phosphoribosyltransferase domain containing protein 1 Martin Welin1,*, Louise Egeblad2,*, Andreas Johansson1, Pa˚l Stenmark1,, Liya Wang2, Susanne Flodin1, Tomas Nyman1, Lionel Tre´saugu
Trang 1phosphoribosyltransferase domain containing protein 1 Martin Welin1,*, Louise Egeblad2,*, Andreas Johansson1, Pa˚l Stenmark1,, Liya Wang2,
Susanne Flodin1, Tomas Nyman1, Lionel Tre´saugues1, Tetyana Kotenyova1, Ida Johansson1, Staffan Eriksson2, Hans Eklund3and Pa¨r Nordlund1
1 Structural Genomics Consortium, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden
2 Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, Uppsala, Sweden
3 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 8 524 868 50; +46 18 55 0762
Tel: +46 8 524 868 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
(Received 15 November 2009, revised 18
August 2010, accepted 30 September 2010)
doi:10.1111/j.1742-4658.2010.07897.x
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 1 (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 nucleo-tide 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 effi-ciency (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 )
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.
Trang 2Human hypoxanthine guanine
phosphoribosyltransfer-ase (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),
pro-ducing 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
hav-ing mutations resulthav-ing 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]
Further-more, 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,
phos-phoribosyltransferase domain containing protein 1
(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
pro-moter 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 do
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
phosphoribosyl-transferase activity [10]
The structural characterization of numerous
com-plexes 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 dif-ferential static light scattering (DSLS) thermal-melt assay PRPP, IMP and GMP were the top hits and therefore were identified as potential substrates, prod-ucts 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 struc-tural 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
Trang 3not 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
car-bonyl 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
spectro-photometer The UV-absorption spectrum of the solute
from the precipitated protein displayed a similar
UV-absorption spectrum as a GMP solution,
suggest-ing that GMP was bound to the protein A similar
GMP bound enzyme was observed in a xanthine
phos-phoribosyltransferase family member in Bacillus
subtil-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 hydro-gen 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 crys-tallization 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
A
C
B
Fig 1 The structure of PRTFDC1 (A) mono-mer and (B) tetramono-mer generated using sym-metry-related molecules Interactions with GMP (C) GMP, phosphate and calcium ions are shown as sticks and spheres colored pink and black, respectively.
Trang 4additional density, suggesting that it may be a
degra-dation product of PRPP from the crystallization
solution
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
com-pared (Protein Data Bank code: 1Z7G, 1D6N, 1BZY,
1HMP) The overall structures are very similar with
the exception of loop II, which, in some HPRT
com-plexes 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
confor-mation 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]
Screening of nucleotide metabolome library to
identify PRTFDC1 substrates
To investigate the potential function of PRTFDC1 a
DSLS thermal-melt assay was used to screen for
inter-actions with a nucleotide metabolome library
contain-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 com-pounds producing the largest increase in calculated
DTagg (i.e the difference in midpoint of the aggrega-tion process measured by DSLS) were PRPP, GMP and IMP Large increase in thermal shift is an indica-tion 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 hypo-xanthine, guanine, cytidine, uracil, adenine and xan-thine (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 pres-ence 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
Steady-state kinetic analysis
To examine whether PRTFDC1 possessed any phos-phoribosyltransferase activity, a radiochemical method was used with tritium-labeled bases and PRPP as strates Reaction products were separated from sub-strate and quantified by using the DE-81 filter paper
S103 Y112
R207 / 199 K173 / 165
G145 / D137
D142 / 134 E141 / 133
L75 / 67 D201 / 193
G77 / 69 G197 / 189
G145 / D137
Fig 2 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 struc-tures are colored brown, white and pink The water molecule in proximity to the Asp137 is colored in blue.
Trang 5technique taking the advantage of charge difference
between substrate and product
Phosphoribosyltransfer-ase activity was detected with hypoxanthine and guanine
and there was no detectable activity with adenine
Sub-sequently, 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 lmolÆmin)1Æmg)1, whereas the
val-ues for HPRT were 3.9 ± 1.5 lm and 23.3 ± 2.0 lmolÆ
min)1Æmg)1, respectively (Fig 3A,C andTable 2) With
hypoxanthine as a substrate, the Kmvalue is
approxi-mately six-fold higher for PRTFDC1, and Vmaxis
appro-ximately 70-fold lower compared to those of HPRT
Thus, the kcat⁄ Kmvalue with PRTFDC1 is only 0.26%
of that of HPRT The Km (guanine) value for
PRTFDC1 was 36.1 ± 14.3 lm, and the Vmaxwas value
2.9 ± 0.7 lmolÆmin)1Æmg)1, whereas the values for
HPRT were 9.9 ± 0.2 lm and 899 ± 117 lmolÆmin)1Æ
mg)1, respectively (Fig 3B,D and Table 2) This
indi-cates that, with guanine as a substrate, the Kmvalue 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
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 confor-mation 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 ther-mal shift Kinetic studies of PRTFDC1 showed that this enzyme had similar substrate specificity as HPRT, with guanine as the best substrate However, the catalytic effi-ciency (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 situa-tion 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 hypoxan-thine 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 lmolÆmin)1Æmg)1, which is 20-fold higher than reported previously [45] This
Table 1 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 l Ma DTagg(C) 10 m Mb
Ligand DTagg(C) 500 l Mc DTagg(C) 50 l Md
Hypoxanthine ⁄ PRPP 4.7 ± 1.2
Cytosine ⁄ PRPP )0.1 ± 0.1
Adenine ⁄ PRPP )0.2 ± 0.1
Xanthine ⁄ PRPP 0.0 ± 0.1
a Condition; [ligand]: 500 l M in 20 m M Hepes, 300 m M NaCl, 1 m M
MgCl 2 DT agg calculated using control T agg ; 53.0 ± 0.7 C b
Condi-tion; [ligand]: 10 m M in 20 m M Hepes, 300 m M NaCl, 10 m M MgCl2.
DTaggcalculated using control Tagg; 52.5 ± 0.6 C c [PRPP]: 50 l M ;
[base]: 500 l M in 20 m M Hepes, 300 m M NaCl, 10 m M MgCl 2 ,
1.25% dimethylsulfoxide DTagg calculated using control Tagg;
52.7 ± 0.4 C d [PRPP]: 50 l M in 20 m M Hepes, 300 m M NaCl,
10 m M MgCl 2 , 1.25% dimethylsulfoxide.
Trang 6discrepancy might be explained by the different methods
used We used a radiochemical method in which the
amount of products was quantified directly, and
there-fore it is a more accurate and sensitive method
com-pared 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 car-rying mutations in HPRT might lead to a milder dis-ease 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 cata-lytic role in the nucleotide salvage pathway When PRTFDC1 has been shown to interact with HPRT
PRTFDC1
PRTFDC1
HPRT
HPRT
0.4
0.3
–1 )
–1 )
0.2
0.1
0.0
–50 200 400 600 800 1000
0 50 100150 200 250
2 4 6 8 10 12
50 100 150 200 250
S
0 –10
0.10 0.08 0.06 0.04 0.02
10 20 30 40 50 60
S S
60 50 40 30 20 10 –40 –20 0 20 40 60 80 100 120
0 120
100 80 60 40 20
25
20
15
10
5
0
1000 800 600 400 200 0
1.5
1.0
0.5
0.0
Fig 3 Characterization of PRTFDC1 and
HPRT with hypoxanthine and guanine.
Michaelis–Menten and Hanes–Woolf plots
illustrate the kinetic pattern The
experi-ments were repeated three to four times.
(A) PRTFDC1 and hypoxanthine (B) HPRT
and hypoxanthine (C) PRTFDC1 and
guanine (D) HPRT and guanine.
Table 2 Kmand Vmaxvalues for Hx and G in the presence of 1 m M 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 k cat was calculated using
M w (HPRT) = 27132 Da and M w (PRTFDC1) = 28226 Da The k cat ⁄ K m for HPRT was set to 100% as a reference for both substrates, and
kcat⁄ K m for PRTFDC1, shown in parenthesis, was calculated in relation to this.
Km(l M )
Vmax (lmolÆmin)1Æmg)1) kcat(s)1) kcat⁄ K m (s)1Æ M )1)
Hypoxanthine
Guanine
Trang 7[29], one possibility is that it participates in forming
heterooligomers containing subunits of both
PRTFDC1 and HPRT, thereby providing an
addi-tional 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
expres-sion 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
eluci-dated 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
struc-ture 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
(accession numbers: BC008662 and BC000578) The sequences encoding residues 1–225 (PRTFDC1) and 1–218 (HPRT) were amplified by PCR and inserted into pNIC28-Bsa4 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 lgÆmL)1kanamycin was inoculated and grown at 37C overnight; 40 mL of this culture were used to inoculate 3 L
of TB supplemented with 50 lgÆmL)1 kanamycin and approximately 50 lL of Breox antifoam (Cognis Perfor-mance Chemical UK Ltd) in glass flasks using the large scale expression system Cells were grown at 37C until
D600 of 1.2 was reached followed by down-tempering to
18C for 1 h in water bath Expression was induced by the addition of isopropyl thio-b-d-glactoside with a concentra-tion of 0.5 mm and growth was allowed to continue over-night Cells were harvested by centrifugation at 3500 g for
20 min 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-carboxy-ethyl)phosphine (TCEP) supplemented with one tablet per
50 mL of Complete EDTA-free protease inhibitor tablet (Santa Cruz Biotechnlogy, Santa Cruz, CA, USA) and
4 lL per 50 mL of benzonase Cells were disrupted by high
Fig 4 Sequence alignment of PRTFDC1 (accession number: NP_064585), HPRT (accession number: AAA36012), Plasmo-dium falciparum hypoxanthine-guanine-xan-thine 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.
Trang 8pressure homogenization run three times at 10 000 p.s.i.
and samples were centrifuged for 40 min at 50 228 g The
soluble fraction was filtered through 0.2 lm filters and
sub-jected to further purification on an A¨KTAprime system
(GE Healthcare)
Columns used were HiTrap Chelating 1 mL and
HiLoad 16 ⁄ 60 Superdex 200 Prep Grade (GE Healthcare)
The 1 mL HiTrap chelating HP column was equilibrated
with IMAC buffer 1 [50 mm Hepes (pH 7.5), 10 mm
imidaz-ole, 500 mm NaCl, 10% glycerol, 0.5 mm TCEP], washed
with IMAC buffer 1 followed by IMAC buffer 2 [50 mm
Hepes (pH 7.5), 30 mm imidazole, 500 mm NaCl, 10%
glyc-erol, 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, 2 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
purification was conducted on an A¨KTAxpress system
(GE Healthcare)
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
cal-cium acetate, 40% PEG 300] co-crystallized with 10 mm
PRPP and 10 mm MgCl2at room temperature
Crystalliza-tion experiments were set up using the Phoenix
crystalliza-tion 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 mgÆmL)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
inte-grated using mosflm [29] and scaled using scala from the
ccp4software suit [30] The structure was solved with the
molecular replacement software molrep [31] using
coordi-nates 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 3
[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)cys-teine 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 per-formed using clustalw [39] and ESPript [40] Superposi-tions 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-phos-phate), 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 Biotech-nology and Engineering Corporation, Markham, Ontario, Canada) Assay conditions were: 500 lm compound, 0.2 mgÆmL)1 (7.1 lm) protein, 20 mm Hepes (pH 7.5),
Table 3 Data collection and refinement statistics Values in paren-theses refer to the highest resolution shell data.
PRTFDC1-GMP European Synchrotron Radiation Facility beam line
ID29
c = 52.1
Refinement
Rmsd
Ramachandran plot (%) c
a Rworkis defined as R||Fobs| ) |F calc || ⁄ R|F obs |, where Fobs and Fcalc are observed and calculated structure-factor amplitudes, respec-tively. bR free is the R factor for the test set (5% of the data).
c According to MOLPROBITY [35].
Trang 9300 mm NaCl, and 1 mm MgCl2 Following initial
screen-ing, a subset consisting of IMP, GMP, XMP, AMP, CMP
and PRPP were screened at 500 lm and 10 mm, with 1 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, 1 mm MgCl2 and 1.25%
dimethylsulfoxide All StarGazer-384 screening was
con-ducted 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 1CÆmin)1, and
images take every 0.5C 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
condi-tion 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
de-oxynucleoside 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 mgÆmL)1 BSA, 1 mm PRPP, 1–200 lm
[3H]hypoxanthine (13.8 CiÆmmol)1; Perkin Elmer, Boston,
MA, USA), 1–100 lm [3H]guanine (10.7 CiÆmmol)1;
Mor-avek, Brea, CA, USA) and [3H]adenine (27.2 CiÆmmol)1;
Perkin Elmer) The assay mix was preheated at 37C for
5 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, 8 and 12 min The
nega-tively-charged products will bind to the positively charged
DE-81 filter papers Nonreacted substrates were removed
by washing the filter papers 3· 5 min in 5 mm ammonium
formate solution and once in deionized water The
tritium-labeled products on DE-81 filter paper were eluted for
30 min in 0.5 mL of 0.2 m KCl⁄ 0.1 m HCl; subsequently,
2 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 = VmaxÆ[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
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 Founda-tion for Strategic Research and the Wellcome Trust
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