA), with an assay volume of 50 lL per well The plate was heated at °CỈmin)1, and images take every 0.5 °C in the range 25–80 °C Intensities (as a measure of light scattering from protein aggregation) were converted from the images, and the intensities were plotted as a function of temperature and the midpoint of transition in aggregation (Tagg) calculated [42,43] using the manufactuter’s software (Harbinger Biotech) The reported DTagg represents the calculated difference between Tagg in the presence of compound to be tested and a control condition without compound Enzyme activities were determined by initial velocity measurements based on four time samples using the DE-81 (DEAE-cellulose) filter paper assay adopted from the deoxynucleoside kinase assay method [44] with tritium-labeled bases as substrates The standard assay mixture contained in a reaction volume of 50 lL: 100 mm Tris-HCl (pH 8.0), 10 mm MgCl2, 0.5 mgỈmL)1 BSA, mm PRPP, 1–200 lm [3H]hypoxanthine (13.8 CiỈmmol)1; Perkin Elmer, Boston, MA, USA), 1–100 lm [3H]guanine (10.7 CiỈmmol)1; Moravek, Brea, CA, USA) and [3H]adenine (27.2 CiỈmmol)1; Perkin Elmer) The assay mix was preheated at 37 °C for min, and the reactions were started by adding 10 lL of enzyme (HPRT: 0.07 or 0.7 ng per assay; PRTFDC1: 39 ng per assay), and a 10 lL aliquot was withdrawn and spotted onto DE-81 filter paper at 0, 4, and 12 The negatively-charged products will bind to the positively charged DE-81 filter papers Nonreacted substrates were removed by washing the filter papers · in mm ammonium formate solution and once in deionized water The tritiumlabeled products on DE-81 filter paper were eluted for 30 in 0.5 mL of 0.2 m KCl ⁄ 0.1 m HCl; subsequently, mL of scintillation liquid (Optiphase ‘hisafe’ 3; Perkin Elmer) was added to each vial, and radioactivity was counted in a liquid scintillation counter (Beckman Coulter, Fullerton, CA, USA) The kinetic data were fitted into the Michaelis–Menten equation v = VmaxỈ[S] ⁄ (Km + [S]) using the sigmaplot enzyme kinetic model, version 2.1 (SPSS Inc., Chicago, IL, USA) Acknowledgements This work was supported by grants from the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (to L.W and S.E.), the 4928 Swedish Research Council (to H.E and P.N.) and the Swedish Cancer Foundation (to H.E and P.N.) The Structural Genomics Consortium is a registered charity (number 1097737) that receives funds from the Canadian Institutes for Health Research, the Canadian Foundation for Innovation, Genome Canada through the Ontario Genomics Institute, GlaxoSmithKline, Karolinska Institutet, the Knut and Alice Wallenberg Foundation, the Ontario Innovation Trust, the Ontario Ministry for Research and Innovation, Merck & Co., Inc., the Novartis Research Foundation, the Swedish Agency for Innovation Systems, the Swedish Foundation for Strategic Research and the Wellcome Trust References Jinnah HA, De Gregorio L, Harris JC, Nyhan WL & O’Neill JP (2000) The spectrum of inherited mutations causing HPRT deficiency: 75 new cases and a review of 196 previously reported cases Mutat Res 463, 309–326 Jinnah HA, Harris JC, Nyhan WL & O’Neill JP (2004) The spectrum of mutations causing HPRT deficiency: an update Nucleosides Nucleotides Nucleic Acids 23, 1153–1160 Sculley DG, Dawson PA, Emmerson BT & Gordon RB (1992) A review of the molecular basis of hypoxanthineguanine phosphoribosyltransferase (HPRT) deficiency Hum Genet 90, 195–207 Lesch M & Nyhan WL (1964) A familial disorder of uric acid metabolism and central nervous system function Am J Med 36, 561–570 Song S & Friedmann T (2007) Tissue-specific aberrations of gene expression in HPRT-deficient mice: functional complexity in a monogenic disease? Mol Ther 15, 1432–1443 Jinnah HA (2009) Lesch-Nyhan disease: from mechanism to model and back again Dis Model Mech 2, 116–121 Wang CC (1984) Parasite enzymes as potential targets for antiparasitic chemotherapy J Med Chem 27, 1–9 Cai LY, Abe M, Izumi S, Imura M, Yasugi T & Ushijima T (2007) Identification of PRTFDC1 silencing and aberrant promoter methylation of GPR150, ITGA8 and HOXD11 in ovarian cancers Life Sci 80, 1458–1465 Suzuki E, Imoto I, Pimkhaokham A, Nakagawa T, Kamata N, Kozaki KI, Amagasa T & Inazawa J (2007) PRTFDC1, a possible tumor-suppressor gene, is frequently silenced in oral squamous-cell carcinomas by aberrant promoter hypermethylation Oncogene 26, 7921–7932 10 Keebaugh AC, Sullivan RT & Thomas JW (2007) Gene duplication and inactivation in the HPRT gene family Genomics 89, 134–142 11 Nicklas JA (2006) Pseudogenes of the human HPRT1 gene Environ Mol Mutagen 47, 212–218 FEBS Journal 277 (2010) 4920–4930 ª 2010 The Authors Journal compilation ª 2010 FEBS M Welin et al 12 Xu Y & Grubmeyer C (1998) Catalysis in human hypoxanthine-guanine phosphoribosyltransferase: Asp 137 acts as a general acid ⁄ base Biochemistry 37, 4114–4124 13 Balendiran GK, Molina JA, Xu Y, Torres-Martinez J, Stevens R, Focia PJ, Eakin AE, Sacchettini JC & Craig SP III (1999) Ternary complex structure of human HGPRTase, PRPP, Mg2+, and the inhibitor HPP reveals the involvement of the flexible loop in substrate binding Protein Sci 8, 1023–1031 14 Eads JC, Scapin G, Xu Y, Grubmeyer C & Sacchettini JC (1994) The crystal structure of human hypoxanthine-guanine phosphoribosyltransferase with bound GMP Cell 78, 325–334 15 Focia PJ, Craig SP III, Nieves-Alicea R, Fletterick RJ & Eakin AE (1998) A 1.4 A crystal structure for the hypoxanthine phosphoribosyltransferase of Trypanosoma cruzi Biochemistry 37, 15066–15075 16 Shi W, Li CM, Tyler PC, Furneaux RH, Cahill SM, Girvin ME, Grubmeyer C, Schramm VL & Almo SC (1999) The 2.0 A structure of malarial purine phosphoribosyltransferase in complex with a transition-state analogue inhibitor Biochemistry 38, 9872–9880 17 Shi W, Li CM, Tyler PC, Furneaux RH, Grubmeyer C, Schramm VL & Almo SC (1999) The 2.0 A structure of human hypoxanthine-guanine phosphoribosyltransferase in complex with a transition-state analog inhibitor Nat Struct Biol 6, 588–593 18 Scapin G, Grubmeyer C & Sacchettini JC (1994) Crystal structure of orotate phosphoribosyltransferase Biochemistry 33, 1287–1294 19 Keough DT, Brereton IM, de Jersey J & Guddat LW (2005) The crystal structure of free human hypoxanthine-guanine phosphoribosyltransferase reveals extensive conformational plasticity throughout the catalytic cycle J Mol Biol 351, 170–181 20 Johnson GG, Eisenberg LR & Migeon BR (1979) Human and mouse hypoxanthine-guanine phosphoribosyltransferase: dimers and tetramers Science 203, 174– 176 21 Gayathri P, Sujay Subbayya IN, Ashok CS, Selvi TS, Balaram H & Murthy MR (2008) Crystal structure of a chimera of human and Plasmodium falciparum hypoxanthine guanine phosphoribosyltransferases provides insights into oligomerization Proteins 73, 1010–1020 22 Strauss M, Behlke J & Goerl M (1978) Evidence against the existence of real isozymes of hypoxanthine phosphoribosyltransferase Eur J Biochem 90, 89–97 23 Arent S, Kadziola A, Larsen S, Neuhard J & Jensen KF (2006) The extraordinary specificity of xanthine phosphoribosyltransferase from Bacillus subtilis elucidated by reaction kinetics, ligand binding, and crystallography Biochemistry 45, 6615–6627 24 Raman J, Sumathy K, Anand RP & Balaram H (2004) A non-active site mutation in human hypoxanthine Studies of the human PRTFDC1 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 guanine phosphoribosyltransferase expands substrate specificity Arch Biochem Biophys 427, 116–122 Xu Y, Eads J, Sacchettini JC & Grubmeyer C (1997) Kinetic mechanism of human hypoxanthine-guanine phosphoribosyltransferase: rapid phosphoribosyl transfer chemistry Biochemistry 36, 3700–3712 Canyuk B, Focia PJ & Eakin AE (2001) The role for an invariant aspartic acid in hypoxanthine phosphoribosyltransferases is examined using saturation mutagenesis, functional analysis, and X-ray crystallography Biochemistry 40, 2754–2765 Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding Anal Biochem 72, 248–254 Page R & Stevens RC (2004) Crystallization data mining in structural genomics: using positive and negative results to optimize protein crystallization screens Methods 34, 373–389 Leslie AGW (1992) Joint CCP4 + ESF-EAMCB Newsletter on Protein Crystallography, No 26 CCP4 (1994) The CCP4 suite: programs for protein crystallography Acta Crystallogr D Biol Crystallogr 50, 760–763 Vagin A & Teplyakov A (2000) An approach to multicopy search in molecular replacement Acta Crystallogr D Biol Crystallogr 56, 1622–1624 Brunger AT, Adams PD, Clore GM, DeLano WL, Gros P, Grosse-Kunstleve RW, Jiang JS, Kuszewski J, Nilges M, Pannu NS et al (1998) Crystallography & NMR system: a new software suite for macromolecular structure determination Acta Crystallogr D Biol Crystallogr 54, 905–921 Emsley P & Cowtan K (2004) Coot: model-building tools for molecular graphics Acta Crystallogr D Biol Crystallogr 60, 2126–2132 Murshudov GN, Vagin AA & Dodson EJ (1997) Refinement of macromolecular structures by the maximum-likelihood method Acta Crystallogr D Biol Crystallogr 53, 240–255 Lovell SC, Davis IW, Arendall WB III, de Bakker PI, Word JM, Prisant MG, Richardson JS & Richardson DC (2003) Structure validation by Calpha geometry: phi,psi and Cbeta deviation Proteins 50, 437–450 Kleywegt GJ & Jones TA (1998) Databases in protein crystallography Acta Crystallogr D Biol Crystallogr 54, 1119–1131 Schuttelkopf AW & van Aalten DM (2004) PRODRG: a tool for high-throughput crystallography of proteinligand complexes Acta Crystallogr D Biol Crystallogr 60, 1355–1363 DeLano WL (2002) The PyMOL Molecular Graphics System DeLano Scientific, Palo Alto Chenna R, Sugawara H, Koike T, Lopez R, Gibson TJ, Higgins DG & Thompson JD (2003) Multiple sequence FEBS Journal 277 (2010) 4920–4930 ª 2010 The Authors Journal compilation ª 2010 FEBS 4929 Studies of the human PRTFDC1 40 41 42 42 44 M Welin et al alignment with the Clustal series of programs Nucleic Acids Res 31, 3497–3500 Gouet P, Robert X & Courcelle E (2003) ESPript ⁄ ENDscript: extracting and rendering sequence and 3D information from atomic structures of proteins Nucleic Acids Res 31, 3320–3323 Krissinel E & Henrick K (2004) Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions Acta Crystallogr D Biol Crystallogr 60, 2256–2268 Senisterra GA, Markin E, Yamazaki K, Hui R, Vedadi M & Awrey DE (2006) Screening for ligands using a generic and high-throughput light-scatteringbased assay J Biomol Screen 11, 940–948 Vedadi M, Niesen FH, Allali-Hassani A, Fedorov OY, Finerty PJ Jr, Wasney GA, Yeung R, Arrowsmith C, Ball LJ, Berglund H, et al (2006) Chemical screening methods to identify ligands that promote protein stability, protein crystallization, and structure determination Proc Natl Acad Sci USA 103, 15835–15840 Tyrsted G & Munch-Petersen B (1977) Early effects of phytohemagglutinin on induction of DNA polymerase, thymidine kinase, deoxyribonucleoside triphosphate 4930 pools and DNA synthesis in human lymphocytes Nucleic Acids Res 4, 2713–2723 45 Keough DT, Ng AL, Winzor DJ, Emmerson BT & de Jersey J (1999) Purification and characterization of Plasmodium falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase and comparison with the human enzyme Mol Biochem Parasitol 98, 29–41 Supporting information The following supplementary material is available: Table S1 Nucleotide metabolome library This supplementary material can be found in the online version of this article Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset Technical support issues arising from supporting information (other than missing files) should be addressed to the authors FEBS Journal 277 (2010) 4920–4930 ª 2010 The Authors Journal compilation ª 2010 FEBS ... S103 G145/D137 Y 112 D2 01/ 193 L75/67 G145/D137 E1 41/ 133 K173 /16 5 R207 /19 9 G77/69 D142 /13 4 G197 /18 9 Fig Superposition of PRTFDC1 with HPRT-ImmGP (Protein Data Bank code: 1BZY) (A) Residues in the. .. Welin et al Studies of the human PRTFDC1 B A PRTFDC1 25 HPRT 20 10 00 0.2 800 600 400 0 .1 200 –50 50 10 10 50 10 015 0 200 250 S 200 –50 250 50 12 00 10 0 15 0 [Hx] (µM) 2.0 PRTFDC1 50 10 0 15 0 200 250... 2.0 0 .16 ± 0.02 10 .5 ± 0.9 7.4 · 10 3 ± 1. 9 · 10 3 (0.26%) 2.9 · 10 6 ± 1. 0 · 10 6 (10 0%) 36 .1 ± 14 .3 9.9 ± 0.2 2.9 ± 0.7 899 ± 11 7 1. 36 ± 0.34 406 ± 53 3.9 · 10 4 ± 9.3 · 10 3 (0.09%) 4.5 · 10 7 ± 1. 0