Báo cáo khoa học: Structure of the substrate complex of thymidine kinase from Ureaplasma urealyticum and investigations of possible drug targets for the enzyme pdf

Structure of the substrate complex of thymidine kinase from Ureaplasma urealyticum and investigations of possible drug targets for the enzyme Urszula Kosinska 1 *, Cecilia Carnrot 2 *, Staffan Eriksson 2 , Liya Wang 2 and Hans Eklund 1 1 Department of Molecular Biology, Swedish University of Agricultural Sciences, Uppsala Biomedical Centre, Sweden 2 Department of Molecular Biosciences, Swedish University of Agricultural Sciences, Uppsala Biomedical Centre, Sweden Two potential nucleoside kinase genes coding for a thymidine kinase (TK) (EC 2.7.1.21) and a deoxyadeno- sine kinase (EC 2.7.1.74) are found in all sequenced Mollicute genomes [1–9]. Deoxyadenosine kinase from Mycoplasma mycoides ssp. mycoides and TK from Ureaplasma urealyticum (Uu-TK) have previously been cloned and characterized [10,11]. U. urealyticum (also called Ureaplasma parvum) is a human pathogen colon- izing the urogenital tract and it is associated with several pregnancy complications, e.g. infertility, altered sperm motility, chorioamnionitis and pneumonia in the neonate [12]. Bacterial TKs show moderate sequence identity with human TK1 (hTK1) and mollicute TKs, e.g. Uu-TK shares 29% sequence identity with hTK1. The initial characterization demonstrated that Uu-TK also has similar enzyme kinetic properties to hTK1, with specificity for pyrimidine deoxynucleosides and with dTTP serving as a feedback inhibitor [11,13]. Uu-TK is less fastidious than hTK1 with regard to phosphate donors, using all nucleoside triphosphates with similar efficiency [11,13]. No genes encoding the enzymes for the de novo pathway of deoxynucleotide biosynthesis have been found in U. urealyticum, strongly suggesting Keywords: bacterial; crystallography; deoxythymidine; nucleoside analogues; thymidine kinase Correspondence H. Eklund, Department of Molecular Biology, Swedish University of Agricultural Sciences, PO Box 590, Biomedical Centre, S-751 24 Uppsala, Sweden Fax: +46 18 53 69 71 Tel: +46 18 4754559 E-mail: hasse@xray.bmc.uu.se *Note These authors contributed equally to this work. (Received 22 August 2005, revised 14 October 2005, accepted 21 October 2005) doi:10.1111/j.1742-4658.2005.05030.x Thymidine kinases have been found in most organisms, from viruses and bacteria to mammals. Ureaplasma urealyticum (parvum), which belongs to the class of cell-wall-lacking Mollicutes, has no de novo synthesis of DNA precursors and therefore has to rely on the salvage pathway. Thus, thymi- dine kinase (Uu-TK) is the key enzyme in dTTP synthesis. Recently the 3D structure of Uu-TK was determined in a feedback inhibitor complex, dem- onstrating that a lasso-like loop binds the thymidine moiety of the feed- back inhibitor by hydrogen bonding to main-chain atoms. Here the structure with the substrate deoxythymidine is presented. The substrate binds similarly to the deoxythymidine part of the feedback inhibitor, and the lasso-like loop binds the base and deoxyribose moieties as in the com- plex determined previously. The catalytic base, Glu97, has a different posi- tion in the substrate complex from that in the complex with the feedback inhibitor, having moved in closer to the 5¢-OH of the substrate to form a hydrogen bond. The phosphorylation of and inhibition by several nucleo- side analogues were investigated and are discussed in the light of the sub- strate binding pocket, in comparison with human TK1. Kinetic differences between Uu-TK and human TK1 were observed that may be explained by structural differences. The tight interaction with the substrate allows minor substitutions at the 3 and 5 positions of the base, only fluorine substitu- tions at the 2¢-Ara position, but larger substitutions at the 3¢ position of the deoxyribose. Abbreviations AZMT, 3¢-azido-methyl-dT; Ca-TK, Clostridium acetobutylicum thymidine kinase; dNK, deoxynucleoside kinase; FCPU, 3¢-fluoro-5-cyclopropyl- dU; FLT, 3¢-fluoro-dT; hTK, human thymidine kinase; TK, thymidine kinase; Uu-TK, Ureaplasma urealyticum thymidine kinase. FEBS Journal 272 (2005) 6365–6372 ª 2005 The Authors Journal Compilation ª 2005 FEBS 6365 that it has to rely solely on the salvage pathway for synthesis of DNA precursors. Thus, Uu-TK is a gate- way for the biosynthesis of dTTP, suggesting that Uu-TK is a good target for drug development. Recently the 3D structures of Uu-TK and cytosolic hTK1 were determined [14,15]. The structure of these thymidine kinases differs significantly from the earlier known deoxyribonucleoside kinases and form a separ- ate structural family. Humans carry four deoxyribonu- cleoside kinases: cytosolic TK1 and deoxycytidine kinase, and mitochondrial TK2 and deoxyguanosine kinase. Deoxycytidine kinase, TK2, and deoxyguano- sine kinase form a homologous deoxynucleoside kinase (dNK) family with similar structures. This family also includes the herpes viral TKs and the insect multisub- strate deoxyribonucleoside kinase showing high activity with dT [13]. The phosphate donor binds in both fam- ilies to an a ⁄ b domain, but this domain in the TK1 family is more similar to other ATP-binding structures in the RecA family [14]. Furthermore, instead of the helical part forming the substrate site and the LID region being involved in binding of the phosphate donor in the dNK family, there is a domain in hTK1 and Uu-TK that contains a structural zinc atom and lasso-like loop. In the feedback inhibitor complexes of Uu-TK and hTK1, the lasso-like loop binds the thymi- dine moiety of the feedback inhibitor by hydrogen bonding by main-chain atoms [14]. The structure of the bacterial TK from Clostridium acetobutylicum (Ca-TK) is very similar to the Uu-TK, but, in the absence of substrate or feedback inhibitor, the substrate site is open and the lasso-like loop is disordered [16]. In the absence of a true substrate complex for any member of the TK1 family, we have now determined the first structure of a member of the TK1 family in complex with the substrate dT. The possibilities for drug design have been investigated by enzyme kinetics and analyzed in view of substrate binding. It appears that a combination of substitutions at several positions of the nucleoside can pick up the small differences between mycoplasmic and human TK1, which suggests the route for further advances. Results and discussion Overall structure The overall tetrameric structure of Uu-TK in the sub- strate complex is very similar to that in the complex with the feedback inhibitor dTTP. Each subunit can be superimposed, with rmsds for Ca atoms of 0.3–0.6 A ˚ . The main differences are located close to the phosphate binding sites, where a flexible loop conformation differs among the four subunits of the tetramer and among subunits in the two complexes (Fig. 1A). In the present substrate complex structure, only subunit A in the tetramer has a completely visible loop, whereas in the dTTP complex, only subunits B and D have visible loops [14]. In all other cytosolic TK structures determined so far, this loop is visible in only a few subunits. For example, in one of the inde- pendent structure determinations of hTK1, this loop is fully visible in one of eight subunits in the asymmetric unit [15]. It may be that this loop is involved in phos- phate donor interactions, but no such complex has so far been determined. In the Ca-TK structure, the prod- uct ADP is bound at the phosphate donor site. In spite of this, only part of the loop is ordered in one of the subunits [16]. This conformation is similar to that observed in the Uu-TK–dTTP complex. The phosphate donor site in the present Uu-TK–dT structure is occu- pied by water molecules and, in one subunit, a Tris molecule. Although the enzyme was crystallized in the presence of the ATP analogue adenosine 5¢-[b,c-methy- lene]-triphosphate (p[CH 2 ]ppA; AMP-PCP), there was no density for this molecule. Substrate binding The substrate dT binds to the enzyme in a similar way to the dT moiety of the feedback inhibitor dTTP (Figs 1B and 2) and has the same interactions with the main chain of the lasso-like loop. This loop has the same conformation in both complexes in contrast to Ca-TK where no substrate was present in the crystals. There, the lasso-like loop was disordered [16]. The sub- strate site is obviously induced by substrate binding. The base is hydrogen-bonded to main-chain atoms: O2 to N in residue 180, N3 to O in residue 178, and O4 to N in residue 128 (Fig. 2). The methyl group is posi- tioned in a hydrophobic pocket lined by Cb of Ser163, Sd in Met21, and Cd1 in Leu124. The closest polar atom is the carbonyl oxygen of residue 126. O3¢ of the deoxyribose is hydrogen-bonded to the main-chain amino group of Gly182. Glu97 has different conformations in the substrate complex and the previously determined inhibitor com- plex. In the dT complex, O5¢ is hydrogen bonded to Glu97, in good agreement with its role as the catalytic base for the phosphoryl transfer reaction (Fig. 1B). In the inhibitor complex, the phosphates of the feedback inhibitor repel the glutamate side chain. A shift in the catalytic base was observed when the substrate and feedback inhibitor complex of Drosophila dNK were compared, but the conformational change was larger in that case [17]. In Uu-TK, only the side chain chan- Thymidine kinase in Ureaplasma urealyticum U. Kosinska et al. 6366 FEBS Journal 272 (2005) 6365–6372 ª 2005 The Authors Journal Compilation ª 2005 FEBS ges conformation, whereas in Drosophila dNK the shift is accompanied by main-chain movements. Substrate specificity, nucleoside analogues With radiolabelled ATP and a fixed concentration (100 lm) of various nucleoside analogues, Uu-TK activity was measured by a TLC assay. Table 1 shows that 5-halogenated analogues are good substrates and have the highest activities. The iodo atom has the same van der Waals radius as the methyl group of thymine, and the corresponding analogue has an activity com- parable to that of dT. The analogue with the smaller fluorine in the 5 position has a lower activity, slightly lower than that of dU, which has a hydrogen atom in the 5 position. The chlorine substitution is an outlier in the halogen substitution series, as it has the highest activity of the halogenated analogues. There is no cor- relation between the electronegativity of the substituent and its activity in the phosphorylation reaction. From these investigations, it appears that substitu- tions at the 5 position as large as a cyclopropyl group are tolerated, and for an ethyl group the activity is decreased to about half of that of dT. Larger substitu- Fig. 2. Interactions between thymidine and Uu-TK. Hydrogen bonds are shown as dotted lines. The tight binding site for the 2¢ position between the main chain of Lys180-Ile181 and Met21. Any substitu- tion at the 2¢ position hinders proper closure of the lasso, and thereby weakens substrate co-ordination. A B Fig. 1. (A) The structure of one subunit of Uu-TK (yellow) with con- formations of the flexible loop as found in Uu-TK in complex with dT in subunit A (orange, A), Uu-TK in complex with dTTP (green, B) and in hTK1 in complex with dTTP (grey, C). The conformation of the loop shown in green (B) is very similar to that found in Ca-TK in complex with ADP as well as the loops in chains C and D of the Uu-TK–dT structure. (B) Superimposition of the nucleotide-binding region in the substrate complex (orange) and the inhibitor complex (olive). The side chain of the catalytic Glu97 has different positions in the two complexes. In the substrate complex, it is in a catalyti- cally favourable position pointing inwards the active site. In the inhibitor complex, the side chain is repelled by the phosphates of the inhibitor. U. Kosinska et al. Thymidine kinase in Ureaplasma urealyticum FEBS Journal 272 (2005) 6365–6372 ª 2005 The Authors Journal Compilation ª 2005 FEBS 6367 tions clash sterically with neighbouring residues in the 5 position binding pocket (Fig. 2). With bulkier modi- fications, the activity decreased and no activity was seen with, e.g. 5-(2-bromovinyl)-dU (data not shown), which is also the case with hTK1 [18,19]. This steric hindrance in the 5 position correlates well with the small hydrophobic binding pocket in the enzyme. Analogues with modifications in the N3 position showed much lower activity than dT. The compounds had one to three carbons added in different configura- tions and gave 15–20% activity (Table 1). The hydro- gen-bonding between the N3 nitrogen in dT and the main-chain carbonyl of residue 178 is lost with alkyl substitutions in the N3 analogues tested. A substitution in this position will probably hinder the tight spacing of the lasso-like loop and disturb proper binding. The 3¢-OH is in an exposed position that can tolerate large substitutions (Table 1). The 3¢-OH of the deoxy- ribose forms a hydrogen bond with the main-chain amino group of Gly182 that is lost when the hydroxy group is replaced. Still, an electronegative substituent, such as fluorine, retains 50% of the activity. For the analogue with no hydroxy group in the 3¢ position, 2¢,3¢-didehydro-T, the activity was  3% of that with dT [11]. 3¢-Modified analogues that contain polar groups, e.g. 3¢-fluoro-dT (FLT) and 3¢-azido-dT (AZT) can still form a hydrogen bond with the amino group of Gly182, whereas analogues with nonpolar atoms bound to the 3¢-carbon, such as 3¢-fluoro-methyl-dT (FMT) and 3¢-azido-methyl-dT (AZMT), cannot form any hydrogen bond. The latter two showed 4–7-fold lower activity than with dT (Table 1). The correspond- ing a form of FMT and AZMT were inactive (data not shown). Such substitutions would clash with Met21. Analogues with modifications at both the 3¢ and 5 position had lower activity than their corresponding analogue with only one modification, e.g. 3¢-fluoro- 5-fluoro-dU (FFU) vs. FLT. This is probably also the case with analogues modified at both the 3¢ and 3N positions. Analogues with modifications at the 2¢ position showed the lowest activity (< 2%) of all analogues tested, e.g. arabinosyl-dT, 2¢-difluoro-dU and 2¢- chloro-dU (data not shown). This agrees well with the tight binding site that is crowded on both sides of the 2¢ position (Fig. 2). The OH group in arabinosyl-dT would interact sterically with Tyr187, which is one of the important residues that keep the lasso in place. The smaller fluorine in the 2¢-Ara position was accepted and 2¢-fluoro-arabinosyl-5-iodo-dU (FIAU) and 2¢- fluoro-arabinosyl-5-methyl-dU (FMAU) showed 44% and 34% activity, respectively. Any substitution on the other side, such as 2¢-difluoro-dU and 2¢-chloro-dU, would interact with the main-chain carbonyl of residue 180 (Fig. 2). Analogues as inhibitors Some analogues were chosen for further analysis as inhibitors of dT phosphorylation. The IC 50 values are presented in Table 2. dU, 5-fluro-dU (FdU) and Table 1. Phosphorylation of nucleoside analogues by Uu-TK and hTK1. The values are from one experiment repeated with similar results (< 20% variation). The specific activity with dT was set to 100% (1900 units), and 100 l M [c- 32 P]ATP was used. Substrate (100 l M) Activity (%) ReferenceUu-TK hTK1 5-Chloro-dU (CldU) a 122 196 5-Iodo-dU (IdU) a 113 170 5-Fluoro-dU (FdU) a 5-Ethyl-dU (EtdU) b 61 50 95 80 [11,19], [19] dU a 3-Methyl-dT (MeT) c 46 21 77 43 [11,19], [19] 3-(2-Propynyl)-dT (PropT) c 18 21 3-Isopropyl-dT (IsoT) c 14 17 [19] 3¢-Fluoro-dT (FLT) b 52 30 [29] 3¢-Azido-dT (AZT) a 3¢-Azido-methyl-dT (AZMT) b 35 8 52 15 [11,19], [29] 3¢-Fluoro-methyl-dT (FMT) b 715 2¢-Fluoro-arabinosyl-5-iodo-dU (FIAU) d 44 76 [19] 2¢-Fluoro-arabinosyl-5-methyl-dU (FMAU) d 34 48 [19] 3¢-Fluoro-5-cyclopropyl-dU (FCPU) b 52 33 3¢-Azido-5-iodo-dU (AZIU) b 22 70 3¢-Fluoro-5-fluoro-dU (FFU) b 19 20 3¢-Fluoro-5-ethynyl-dU (FEU) b 15 10 Source of the compounds: a Sigma-Aldrich; b N. G. Johansson, Medivir, Stockholm, Sweden; c W Tjarks, College of Pharmacy, The Ohio State University, Columbus, Ohio; d J Fox, Memorial Sloan Kettering Cancer Institute, New York. Table 2. IC 50 values of selected nucleoside analogues with Uu-TK and hTK1. Substrate concentrations were 1 l M dT and 2 mM ATP for Uu-TK and 0.2 l M dT and 2 mM ATP for hTK1. Substrate IC 50 (lM) Uu-TK hTK1 dU 484 ± 24 15 ± 0.8 FdU 273 ± 13 10 ± 0.5 MeT 234 ± 12 100 ± 5 EtdU 47 ± 2 8 ± 1 FIAU 16±0.8 78±2 FLT 14 ± 0.7 3.5 ± 0.2 FCPU 11 ± 0.6 4 ± 0.2 AZMT 11 ± 0.6 62 ± 3 AZIU 6 ± 0.3 < 1 Thymidine kinase in Ureaplasma urealyticum U. Kosinska et al. 6368 FEBS Journal 272 (2005) 6365–6372 ª 2005 The Authors Journal Compilation ª 2005 FEBS 3-methyl-dT (MeT) showed low ability to inhibit Uu-TK, with IC 50 values of 200–500 lm, using 1 lm [ 3 H]dT as substrate. 5-Ethyl-dU (EtdU) had an inter- mediate IC 50 value (47 lm), whereas 2¢ and 3¢ ana- logues containing fluorine or azido substitutions were relatively efficient inhibitors (IC 50 values < 20 lm), with 3¢-azido-5-iodo-dU (AZIU) having the lowest IC 50 value (6 lm) (Table 2). IC 50 values for hTK1, using 0.2 lm [ 3 H]dT as substrate, were also determined with the above analogues (Table 2). A lower dT con- centration was used for hTK1 to compensate for the lower K m value observed with hTK1 and dT (in the presence of 2 mm ATP) [11,20]. The results with hTK1 showed a pattern, with MeT, FIAU and AZMT form- ing a group of quite poor inhibitors with IC 50 values of 60–100 lm. The other analogues tested with hTK1 fell into a group with relatively high capacity to inhi- bit, and their IC 50 values ranged from 15 lm down to below 1 lm for 3¢-azido-5-iodo-dU (AZIU), the best inhibitor (Table 2). FLT and 3¢-fluoro-5-cyclopropyl-dU (FCPU), with IC 50 values of 14 and 11 lm, respectively, were the only analogues that showed higher activity with Uu-TK than with hTK1 (Table 1). Kinetic studies were performed with Uu-TK and hTK1 using FLT and FCPU as variable substrates together with 0.5 mm [c- 32 P]ATP. These analogues had about 3.5–4-fold higher k cat values than dT, but at the same time five- fold higher K m values, resulting in a slightly lower effi- ciency than dT (Table 3). In the case of hTK1, FLT showed in this experiment higher efficiency than FCPU and dT. Comparison with hTK1 Overall, the relative phosphorylation rates of the ana- logues tested with Uu-TK are equal to or lower than the corresponding rates with hTK1. The exceptions are FCPU and FLT, which show higher relative activity with Uu-TK than with hTK1. FCPU has a cyclopropyl substitution in the 5¢ position of the base, and its bet- ter activity with Uu-TK is probably due to the slightly larger binding pocket of Uu-TK. This enzyme has a serine at position 163 where hTK1 has a threonine, and the extra methyl group makes the site narrower. FCPU and FLT also have fairly low IC 50 values, which should be an advantage for a potential inhibitor. However, the corresponding IC 50 values with hTK1 were still lower, and this was the case with seven out of the nine nucleosides tested as inhibitors of dT phos- phorylation. Only FIAU and AZMT had higher IC 50 values with hTK1 than with Uu-TK. Still, they were not very efficiently phosphorylated by Uu-TK. These results show that the capacity of a nucleoside to be a good inhibitor does not directly correlate with its rate of phosphorylation. Despite the high struc- tural similarities between Uu-TK and hTK1, the latter is much easier to inhibit, as shown by its lower IC 50 values. Furthermore, these two enzymes show a 10-fold difference in K m values for dU and dU ana- logues [11,21]. The reasons for these differences in function may be related to the differences around the 5 position of the substrate, where Uu-TK has a serine and hTK1 has the more hydrophobic threonine. The analogues investigated so far may not be direct lead compounds for the further development of selective Uu-TK inhibitors, but this study demon- strates the necessity to use both a structural and functional approach for identification of new inhibi- tors and alternative substrates when nucleoside kinas- es are the targets. Such inhibitors would be desirable, as they could serve as efficient antibiotics because U. urealyticum lacks the capacity to synthes- ize DNA precursors de novo. The most promising route for further drug development for Uu-TK from this study seems to be to explore further substitu- tions at the 5 position and 3¢ position and combina- tions thereof. Experimental procedures Materials The radiolabelled substances [ 3 H]dT (25 CiÆmmol )1 ) and [c- 32 P]ATP ( 3000 CiÆmmol )1 ) were purchased from Amersham Biosciences (Uppsala, Sweden). Recombinant Uu-TK and hTK1 were prepared as previously described [11,22]. Enzyme assay TK activity was determined by using a DE-81 filter paper technique with [ 3 H]Thd or by a phosphoryl-transfer assay with [c- 32 P]ATP, as previously described [11]. The standard reaction mixture contained 50 mm Tris ⁄ HCl, pH 7.6, 2 mm Table 3. Kinetic parameters of FLT, FCPU and dT with Uu-TK and hTK1. A fixed [c- 32 P]ATP concentration (0.5 mM) was used. k cat values were calculated based on a subunit molecular mass of 27.5 kDa (Uu-TK) and 25.5 kDa (hTK1), respectively. Substrate K m (lM) k cat (s )1 ) k cat ⁄ K m (s )1 ÆM )1 ) Uu-TK hTK1 Uu-TK hTK1 Uu-TK hTK1 FLT 47 5 0.67 0.19 1.4 · 10 4 3.6 · 10 4 FCPU 42 8 0.80 0.09 1.9 · 10 4 1.1 · 10 4 dT 9 6 0.19 0.16 2.1 · 10 4 2.8 · 10 4 U. Kosinska et al. Thymidine kinase in Ureaplasma urealyticum FEBS Journal 272 (2005) 6365–6372 ª 2005 The Authors Journal Compilation ª 2005 FEBS 6369 MgCl 2 ,2mm ATP, 0.5 mgÆmL )1 BSA, 5 mm dithiothreitol, 1 lm [ 3 H]dT and 0.5 ng Uu-TK or 0.2 lm [ 3 H]dT and 1 ng hTK1 in a total volume of 50 lL. When the reaction was finished, the filters were washed three times in 5 mm ammo- nium formate and once in water. Reaction products were then eluted with 0.5 mL 0.1 m HCl ⁄ 0.2 m KCl, and the radioactivity was determined by liquid scintillation counting (Beckman). The results were analysed by the SigmaPlot Enzyme Kinetic Module version 1.1 (SPSS Science, Chi- cago, IL, USA). The phosphoryl-transfer assay was per- formed with 100 lm or 500 lm [c- 32 P]ATP in the same buffer as above with variable concentrations of nucleosides and 10 ng Uu-TK in a total volume of 25 lL. The phos- phorylated products were separated by TLC and quantified by phosphorimaging analysis (Fujifilm Image Gauge, ver- sion 3.3). One unit of kinase activity was defined as the formation of 1 nmol deoxyribonucleoside 5¢-monophosphate per mg protein per min. Crystallization, data collection and refinement The His-tag of Uu-TK was cleaved off overnight with 10 UÆmg )1 thrombin purchased from Amersham Bio- sciences. The protein was crystallized by the hanging drop vapour diffusion method at 14 °C. The protein solution consisted of 10 mgÆmL )1 Uu-TK, 5 mm Thd and 3 mm AMP-PCP, whereas the reservoir solution consisted of 15% poly(ethylene glycol) 3350 and 0.3 m ammonium for- mate. A small petri dish (diameter 5.5 cm) was filled with 0.5 mL of the crystallization solution. 2 lL protein solu- tion together with 2 lL crystallization solution was applied to the lid of the Petri dish. Two different crystal forms appeared after 2–5 days. Before flash-freezing in liquid nitrogen, the crystals were swept through cryo-pro- tecting solution consisting of 15% poly(ethylene glycol) 3350, 0.3 m ammonium formate and 20% glycerol. Data were collected at ID14-3 ESRF, processed with mosflm [23], and scaled with scala from the CCP4 pro- gram suit [24]. The statistics from data reduction are pre- sented in Table 4. The structure was solved with molrep [25]. As search model, the tetramer of Uu-TK from the dTTP complex, PDB code 1XMR, was used. Residues 50– 67 were omitted from each chain of the search model. The omitted region or parts of the omitted region, depending on the chain, could be traced with ARPwARP [26]. After simulated annealing performed in CNS, subse- quent refinement was performed in REFMAC5 [27]. Dur- ing the whole refinement, NCS restraints were applied to residues 12–49 and 69–213 for each protein chain. By the end of the refinement, the restraints were loosened to medium for main-chain atoms and loose for side-chain atoms. A TLS model consisting of 15 TLS groups was applied during the final steps of the refinement. Each monomer was divided into four domains [the a ⁄ b domain, the flexible loop (missing in subunit B), the lasso domain, and the C-terminus), together creating 15 TLS groups. Table 4 shows the statistics from data refinement. All model building was carried out in O [28]. Each monomer contains 223 residues, although the N-terminus and C-ter- minus are disordered, and between 6–11 residues depend- ing on the chain are omitted from the model. In addition, there is a disordered region between residues 51 and 66. Only in chain A could the whole region be traced; in chains C and D parts of the region are modelled, and in chain B the entire region is missing. As observed previ- ously [14–16], this part of the structure takes different conformations. The structure has been deposited with PDB code 2B8T. Acknowledgements This work was supported by grants from the Swedish Research Council for the Environment, Agricultural Sciences and Spatial Planning (to L.Y. and S.E.), the Swedish Research Council (to H.E. and S.E.), and the Swedish Cancer Foundation (to H.E.). References 1 Fraser CM, Gocayne JD, White O, Adams MD, Clay- ton RA, Fleischmann RD, et al. (1995) The minimal gene complement of Mycoplasma genitalium. Science 270, 397–403. Table 4. Data collection and refinement statistics. Numbers in par- entheses refer to the outer resolution bin. Space group P2 1 Cell dim. (A ˚ , °)a¼ 57.16 b ¼ 115.65 c ¼ 64.47 b ¼ 101.02 Content of the asymmetric unit 1 tetramer Resolution (A ˚ ) 2.00 (2.11–2.00) Completeness (%) 99.1 (98.5) R meas (%) 9.0 (48.4) I ⁄ rI 16.4 (2.9) Redundancy 5 No. of observed reflections 274 598 No. of unique reflections 54 312 Beam line ESRF, ID14eh3 Wavelength (A ˚ ) 0.931 Temperature (K) 100 R (%) 19.5 R free (%) 23.5 R.m.s.d. Bond length (A ˚ ) 0.008 Bond angle (°) 1.04 Mean B-value (A ˚ 2 )32.9 Thymidine kinase in Ureaplasma urealyticum U. Kosinska et al. 6370 FEBS Journal 272 (2005) 6365–6372 ª 2005 The Authors Journal Compilation ª 2005 FEBS 2 Glass JI, Lefkowitz EJ, Glass JS, Heiner CR, Chen EY & Cassell GH (2000) The complete sequence of the mucosal pathogen Ureaplasma urealyticum. Nature 407, 757–762. 3 Chambaud I, Heilig R, Ferris S, Barbe V, Samson D, Galisson F, et al. (2001) The complete genome sequence of the murine respiratory pathogen Mycoplasma pul- monis. 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Structure of the substrate complex of thymidine kinase from Ureaplasma urealyticum and investigations of possible drug targets for the enzyme Urszula Kosinska 1 *,. posi- tion in the substrate complex from that in the complex with the feedback inhibitor, having moved in closer to the 5¢-OH of the substrate to form a hydrogen bond. The phosphorylation of and inhibition. any member of the TK1 family, we have now determined the first structure of a member of the TK1 family in complex with the substrate dT. The possibilities for drug design have been investigated by enzyme