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1 Laboratoire de Chimie Structurale des Macromolécules, Institut Pasteur, 75724 Paris Cedex 15, France
2 Unité de Biochimie Cellulaire, Institut Pasteur, 75724 Paris Cedex 15, France
3 Unité de Chimie Organique, Institut Pasteur, 75724 Paris Cedex 15, France
4 Centre de Biochimie Structurale, Faculté de Pharmacie, Université de Montpellier I, 34000 Montpellier, France
Reprint requests to: Héléne Munier-Lehmann, Laboratoire de Chimie Structurale des Macromolécules, Institut Pasteur, 28, Rue du Dr Roux, 75724 Paris Cedex 15 - France; e-mail: hmunier{at}pasteur.fr; fax: 33 1 40 61 39 63.
(RECEIVED November 2, 2000; FINAL REVISION March 9, 2001; ACCEPTED March 20, 2001)
Article and publication are at www.proteinscience.org/cgi/doi/10.1110/ps.45701.
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Keywords: Tuberculosis; circular dichroism; fluorescence spectroscopy; molecular modeling; structure-function relationship
Abbreviations: TMPK, thymidylate kinase • TMPKec, thymidylate kinase from E. coli • TMPKmt, thymidylate kinase from M. tuberculosis • TMPKy, thymidylate kinase from yeast • NMPK, nucleoside monophosphate kinase • AZT-MP, 3'-azido-3'-deoxythymidine monophosphate
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Introduction |
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We have cloned the tmk gene of M. tuberculosis and characterized the overexpressed protein by various biochemical and physico-chemical methods. Kinetic studies revealed similarities, but also significant differences between TMPKmt and its counterpart from yeast or from E. coli. The most surprising observation is the fact that 3'-azido-3'-deoxythymidine monophosphate (AZT-MP), which is substrate for TMPKec and TMPKy, behaves as a competitive inhibitor for TMPKmt. Based on the available three-dimensional structure of TMPKy and TMPKec (Lavie et al. 1997; 1998a, b), we propose a structural model of TMPKmt, which could give clues for understanding the differences in substrate-analog phosphorylation between these enzymes. This model enabled us to design new substrate analogs or competitive inhibitors, which behaved as predicted.
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Results |
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). The conservation of a similar fold in TMPKmt was confirmed further through hydrophobic cluster analysis comparison (Callebaut et al. 1997) and molecular modeling using the structures of TMPKy and TMPKec in complex with P 1–(5'-adenosyl)-P 5–(5'-thymidyl)pentaphosphate (TP5A) (Lavie et al. 1998a, b; codes PDB3TMK and PDB5TMP). The pseudo-energy derived from TITO (Labesse and Mornon 1998), PROSA (Sippl and Weitckus 1992) and the score from Verify3D (Eisenberg et al. 1997) validate the model (-50.5, -1.2 and +0.3, respectively). The values of the different validation scores compared favorably with the ones obtained for the two crystal structures of TMPKy and TMPKec (TITO: -58.8 and -63.2 for PDB3TMK and PDB5TMP respectively; PROSA: -1.7 and Verify3D: +0.4 for both PDB3TMK and PDB5TMP). The dimer interface described in the crystal structure of TMPKy was predicted to be conserved in TMPKmt. The 
3 and 6 helices involved in this interface possess mainly hydrophobic side-chains.
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). The shorter acidic side chain in TMPKy and TMPKmt is correlated with the close contact with Gln172 in TMPKmt instead of Phe167 in TMPKec. The TMPKmt P-loop possesses a unique substitution (Arg14) instead of an otherwise very well conserved Thr in TMPKy and TMPKec (Fig. 1), and other NMPKs (Chenal-Francisque et al. 1999; Lavie et al. 1998a). A particularity of TMPKmt is that the Arg residues, underlined in Figure 1, which participate in stabilizing the presumed transition state, are found both in the P-loop (as in TMPKy) and in the LID region (as in TMPKec). By similarity with TMPKec, the LID region (Schulz et al. 1990) of TMPKmt is predicted to adopt a helical conformation and would harbor three arginines, two of them (Arg149 and Arg153) likely involved in ATP binding (Lavie et al. 1998b). The LID domains are solvent-exposed segments observed in various NMPKs, whose conformation changes upon binding of ATP, leading to closure of the catalytic site (Schulz et al. 1990). In this domain, the backbone conformation of TMPKy differs considerably in correlation with the presence of a bulky Arg15 pointing toward the LID. This large hydrophilic residue is replaced by a glycine in TMPKmt and TMPKec.
Two other structural motifs, including residues directly involved in the active site, also suggested the "chimeric" nature of TMPKmt when compared with TMPKy and TMPKec. The stretch harboring the 91///DRxx/SxxAYs104 signature (where `/' stands for any hydrophobic amino acid, x for any other amino acid, upper case for strictly conserved residues and lower case for position allowing conservative mutations) is better conserved between TMPKmt and TMPKy (Fig. 1). It comprises residues lying in the catalytic site such as Arg95 (Fig. 2A). By contrast, the consensus sequence 175rtxaxy/eLaaq185 found in the 7-helix was more similar in TMPKmt and TMPKec (Fig. 1). We predict that Tyr179 would be involved in hydrogen bonding with two strictly conserved catalytic residues Arg95 and Ser99 (Fig. 2A).
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) and Thr105 in TMPKec (Fig. 2C). The Thr105 side chain in TMPKec is hydrogen-bonded to the O4 carbonyl group of dTMP and to the guanidinium group of the conserved Arg74 in TMPKmt. The Asn100 side chain of TMPKmt might thus participate in a hydrogen bond network involving the base moiety of dTMP and/or the neighboring Ser104.
Overexpression, purification, and molecular characterization of TMPK from M. tuberculosis
TMPKmt (Rv3247c) expressed in E. coli represented over 30% of total bacterial proteins. The recombinant enzyme, recovered from the supernatant after E. coli breakage and centrifugation, was purified by a two-step procedure. Chromatography on Blue-Sepharose and gel permeation yielded over 95% pure enzyme as revealed by SDS-PAGE (Fig. 3). The molecular mass of TMPKmt, determined by electrospray ionisation mass spectrometry (22635.89 ± 2.23 Da), corresponded to that calculated from the deduced protein sequence (22634.58 Da). The molecular mass of the native protein determined by sedimentation equilibrium ultracentrifugation (46600 ± 1300 Da) is consistent with a dimer.
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Aliquots of 1 mg/mL protein in 50 mM Tris-HCl pH 7.4 were heated for 10 minutes at temperatures between 35°C and 70°C, after which residual activity was determined. TMPKmt was half inactivated at 65°C. The thermal unfolding of TMPKmt examined by microcalorimetry is irreversible (Fig. 4). The midtemperature (Tm) of thermal denaturation of TMPKmt alone (68°C) was not affected by 0.5 mM ATP, whereas addition of 0.5 mM dTMP increased the Tm of the protein by 5.7°C.
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). To discriminate between the two models, we studied the effect of protein concentration on the transition curve characteristics. The experiments were repeated at 2.10–7, 10–6 and 4.10–6 M protein concentrations and the parameters obtained from individual fits to equations 2 and 3 are shown in Table 1.
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and Table 1). However, when using the transition including the monomeric intermediate, we observed a remarkable correlation between the sets of parameters deduced from the experiments monitored by CD and by fluorescence.
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G0,1 and G0,2, are 18.1 ± 0.9 and 6.1 ± 0.3 kcal/mol, respectively, and the corresponding equilibrium constants are (6 ± 5).10–14 M and (3.5 ± 1.6).10–5 M.
Catalytic properties of TMPK from M. tuberculosis
The specific activity of TMPKmt is four- and six-fold lower than that of TMPKec and TMPKy, respectively. The Km for dTMP and ATP are of the same order of magnitude in TMPKmt, TMPKec and TMPKy (Table 2). TMPKmt has a broader specificity for nucleoside triphosphates (Table 3), than that observed with various other NMPKs (Chenal-Francisque et al. 1999; Munier-Lehmann et al. 1999; Bucurenci et al. 1996). The reaction rates with ATP or dATP as phosphate donors were similar. ITP, GTP, CTP, and UTP also could serve as efficient phosphate donors (order of efficacy: ITP > GTP > CTP > UTP). Even dGTP, dCTP, and dTTP act as phosphate donors, even though much less efficiently (1% for dTTP, 8% for dCTP, and 35% for dGTP compared to ATP).
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). Replacement of the methyl group in the pyrimidine ring by a bromine (5Br-dUMP) does not affect the Km and the Vm of TMPKmt, whereas substitution by an iodine (5I-dUMP), which is larger than the Br atom, affects slightly both parameters (Km value 3.5-fold higher than that with dTMP and reaction rate 70% that with dTMP). On the other hand, replacement of the methyl group by F (5F-dUMP), which is smaller than Br, or removal of the methyl group (dUMP) drastically affect the affinity (Km value 10-fold and 50-fold higher than that with dTMP, respectively) and to a lesser extent the reaction rates (44% and 30% that with dTMP, respectively).
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). 2N-acetyl 5-methyl iso dCMP is substrate for TMPKmt (at ATP 0.5 mM, 60% reaction rate). On the other hand, 5-methyl iso dCMP is a competitive inhibitor with a Ki of 130 µM.
Modification of the 3'-OH group on the ribose moiety affected only slightly the affinity of different analogs of dTMP for the enzyme (Table 4). However, catalysis was found to be drastically reduced to about 1% for 3'F-dTMP and dehydro-TMP. Surprisingly, AZT-MP was found to be a competitive inhibitor of TMPKmt with a Ki of 20 µM (Fig. 6). The presence of an azido group abolished catalysis without changing the affinity. Nonetheless, AZT-MP is a substrate for the TMPKec and TMPKy, with only a 2-fold reduction of kcat for the bacterial enzyme and 200-fold for the yeast enzyme (Lavie et al. 1998a).
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Discussion |
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Lavie et al. (1998b) proposed a classification based on the capacity to phosphorylate AZT-MP: type I with TMPKy as representative and type II with TMPKec as representative, which differ in the way they stabilize the transition state by the presence of key arginine residues in the P-loop (type I) or in the LID region (type II). The larger AZT-MP compared to dTMP is well accommodated in TMPKec (type II), but in TMPKy (type I), the shift of a catalytic residue results in a substantial loss of activity. According to this classification, TMPKmt and TMPK from Yersinia pestis (TMPKyp), which possesses a LID domain very similar or identical to that of TMPKec, should belong to type II TMPKs and thus should phosphorylate AZT-MP with a good kcat. However, we found that TMPKmt does not phosphorylate AZT-MP and TMPKyp had a very low AZT-MP phosphorylating activity (Chenal-Francisque et al. 1999). The classification should thus be revised and residues other than arginines should be included to explain the different capacity of TMPKs in their AZT-MP phosphorylation.
In this context, we proposed a structural model of TMPKmt, based on the solved three-dimensional structures of TMPKec and TMPKy (Lavie et al. 1997; 1998ab), in order to explain at least in part our results and to provide a basis for developing potent inhibitors of TMPKmt. The overall sequence conservation suggested that the TMPKmt structure likely deviates from the already known TMPK structures by an estimated root-mean-square deviation on the common C atom positions of 
1 Å. However, our current model is in agreement with all our experimental data, especially those derived from trypsin digestion, stabilisation effects by ATP (low) and dTMP (high), CD and fluorescence spectroscopy, as well as the specificity for the various substrate analogs tested here. Analysis of the modeled active site leads us to predict the role of some residues lying within the phosphate donor or substrate binding pockets and Figure 2A presented the residues of TMPKmt involved in ATP and dTMP binding. According to the modeled structure, the methyl group of the dTMP base moiety would interact closely with the side chains of Phe36, Pro37, Arg74, and Arg95, which are in the same environment as in TMPKy (Fig. 2B) and TMPKec (Fig. 2C). The presence of the guanidinium group of Arg74 (distance to the methyl 3.9 Å) in an otherwise hydrophobic pocket would explain the measured affinity of the halogenated substrates. The corresponding substitution of Glu12 and Phe167 in TMPKec (Fig. 2C) to Asp9 and Gln172 in TMPKmt and Asp14 and Gln157 in TMPKy (Fig. 2B), respectively, might explain the much lower activity of TMPKy and TMPKmt on AZT-MP. While TMPKmt is already slightly less active on natural dTMP, some other structural factors might further decrease and finally abolish the activity on AZT-MP. Similarly, 3' halogenated or deoxygenated dTMP analogs also are poor substrates for TMPKmt. Based on structural comparison, we suggest three possibilities to explain these catalytic properties of TMPKmt: the presence of specific substitutions (e.g., Arg14 and Asn100) in the active site of TMPKmt compared to the two other TMPKs, the overall low sequence similarity (25%; greater with hyperthermophilic TMPKs) and its enhanced thermal stability, suggesting a lower flexibility at 37°C.
New substitutions of the 3' hydroxyl of dTMP already can be proposed based on the predicted close interaction of this ribose hydroxyl with Asp9, which carboxyl group repels any negatively charged fluoro or azido group. Furthermore, our model indicated that TMPKmt might accomodate in its dTMP binding site bulky substituants linked to C2 of the pyrimidine ring. Enough space is left in between Ser104 and the base moiety to be filled by groups such as urea or guanidium. Thus two different substitutions at position 2 on the base moiety were tested. For 5-methyl iso dCMP, the carbonyl group of dTMP is replaced by a polar group, an amine group, which would interact unfavorably with the surrounding aromatic side chains from residues Phe70, Tyr103, and Tyr165, normally involved in the interaction with dTMP (Fig. 2A). On the contrary, the bulkier acetyl group in 2N-acetyl 5-methyl iso dCMP would not alter the interaction with the surrounding aromatic chains and would fit nicely in the pocket formed by Ser104 and Asn100. As predicted by the model, 5-methyl iso dCMP is a competitive inhibitor for TMPKmt whereas 2N-acetyl 5-methyl iso dCMP is a substrate for TMPKmt.
Local variation and fine structural divergence in essential enzyme such as dihydrofolate reductase (Li et al. 2000) or adenosine kinase (Carret et al. 1999) turned out to be attractive features for designing precisely tuned new drugs. Our study suggested that despite their global conservation among living organisms, TMPKs also could be targeted in a species-specific manner. The main advantage of this strategy is to select well-characterized protein families for which known inhibitors might become useful tools for rapid drug discovery as in the successful case of human immunodeficiency virus (HIV) protease (Wlodawer and Vondrasek 1998).
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Materials and methods |
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35S]ATP (1000 Ci/mmol) was a product from the Radiochemical Center (Amersham, UK). The sequencing reactions were performed using the Fidelity DNA Sequencing System from Q.BIOgene. Oligonucleotides were synthesized according to the phosphoramidate method using a commercial DNA synthesizer (Cyclone TM Biosearch). Blue-Sepharose and Ultrogel AcA54 were obtained from Amersham Pharmacia Biotech. L-1–(tosylamino)-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin, 5F-dUMP, 5Br-dUMP, 5I-dUMP and AZT-MP were from Sigma and ultra pure urea from ICN Biochemicals, Inc.
Bacterial strains and growth conditions
The E. coli NM554 (Raleigh et al. 1988) and BLI5 (Serina et al. 1995) strains were used for DNA purification and sequencing, and for production of recombinant TMPKmt encoded by the plasmid pHL50, respectively. Cultures were grown at 37°C in 2YT medium (Sambrook et al. 1989) supplemented with 100 µg/mL ampicillin and 30 µg/mL chloramphenicol. Production of recombinant TMPKmt was induced with isopropyl-1-thio-ß-D-thiogalactoside (1 mM final concentration) when cultures reached an absorbance of 1.5 at 600 nm. Bacteria were harvested by centrifugation 3 h after induction.
Plasmids
The 642 bp fragment corresponding to the tmk gene of M. tuberculosis (MTCY20B11 gene in Genebank) was amplified by PCR (Sambrook et al. 1989) using the cosmid SCY20B11 as the matrix. The two synthetic oligonucleotides used for amplification were 5'-GGGGCATATGCTAATCGCGATTGAGGGCGTTGAC-3' and 5'-CCCCAAGCTTTCAACTTGGCACGTCTGGAGGCG-3'. During amplification, NdeI and HindIII restriction sites (in bold letters in the oligonucleotide sequences) were created at both ends of the amplified fragment. After digestion by NdeI and HindIII, the amplified tmk gene was inserted into the pET22b plasmid (Novagen, Inc.) digested with the same enzymes. Three clones containing the M. tuberculosis tmk gene and overexpressing TMPKmt were characterised and one of them (pHL50) was kept for further studies. The DNA insert was sequenced using the double-stranded dideoxynucleotide sequencing technique (Sanger et al. 1977) in order to verify the absence of any mutational events in the course of amplification.
Purification and activity assay of TMPK from M. tuberculosis
Bacteria suspended in 50 mM Tris-HCl pH 7.4 were disrupted by sonication. After centrifugation at 14000 rpm for 30 minutes, the bacterial extract was loaded onto a Blue-Sepharose column equilibrated with the same buffer (10 mg of protein/mL of swollen gel). The column was washed with 10 volumes of 50 mM Tris-HCl pH 7.4 followed by 1 M NaCl in 50 mM Tris-HCl pH 7.4 (4 volumes). TMPKmt was eluted with 4 volumes of 2 M NaCl in 50 mM Tris-HCl pH 7.4. Fractions containing TMPKmt were pooled, concentrated and loaded onto a 1 x 110 cm Ultrogel AcA54 column equilibrated with 50 mM Tris-HCl pH 7.4 and fractions of 1.8 mL were collected at flow rates of 9 mL/h. TMPKmt activity was determined at 30°C using the coupled spectrophotometric assay at 334 nm (0.5 mL final) on an Eppendorf ECOM 6122 photometer (Blondin et al. 1994). One unit of enzyme activity corresponds to 1 µmole of the product formed in 1 minute at 30°C and pH 7.4.
Equilibrium sedimentation
The experiments were performed at 20°C on a Beckmann Optima XL-A analytical centrifuge using an An-60 Ti rotor and a cell with a 12 mm optical path length. Samples (150 µL) in 50 mM Tris-HCl pH 7.4 at 0.2 mg/mL were centrifuged at 20,000 rpm. Radial scans of absorbance at 280 nm were taken at 2 h intervals. Equilibrium was achieved after 14 h. Data were analyzed by the XL-A program supplied by Beckman. The partial specific volume of TMPKmt (0.718 cm3/g) was calculated from the sequence (Zamyatnin 1984).
Differential scanning microcalorimetry
Differential scanning microcalorimetry was carried out on a Micro Cal MCS calorimeter controlled by the MCS Observer program (MicroCal). Samples were extensively dialyzed against the buffer used in scanning experiments (50 mM Tris-HCl pH 7.4) and were routinely degassed for 5 minutes before they were used for a calorimetric analysis. They were scanned after the actual calorimetric scan for a second time to estimate the reversibility of the unfolding transition. Buffer baselines were measured under identical conditions and were subtracted from the corresponding data of the protein samples.
Equilibrium unfolding experiments
Urea was used as chemical denaturant. Typically, the protein at constant concentration (10–7, 2.10–7, 10–6 and 4.10–6 M) was incubated for 48 h at 25°C with urea at various concentrations ranging from 0.25 to 8 M in 50 mM Tris-HCl pH 7.4. For refolding experiments, protein at 10–6 M was first totally unfolded at 8 M urea for 24 h, then diluted 10-fold to a constant final concentration of 10–7 M in solution containing urea at various concentrations and incubated for 48 h at 25°C before measuring the fluorescence intensity or the ellipticity. For each experiment, a series of samples containing only urea at various concentrations in 50 mM Tris-HCl pH 7.4 was used as baseline controls.
Fluorescence measurements
All fluorescence measurements were carried out using a Perkin-Elmer LS5B spectrofluorometer, with a 1 x 1 cm cell thermostated at 25°C. Emission spectra were acquired between 310 and 410 nm upon excitation at 295 nm (bandwidth 5 nm). The emission bandwidth was set according to the protein concentration. When working at a constant emission wavelength, fluorescence intensity was recorded for 1 min with a sampling period and an integration time of 1 s and then averaged.
Circular dichroism measurements
All CD measurements were acquired with a CD6 spectropolarimeter from Jobin-Yvon. The far UV CD spectra were recorded in a 0.01 cm path length cell to minimize the buffer contribution, with a step of 0.5 nm and an integration time of 1 s. Five successive scans were averaged. For unfolding transitions, the ellipticity was recorded at 225 nm because of the high absorption of urea at lower wavelengths. Standard cells with path lengths of 0.5 cm and 0.2 cm were used for the series at protein concentrations of 10–6 and 4.10–6 M, respectively. Ellipticity was measured for 1 min with a sampling period and time constant of 1 s. The final ellipticity was calculated by averaging the 60 recorded data values.
Data analysis
Nonlinear least square fittings of the equilibrium transitions were achieved using equations derived from three different models: the 2-state dissociation equilibrium of a dimer into monomers; the 3-state double equilibrium coupling the dissociation process to the isomerization of the monomeric intermediate state; and the 3-state double equilibrium in which isomerization occurs in the dimeric state.
The corresponding equations have been established as follows. The signal S(C) (fluorescence intensity or maximum emission wavelength, ellipticity) measured at a given denaturant concentration C results from the linear combination of the individual contributions of all the species in equilibrium (2 or 3 species):
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Si and fi are the specific signal and the fraction of the species i, respectively. The nonhorizontality of the signal baselines at low (native state) and high (denatured state) denaturant concentrations is taken into account as a linear dependence upon denaturant concentration with the slope coefficients, positive or negative, n and u respectively:
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Sn,0 and Su,0 are the specific signals of the native and denatured species extrapolated at 0 concentration of denaturant.
The dependence on free energy for each individual equilibrium G is supposed to vary linearly with the denaturant concentration:
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G0 corresponds to the free energy of the individual equilibrium in the absence of denaturant.
2-state dissociation process:
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The signal at a given concentration C of denaturant is given by equation 1:
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(N2)0 is the total protein concentration expressed in terms of dimer.
3-state dissociation process with dimeric intermediate:
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The signal at a given concentration C of denaturant is given by equation 2:
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(N2)0 is the total protein concentration expressed in terms of dimer.
3-state dissociation process with monomeric intermediate:
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The signal at a given concentration C of denaturant is given by equation 3:
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(N2)0 is the total protein concentration expressed in terms of dimer.
Sequence comparison and molecular modeling
Protein sequence database searches were performed with the PSI-BLAST version 2.0.5 program (Altschul et al. 1997) with default parameters. Pairwise and multiple alignments were confirmed by hydrophobic cluster analysis as previously described (Callebaut et al. 1997), in order to delineate structurally conserved regions along the amino acid sequences. Alignment refinement was subsequently performed using the program TITO (Labesse and Mornon 1998) where the TMPKec and TMPKy were used as templates (Lavie et al. 1998a, b). TMPKmt secondary structures (-helix, ß-strand) were assigned during TITO processing and the secondary structure derived by homology for TMPKmt were used as additional restraints in the following modeling steps. Three-dimensional models were built using the TMPKec and TMPKy as combined templates in MODELLER 4.0 (Sali and Blundell 1993) and assessed using Verify3D (Eisenberg et al. 1997). These three-dimensional structures were visualized on a UNIX workstation using XmMol (Tuffery 1995).
Synthesis of 2N-acetyl-5-methyl-2'-deoxyisocytidine 5'-monophosphate and of 5-methyl-2'-deoxyisocytidine 5'-monophosphate
5-methyl-2'-deoxyisocytidine, synthesised from 2,5'-anhydrothymidine according to Jurczyk et al. (1998), was converted into 2N-acetyl-5-methyl-2'-deoxyisocytidine by peracetylation followed by selective saponification (Gait 1984). 5'-Dimethoxytritylation of the N-protected nucleoside, followed by 3'-acetylation and subsequent 5'-deprotection under acidic conditions afforded 3'-O,2N-diacetyl-5-methyl-2'-deoxyisocytidine. Phosphorylation according to classical procedure (Tener 1961) followed by deprotection under mild basic conditions (2% sodium methylate in methanol) afforded 2N-acetyl-5-methyl-2'-deoxyisocytidine 5'-monophosphate. Complete deprotection by heating at 55°C overnight yielded 5-methyl-2'-deoxyisocytidine 5'-monophosphate. Both nucleotides were purified by reverse phase HPLC (C18 column, gradient of acetonitrile in 10 mM TEAA buffer) and isolated as sodium salt by passing through a Dowex 50WX8 column. Their structures were confirmed by NMR using a 300-MHz Bruker spectrometer. 2N-Acetyl-5-methyl-2'-deoxyisocytidine 5'-monophosphate as triethylammonium salt 1H (D2O): 
1.98 (s, 3H, CH3, J = 0.8 Hz); 2.45 (m, 2H, H2' and H2"); 4.03 (s, 3H, CH3); 4.11 (m, 2H, H5' and H5"); 4.23 (m, 1H, H4'); 4.60 (m, 1H, H3'); 6.36 (t, 1H, H1', J = 6.5 Hz); 7.96 (s, 1H, H6, J = 0.9 Hz). 13C: 13.50 (CH3); 40. 21(C2'); 56.87 (CH3); 64.97 and 65.03 (C5'); 71.52 (C3'); 86.84 and 86.95 (C4'); 87.60 (C1'); 117.48 (C5); 137.23 (C6); 157.17 (C2 and CO); 175.95 (C4). 31P: 0.81 and 5-Methyl-2'-deoxyisocytidine 5'-monophosphate as sodium salt 1H (D2O): 1.94 (d, 3H, CH3, J = 1 Hz); 2.38 (m, 2H, H2' and H2"); 4.06 (m, 2H, H5' and H5"); 4.18 (m, 1H, H4'); 4.60 (m, 1H, H3'); 6.37 (t, 1H, H1', J = 7.2 Hz); 7.81 (d, 1H, H6, J = 1 Hz). 13C: 13.43 (CH3); 39.45 (C2'); 63.91 (C5'); 70.23 (C3'); 85.98 and 86.08 (C4'); 86.67 (C1'); 114.47 (C5); 133.46 (C6); 153.96 (C2); 170.18 (C4). 31P: 1.73. Mass spectrometry by ESI-MS confirmed the presence of M = 321 and M +Na +K for 5-methyl-2'-deoxyisocytidine 5'-monophosphate.
Other analytical procedures
Protein concentration was measured according to Bradford (1976), using a Bio-Rad kit or by amino-acid analysis on a Beckman system 6300 high-performance analyzer after 6 N HCl hydrolysis for 22 h at 110°C. SDS-PAGE was performed as described by Laemmli (1970).
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Acknowledgments |
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This work was supported by grants from the Institut Pasteur, the Institut National de la Santé et de la Recherche Médicale (U414), the Centre National de la Recherche Scientifique (URA 2185 and UMR 5048), and from GIP-HMR.
The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
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References |
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) and 4.10–6 M (
). 8-M unfolded TMPKmt was diluted to a final concentration of 10–8 M or and 4.10–6 M in solutions containing urea at various concentrations and spectra were recorded after 48 h incubation. The solid lines correspond to the best fit to equation 3 using a nonlinear least square analysis. (B) variation of the native (fn), monomeric intermediate (fi) and unfolded (fu) fractions calculated from the parameters deduced from the individual fit of the two transition curves in (A). Solid line: TMPKmt at 10–6 M. Dotted line: TMPKmt at 4.10–6 M.