Báo cáo khoa học: Structural studies of nucleoside analog and feedback inhibitor binding to Drosophila melanogaster multisubstrate deoxyribonucleoside kinase doc

Structural studies of nucleoside analog and feedback inhibitor binding to Drosophila melanogaster multisubstrate deoxyribonucleoside kinase Nils E. Mikkelsen 1 , Birgitte Munch-Petersen 2 and Hans Eklund 1 1 Department of Molecular Biology, Swedish University of Agricultural Sciences, Biomedical Center, Uppsala, Sweden 2 Department of Science, Systems and Models, Roskilde University, Denmark Cells need to keep a balanced pool of dNTPs to sus- tain DNA synthesis and repair. The main source of dNTPs comes from the de novo pathway where ribonu- cleosides are converted to ribonucleotides by the enzyme ribonucleotide reductase [1]. In resting cells, where ribonucleotide reductase activity is low, there is an alternative route for obtaining dNTPs, namely the salvage pathway. Here, nucleosides that originate from dead cells and food are salvaged from the extracellular space and transported into the cell. Once inside, they become phosphorylated by deoxyribonucleoside kinas- es and are thus prevented from leaving the cell [2]. Mammalian cells have four different deoxynucleo- side kinases with distinct, but overlapping, substrate affinities. Thymidine kinase 1 (TK1) and deoxycytidine kinase (dCK) are found in the cytosol, and thymidine kinase 2 (TK2) and deoxyguanosine kinase (dGK) are found in the mitochondria. TK1 has the most restricted substrate specificity and phosphorylates only deoxythymidine (dT) and deoxyuridine, whereas dCK is somewhat more relaxed and phosphorylates both pyrimidine and purine deoxynucleosides. The best sub- strate for dCK is deoxycytidine (dC), but dCK also phosphorylates deoxyadenosine and deoxyguanosine. TK2, which phosphorylates the same substrates as TK1, can also phosphorylate dC and other medically interesting dT, deoxyuridine and dC analogs. dGK only phosphorylates the purine deoxyribonucleosides deoxyadenosine, deoxyguanosine and deoxyinosine. In addition, many pharmacological nucleoside ana- logs (NAs) that are used in both antiviral therapy and cancer therapy need activation by deoxynucleoside Keywords cancer gene therapy; deoxyribonucleoside kinase; nucleoside analogs; pyrimidines; X-ray structures Correspondence H. Eklund, Department of Molecular Biology, Swedish University of Agricultural Sciences, Biomedical Center, S-751 24 Uppsala, Sweden Fax: +46 18536971 Tel: +46 184714559 E-mail: hasse@xray.bmc.uu.se (Received 10 January 2008, revised 27 February 2008, accepted 3 March 2008) doi:10.1111/j.1742-4658.2008.06369.x The Drosophila melanogaster multisubstrate deoxyribonucleoside kinase (dNK; EC 2.7.1.145) has a high turnover rate and a wide substrate range that makes it a very good candidate for gene therapy. This concept is based on introducing a suicide gene into malignant cells in order to activate a prodrug that eventually may kill the cell. To be able to optimize the func- tion of dNK, it is vital to have structural information of dNK complexes. In this study we present crystal structures of dNK complexed with four dif- ferent nucleoside analogs (floxuridine, brivudine, zidovudine and zalcita- bine) and relate them to the binding of substrate and feedback inhibitors. dCTP and dGTP bind with the base in the substrate site, similarly to the binding of the feedback inhibitor dTTP. All nucleoside analogs investigated bound in a manner similar to that of the pyrimidine substrates, with many interactions in common. In contrast, the base of dGTP adopted a syn- conformation to adapt to the available space of the active site. Abbreviations 5FdU, floxuridine: 5-fluoro-2¢-deoxyuridine; AZT, zidovudine: 3¢-azidothymidine; BVDU, brivudin: (E)-bromvinyl-2¢-deoxyuridine; BVU, (E)-5- (2-bromovinyl)-uracil; dC, deoxycytidine; dCK, cytosolic deoxycytidine kinase; ddC, zalcitabine: 2¢,3¢-dideoxycytidine; dGK, mitochondrial deoxyguanosine kinase; dNK, Drosophila melanogaster deoxyribonucleoside kinase; dT, deoxythymidine; HSV-1, herpes simplex virus 1; NA, nucleoside analog; TK, thymidine kinase; TK1, thymidine kinase 1; TK2, thymidine kinase 2; VZV, varicella zoster virus. FEBS Journal 275 (2008) 2151–2160 ª 2008 The Authors Journal compilation ª 2008 FEBS 2151 kinase-catalyzed phosphorylation. In humans, the main activators of the NAs are the deoxynucleoside kinases, which phosphorylate the NAs, thereby trapping them inside the cell. This is regarded as the rate-limiting step and makes the deoxynucleoside kinases important actors in combating malignant cells. One approach in this battle is gene therapy, where a suicide gene is introduced into a malignant cell followed by the addi- tion of a NA specifically activated by the enzyme encoded by this gene. The activated NA is then expected to kill the malignant cell. This can occur either by incorporation of the triphosphorylated form of the NA into cellular DNA, causing chain break or termination, or by other inhibitory effects that ulti- mately inhibit viral replication or kill the recipient cell [3] by inducing apoptosis [4]. Examples of NAs targeted towards deoxynucleoside kinases are 1-b- d-arabinofuranosylguanosine and 2-chloro-2¢-deoxyad- enosine, which are phosphorylated by dCK and dGK, respectively. The Drosophila melanogaster multisubstrate deoxyri- bonucleoside kinase (dNK; EC 2.7.1.145) can phos- phorylate all natural substrates and a wide range of medically important NAs with outstanding efficiency, as shown in Table 1 [5–9]. This makes it a very prom- ising candidate as a suicide gene in gene therapy and it has also been shown to be transducible into human cancer cell lines [10]. dNK mutants have given some remarkable results by sensitizing different cancer cell lines towards different NAs by more than 18 000-fold compared with the parental cell line [9, W. Knecht et al., unpublished data]. The possibility of tailoring suicide genes with the end result being the almost com- plete elimination of natural substrate affinities and feedback inhibition, can therefore make the enzymes, produced by these mutated genes, highly efficient acti- vators for specific NAs. In this way, the lower amount of NA needed may considerably reduce the toxic side effects that often accompany this type of therapy. The main drawback in gene therapy has been the targeting and successful delivery of suicide genes into the cells of interest. When this obstacle is overcome, we will have an arsenal of very potent suicide genes that are ready for use in anticancer therapies. The 3D structure of dNK has previously been deter- mined in complexes with substrates and a feedback inhibitor [12,13]. It has a structure similar to that of the human dGK and dCK and belongs to a structural family that also contains some viral thymidine kinases (TKs) [14]. These enzymes contain a P-loop and a LID region that binds phosphates of the phosphate donor, usually ATP (Fig. 1), and an LID region that closes down on the phosphates of the phosphate donor (Fig. 1). In this article we describe the crystal structure of dNK with four different NAs. In addition, we investi- gated additional substrate and dNTP complexes. In most cases, a truncated version of dNK lacking the last 20 residues was used. This truncation mutant has kinetic characteristics similar to those of the full-length enzyme, but because the k cat is two- to threefold higher, it is even faster [15]. Table 1. Kinetic parameters for dNK with natural substrates and NA from the crystal structures. K m (lM) V max (lmolÆmin )1 Æmg )1 ) k cat (s )1 ) k cat ⁄ K m (lM )1 Æs )1 ) dT a 1.2 29.5 14.2 12 dC a 2.3 34.2 16.5 7.2 dA a 225 42.7 20.6 0.092 dG a 665 31.3 19 0.029 5-FdU 1.0 29.8 14.2 14 BVDU b 2.2 13.2 5.9 2.7 AZT c 8.3 0.073 0.036 0.0043 ddC c 1124 8.6 4.2 0.0037 a Data are from [15]. b Data are from [16]. c Data are from [6]. LID P-loop ERS α1 α2 α3 α4 α6 α5 α8 α7 β1 β2 β3 β4 β5 Fig. 1. 3D structure of dNK with dCTP bound as a feedback inhibi- tor. The protein structure has a central parallel five-stranded b sheet surrounded by helices. The LID region, P loop and ERS motifs are in red. Nucleoside analog deoxynucleoside kinase complexes N. E. Mikkelsen et al. 2152 FEBS Journal 275 (2008) 2151–2160 ª 2008 The Authors Journal compilation ª 2008 FEBS Results and Discussion Quality of the structures dNK is an enzyme with flexible parts that had to be stabilized to obtain well-diffracting crystals. The phos- phate-binding regions have, in all structures deter- mined to date, been stabilized by sulfate ions or by the phosphates of a feedback inhibitor. Furthermore, the C-terminus is flexible in all structures, such as in both truncated proteins that we mainly used for crystalliza- tion, as well as in the full-length enzyme (see below). The best diffracting crystals have been obtained in the presence of triphosphate inhibitors, where the phosphate-interacting regions are stabilized, whereas the binary complexes with NAs in the best cases dif- fract slightly better than 3 A ˚ resolution. The structures of dNK in complex with the substrates dC and dT have previously been determined [12,13]. We have now been able to determine the dC complex at a slightly higher resolution (2.3 A ˚ ), which is here used as a refer- ence for the discussion of the NA complexes. Although this complex was co-crystallized with the phosphate donor product ADP, this nucleotide was not found at the phosphate donor site. It had been outcompeted by a sulfate ion, as in the other substrate complexes. NA binding We determined the structures of dNK with four pyrimidine NAs: floxuridine (5FdU, 5-fluoro-2¢-deoxy- uridine), zidovudine (AZT, 3¢-azido-2¢,3¢-dideoxythymi- dine), zalcitabine (ddC, 2¢,3¢-dideoxycytidine) and brivudin [BVDU, (E)-5-(2-bromovinyl)-2¢-deoxyuri- dine]. The kinetic parameters for these are given in Table 1. When discussing the binding and the effect of the analogue on dNK, it is presumed, as previously described [16], that the catalytic or preceding step is rate determining, and that the size of the K m reflects the nucleoside binding affinity. All refinement statistics can be found in Table 2. Floxuridine (5FdU) is an oncologic drug most often used in the treatment of breast and colorectal cancer. The nucleotide form of floxuridine (5FdUMP) irrevers- ibly inhibits thymidylate synthase, which leads to a strong reduction of thymine nucleotides in the cell and this, in turn, inhibits DNA synthesis [17]. 5FdU is phosphorylated efficiently by dNK with the same high k cat ⁄ K m of 2 · 10 7 m )1 Æs )1 as with thymidine, and 10-fold higher than with TK1 [14]. The crystal structure of dNK with 5FdU is very sim- ilar to the previously solved substrate structures with dT and dC [12,13]. It contains a sulfate ion bound in the P loop, and the substrates are at nearly identical positions in the active site. The interactions of the deoxyribose and the base are identical to those of the dT complex, except for the fluoride atom replacing the methyl group on the base (Fig. 2A). In the dC complex we find two water molecules occupying this cleft, making an interacting bridge between OE2 on Glu52 and N4 on the dC base, as shown in Fig. 3A. In the 5FdU complex the fluoride occupies this space, Table 2. Data collection and refinement statistics for the dNK ligand complexes. Statistics dC (ADP) 5FdU ddC BVDU AZT dCTP dGTP dNKwt-dTTP Space group P2 1 2 1 2P2 1 P2 1 P2 1 2 1 2P2 1 2 1 2P2 1 P2 1 2 1 2P2 1 2 1 2 Cell dimensions 120.6 70.4 70.5 137.5 140.0 67.9 119.7 119 62.5 70.7 70.8 112.8 111.9 119 65.1 64.9 68.2 225.4 226.0 69.7 71.1 70.5 69.2 69.1 Content au 1 dimer 4 dimers 4 dimers 2 dimers 2 dimers 2 dimers 1 dimer 1 dimer Resolution (A ˚ ) 50–2.3 30–3.0 50–2.9 30–2.9 20–2.8 50–2.2 50–2.5 45–2.2 Completness (%) 98.5 (91.2) 99.3 (99.3) 97.3 (97.1) 83.9 (87.4) 99.4 (99.8) 99.5 (99.5) 99.8 (99.7) 99.6 (99.6) Rsym 0.075 (0.434) 0.084 (0.528) 0.116 (0.583) 0.094 (0.540) 0.114 (0.474) 0.071 (0.370) 0.096 (0.555) 0.069 (0.414) Rmeas 0.089 (0.522) 0.103 (0.655) 0.136 (0.678) 0.116 (0.666) 0.134 (0.555) 0.088 (0.462) 0.103 (0.597) 0.082 (0.486) Mn(I) ⁄ sd 13.1 (2.1) 11.3 (2.0) 13.0 (2.1) 9.7 (2.3) 9.3 (3.1) 11.3 (3.1) 17.4 (4.0) 16 (3.2) Redundancy 3.4 (2.7) 2.9 (3.0) 3.7 (3.8) 2.8 (2.7) 3.6 (3.6) 2.8 (2.9) 7.1 (7.3) 3.4 (3.5) Reflections 22045 46314 50991 19428 26596 53215 18113 26365 R factor (%) 23.4 25.6 24.8 24.2 23.5 19.5 20.7 21.4 Rfree (%) 27.3 28.1 28.7 28.6 27.1 24.9 26.7 25.9 rmsd bond lengths 0.009 0.013 0.015 0.013 0.012 0.012 0.011 0.010 rmsd bond angles 1.151 1.326 1.471 1.398 1.836 1.403 1.421 1.183 Mean B value (A ˚ 2) 39.2 63.1 45.6 54.6 39.7 31.8 34.4 36.1 Beamline ID14-4 ID23-1 ID14-2 ID14-1 ID-14-1 ID-29 ID-29 ID-29 PDB-code 2vp5 2vp6 2vp9 2vqs 2jj8 2vp4 2vp2 2vp0 N. E. Mikkelsen et al. Nucleoside analog deoxynucleoside kinase complexes FEBS Journal 275 (2008) 2151–2160 ª 2008 The Authors Journal compilation ª 2008 FEBS 2153 expelling the two water molecules in a manner similar to that previously reported for dT and its methyl group [13]. 5FdU is phosphorylated efficiently by dNK with the same K m and k cat values as with thymidine (Table 1). This is in agreement with the high similarity observed between the crystal structures obtained with dT and 5FdU. Zalcitabine (ddC) is an NA used in the treatment of HIV infections. The structure of the ddC complex (Fig. 2B) shows that the analog binds similarly as the natural pyrimidine substrates but lacks a hydrogen bond because of the absence of the 3¢-OH. Two water molecules bridge between Glu52 and N4 of the analog, as seen in the dC complex. The K m for ddC is almost 500-fold higher than for dC, whereas the k cat is decreased only by 3.3-fold. Thus, the catalytic step should be expected to be R167 A R169 E172 Y70 M69 M118 Q81 A110 M88 R105 E52 K33 T34 R167 R169 E172 Y70 M69 M118 Q81 A110 M88 R105 E52 K33 T34 B Fig. 3. Initial difference density maps, contoured at 3r, for (A) dC and one sulfate ion and for (B) AZT and two sulfate ions. All hydro- gen bonds are shown as red dotted lines and water molecules are shown as red balls. E172 A Y70 M69 M118 Q81 A110 M88 R105 E52 E172 Y70 M69 M118 Q81 A110 M88 R105 E52 M88 Y70 M69 M118 Q81 A110 S106 R105 E52 B C Fig. 2. Initial difference density maps, contoured at 3r, covering the NAs (A) 5FdU, (B) ddC and (C) BVDU. Water molecules are shown as red balls. Nucleoside analog deoxynucleoside kinase complexes N. E. Mikkelsen et al. 2154 FEBS Journal 275 (2008) 2151–2160 ª 2008 The Authors Journal compilation ª 2008 FEBS affected very little but the binding should be strongly affected. The structure shows that ddC is in the proper position for P transfer, but very poorly bound due to the loss of the hydrogen bonds as a result of the miss- ing 3¢-OH. Brivudine (BVDU) is an NA used in the treatment of herpes simplex virus type 1 (HSV-1) and varicella zoster virus (VZV) infections. BVDU has also shown potential as a cancer drug in gene therapy ⁄ chemother- apy as a result of its cytostatic activity in cancer cells transduced with viral TK genes. BVDU may also enhance the potency of 5-fluorouracil in combined chemotherapy, because BVDU becomes degraded by thymidine phosphorylase to (E)-5-(2-bromovinyl)uracil (BVU). This metabolite, in turn, inactivates dihydro- pyrimidine dehydrogenase, which is the enzyme that initiates the degradative pathway of 5-fluorouracil. Balzarini et al. [18] have also shown some promising results using BVDU as insecticide, where D. melanog- aster and Spodoptera frugiperda embryonic cells showed high sensitivity towards BVDU. The dNK complexes with BVDU (Fig. 2C) and dT have very similar overall structures. However, BVDU is slightly displaced compared with dT to accommo- date the bulky bromovinyl group in the deep cleft sur- rounded by residues Ser109, Ala110, Val84, Trp57 and Arg105. The LID is partly missing, and helix a3 (which interacts with the LID) is displaced similarly as in the AZT complex (see below). There are no signifi- cant conformational changes of the side chains in the active site, as found in HSV-TK where Tyr132, the equivalent to Met88 in dNK, is shifted to make room for the more bulky groups of dT and BVDU. The minor structural changes in the structure with BVDU compared with dT are in agreement with the very simi- lar kinetic values. There are two previously determined structures, with BVDU and brivudine monophosphate (BVDUMP) in the HSV-1-TK + BVDU complex [19] and the VZV + BVDUMP and ADP complex [20]. Zidovudine (AZT) is a potent inhibitor of HIV repli- cation in vitro and at the time of publishing is still included in the standard regimen for treatment of the disease. AZT is also a substrate for dNK, although with a k cat ⁄ K m that is about 2800-fold lower than the k cat ⁄ K m for dT (Table 1). We have determined a structure of dNK complexed with AZT, and the difference density for the thymidine part of AZT in the active site is well defined, as shown in Fig. 3B. Surprisingly, there were two sulfate ions present – one bound in the P loop, as observed in the other substrate complexes, and the other located between the first sulfate ion and the substrate. There was no density for the N 3 azido group of AZT or the part of the LID region ranging from Arg165 to Cys174. This LID usually clamps down interacting with the sub- strate and the sulfate ion bound in the P loop. The lack of density here is probably caused by the N 3 group of AZT, which protrudes into this loop region (Fig. 3B). Superposition of the AZT complex with the dC complex, clearly shows the steric impact that the N 3 group has on this section. The LID is totally distorted and the interacting helix a3 (Fig. 4) on the opposite side on top of the substrate is pushed back a little in a rigid body-like movement, probably to accommodate the azido group on AZT. This widening of the active site probably also provides space for the second sulfate ion to bind (Fig. 3B). There is also a small shift in the P loop and the sulfate ion occupying this position, which is displaced somewhat compared with the sul- phate ion in the dC complex. According to a k cat for AZT that is more than 400- fold lower than with thymidine, and a K m that is increased by eightfold, the catalytic step should be effected considerably more than the binding. This is in agreement with the N 3 group being somewhat of a hindrance for proper binding but the LID being completely distorted, making P transfer very difficult. In yeast thymidylate kinase a similar shift in the P loop was observed when the deoxythymidine mono- phosphate (dTMP) complex was compared with the AZT-monophosphate (AZTMP) complex. It was Fig. 4. Superposition of dNK structures (tube representation) in complex with AZT (red) and dC (grey) picturing the structural differ- ences when the bulkier AZT (yellow) is bound in the active site together with the two sulfate ions. Part of the LID is missing here as there was no traceable density for this region. N. E. Mikkelsen et al. Nucleoside analog deoxynucleoside kinase complexes FEBS Journal 275 (2008) 2151–2160 ª 2008 The Authors Journal compilation ª 2008 FEBS 2155 speculated that the shift was probably a result of the bulkier AZT and that this displacement of the loop was the probable cause for the reduced catalytic activity of the thymidylate kinase towards AZT [21]. The P loop is involved in binding the phosphoryl donor and has evi- dently moved to an unfavorable position, thereby affect- ing the phosphoryl transfer negatively. Later work with human thymidylate kinase [22,23] showed that mutants with mutated amino acids in the LID region gained effi- ciency in AZTMP phosphorylation. It was suggested that the LID has to be in a closed conformation to be able to phosphorylate the substrate efficiently. Earlier work on dNK revealed that a N64D mutant retained efficiency towards AZT, and structures of the N64D mutant complexed with dT and dTTP were investigated [8]. It was found that the increased effi- ciency towards AZT was probably caused by a reduced stability in the LID region, which made the enzyme more relaxed towards the bulkier azido group. Deoxynucleoside triphosphate complex structures Feedback inhibition of deoxynucleoside kinases is a common way of regulating the nucleotide production of these enzymes, and the end products of the pre- ferred substrates are usually the best inhibitors [24]. Kim et al. [25] proposed that dCK was regulated by the end product of the dCK metabolic pathway where dCTP would act as a feedback inhibitor. They further suggested that dCTP could function as a bisubstrate analog where the triphosphate group would bind in the phosphate donor site and the deoxycytidine base in the phosphate acceptor site as a normal substrate. The first structure of such a feedback-inhibited deoxyribo- nucleoside kinase was human dGK, where it was believed that the co-crystallized ATP was bound as a feedback inhibitor, although the density suggested a dATP [12]. Later work on human TK1 showed that although this kinase was co-crystallized with different substrates, there was always a dTTP bound as a feed- back inhibitor [26]. The dTTP was bound so tightly that even the purification process, which contained no dTTP, did not release it. Similar observations were reported for human TK2 where the feedback inhibitor dTTP was strongly bound [27]. A re-investigation and new refinement of the human dGK structure finally convinced the authors that it actually was a dATP molecule bound in dGK (pdb-code: 2ocp). Earlier work of dNK complexed with dTTP had demonstrated that the feedback inhibitor was indeed bound as a bisubstrate inhibitor occupying both the phosphate donor and acceptor sites. Here, a magne- sium ion was bound to the phosphates [13]. The bind- ing of the inhibitor induces a structural change where the catalytically important residue Glu52 is shifted along with the main chain to bind dTTP and coordi- nate magnesium. We have now determined two additional dNTP com- plexes of dNK that bind like the feedback inhibitor dTTP: one with dCTP at 2.2 A ˚ resolution and one with dGTP at 2.5 A ˚ resolution (Fig. 5). The triphosphate part of these dNTPs is nearly identical to the tripho- sphate part of the dTTP structure and for dCTP the base moiety superimposes perfectly with dC in the dNK–dC complex. One difference, though, is that one of the two water molecules bridging OE2 on Glu52 and N4 on the dC base in the dNK–dC structure is now absent. This is a result of the shift of the Glu52 to a similar position as in the dTTP structure. There is no R167 A B R169 E172 Y70 M69 M118 Q81 A110 R105 K33 T34 R167 R169 E172 Y70 M69 M118 Q81 A110 R105 K33 T34 Fig. 5. Initial difference density maps of (A) dCTP (2.2 A ˚ ) and (B) dGTP (2.5 A ˚ ) and their binding in the dNK active site. All hydrogen bonds are shown as red dotted lines and water molecules are shown as red balls. Nucleoside analog deoxynucleoside kinase complexes N. E. Mikkelsen et al. 2156 FEBS Journal 275 (2008) 2151–2160 ª 2008 The Authors Journal compilation ª 2008 FEBS detectable magnesium coordinating Glu52, which in this structure is tilted a little outwards compared with Glu52 in the dNK–dTTP structure, as shown in Fig. 6. In the structure of the dGTP complex, the guanosine base occupies approximately the same geometrical space as the base in the dCTP and dTTP ligands (Fig. 6). The guanosine base is in the syn-conformation, in contrast to the thymine and cytosine bases that are in the anti- conformation in those complexes. There is a water mol- ecule bridging ⁄ anchoring the N2 of the guanosine base to Ser109 located at the bottom of this hydrophobic cleft. Gln81 makes hydrogen bonds to N7 and O6 on the side of the base acting as a clamp, but otherwise it is supported by the same stacking interactions as described previously in both the dC and dT structures. Gln81 has been moved almost 1 A ˚ to be able to accom- modate the slightly more bulky guanosine base, but otherwise there are no significant changes to the overall 3D structure in the active site. This shows how flexible dNK is in having room for many different substrates by using mostly water molecules as bulk material to retain stability around the bound ligand. There are two previ- ously solved structures of a kinase with a guanosine base in the active site, namely the HSV-TK complexed with ganciclovir and penciclovir [19]. In those cases, the base is in the anti-conformation. Full-length dNK–dTTP complex Most crystallographic studies on dNK have been performed on a C-terminally truncated mutant that has catalytic characteristics similar to those of the wild-type enzyme [15] but was easier to crystallize. However, we were finally able to crystallize the full- length enzyme using the feedback inhibitor dTTP, which made it possible to make comparisons with the corresponding structure of the truncated enzyme. This structure, determined at 2.2 A ˚ resolution, did not show any additional traceable density compared with the truncated dNK structures. Several attempts have been made, to obtain a phos- phate donor or a phosphate donor analog co-crystal- lized together with a substrate, but with no success to date. dNK that was crystallized with the substrate dC and the phosphate donor product ADP or CDP showed no density for either ADP or CDP. The pres- ence of sulphate ions obviously hindered binding of ADP or CDP. Preliminary studies of dNK complexed with the substrate analogs AP 4 dT and AP 5 dT indicate that it might be crucial to have the full-length enzyme to accommodate sufficient binding for crystallization of a complex with the phosphate donor to be able to stabilize the structure of the last 32 amino acids suffi- ciently to be visible in electron density maps. Substrate specificity of dNK Earlier crystallographic studies of substrates dT and dC and on the structure of the feedback inhibitor complex with dTTP, as well as mutation studies, have established some of the basic rules for substrate specificity for this enzyme [7,12,13]. Similar studies on human dGK and dCK have confirmed and further complemented these rules [28]. For dNK, the substrate site is formed by an elongated cavity lined on the top and bottom of hydro- phobic residues. Around this cavity, polar residues are positioned to form specific interactions to the sugar and the base of the substrate. The 3¢-oxygen of deoxyribose is hydrogen bonded to Tyr70 and Glu172, and the 5¢-oxygen is hydrogen bonded to Glu52 and Arg105. A key interaction shared by all the investigated NAs is the binding to Gln81, which forms hydrogen bonds to the nitrogen in position 3 and to the carbonyl or nitrogen at position 4 of the pyrimidine ring. In this study, we determined the structure of the com- plexes of four pyrimidine analogs. It has so far not been possible to obtain useful crystals with purine NAs. All pyrimidine nucleotide analogs bind in similar modes in spite of different substitutions. The interactions with Gln81 are present in all analog complexes and the inter- actions with the 5¢-position are preserved. The effect of removing the 3¢-oxygen in ddC resulted in a weaker interaction owing to the loss of hydrogen bonds. The substitution of the 3¢-oxygen with an azide group in AZT apparently destabilized part of the structure. The only substitutions of the pyrimidine ring of the analogs that we investigated were at the 5-position. There is a pocket close to the 5-position that can accommodate different substitutions. The largest one E52 dGTP/dCTP/dTTP Mg Fig. 6. The three triphosphates dTTP (blue), dCTP (green) and dGTP (yellow), superimposed together with Glu52 from each corre- sponding structure. In the dCTP and dGTP structures Glu52 is suc- cessively pointing outwards when compared with the dTTP structure and in both dCTP and dGTP Glu52 makes contact with Arg195 from the adjacent symmetry-related molecules. Magnesium (grey) is only found in the dTTP structure. N. E. Mikkelsen et al. Nucleoside analog deoxynucleoside kinase complexes FEBS Journal 275 (2008) 2151–2160 ª 2008 The Authors Journal compilation ª 2008 FEBS 2157 that we analyzed was the bromovinyl group of BVDU that fits snugly into this pocket. A larger substitution would probably cause steric hindrance. It has been shown, in kinetic measurements, that dTTP is the only really efficient feedback inhibitor for different substrates [29], which is analogous to dT being the best substrate. In our structural studies, high concentrations in the absence of substrate still allowed binding of other dNTPs. The study of the dNTPs enabled us, for the first time, to obtain a complex with a purine bound at the active site – the dGTP structure. To be able to bind to this rather tight substrate site, the protein does not adapt to the larger substrate by conformational changes. Instead, the base adopts a syn-conformation that differs from the anti-conformation in other sub- strates, NAs and feedback inhibitors. Also in this case, it is the pocket close to the 5-position in the pyrimi- dines that accommodates the larger purine base. Gln81 forms hydrogen bonds to the base also in this case. The position of the guanine is probably also present in purine substrate complexes and may explain the considerably larger K m values with these substrates. Experimental procedures Materials Nucleosides and nucleotides were from Sigma (St Louis, MO, USA). Protein purification and kinetic studies The D. melanogaster dNK was overexpressed in Escheri- chia coli using the glutathione S-transferase (GST) gene fusion expression system (Amersham Pharmacia Biotech, Uppsala, Sweden). Filtered cell homogenate of induced BL21 transformants was applied to a glutathione–Sepha- rose column. The expressed protein was cleaved from gluta- thione S-transferase by thrombin. Details of the expression, purification and kinetic investigations of the recombinant wild-type and truncated dNK have been described else- where [6,15]. Crystallization Crystals of all the dNK complexes were grown using the vapor diffusion method with hanging drops. The solutions (described below) were left to equilibrate at 14 °C and crys- tals usually appeared after 1–2 days. After 2–3 weeks they had typically grown to a suitable size and were flash frozen in liquid nitrogen after a quick wash in a cryo-solution and then stored in liquid nitrogen as described below. dGTP Hanging drops consisted of 2 lL of crystallization solution containing 0.1 m Tris, pH 7.5, 0.2 m lithium citrate and 19% poly(ethylene glycol) 3350 added to 2 lL of enzyme solution containing 10 mgÆmL )1 of protein and 5 mm dGTP. The crystals were cryo-protected by a quick wash through the crystallization solution containing 20% glycerol. dCTP Hanging drops consisted of 2 lL of crystallization solution containing 0.1 m MES, pH 6.5, 0.2 m lithium citrate and 18% poly(ethylene glycol) 3350 added to 2 lL of enzyme solution containing 10 mgÆmL )1 of protein and 5 mm dCTP. The crystals were cryo-protected by a quick wash through crystallization solution containing 20% glycerol. AZT Hanging drops consisted of 2 lL of crystallization solution containing 0.1 m MES, pH 6.5, 0.2 m Li 2 SO 4 and 26% polyethylene glycol 2000 monomethylether added to 2L of enzyme solution containing 30 mgÆmL )1 of protein and 5mm AZT. The crystals were cryo-protected by a quick wash through crystallization liquid containing 26% mPEG2000. ddC Hanging drops consisted of 2 lL of crystallization solution containing 0.1 m MES, pH 6.5, 0.2 m Li 2 SO 4 and 22% mPEG2000 M added to 2 lL of enzyme solution contain- ing 10 mgÆmL )1 of protein and 5 mm ddC. The well solu- tion consisted of 30% mPEG2000 and after 1 week the coverslip with the hanging drop was further shifted to 35% mPEG2000 for an additional week. The crystals were flash frozen without further additions. BVDU Hanging drops consisted of 2 lL of crystallization solution containing 0.1 m MES, pH 6.5, and 2.5 m Am 2 SO 4 added to 2 lL of enzyme solution containing 20 mgÆmL )1 of pro- tein and 3.7 mm BVDU. The crystals were cryo-protected by a quick wash through crystallization liquid containing 25% glycerol. 5FdU Hanging drops consisted of 2 lL of crystallization solution containing 0.1 m MES, pH 6.5, 0.2 m Li 2 SO 4 and 22% mPEG2000 M added to 2 lL of enzyme solution contain- ing 10 mgÆmL )1 of protein and 5 mm 5FdU. The well Nucleoside analog deoxynucleoside kinase complexes N. E. Mikkelsen et al. 2158 FEBS Journal 275 (2008) 2151–2160 ª 2008 The Authors Journal compilation ª 2008 FEBS solution consisted of 30% mPEG2000 and after 1 week the cover slip with the hanging drop was transferred to 35% mPEG2000 for an additional week. The crystals were flash frozen without further additions. dC+ADP Hanging drops consisted of 2 lL of crystallization solution containing 0.2 m K 2 SO 4 , 20% poly(ethylene glycol) 3350, pH 6.8 (Hampton Research PEG ⁄ Ion Screen condition #34), added to 2 lL of enzyme solution containing 15 mgÆmL )1 of protein, 5 mm dC and 5 mm ADP. After 1 week the cover slip with the hanging drop was shifted to 30% poly(ethylene glycol) 3350. The crystals were cryo-pro- tected by a quick wash through a mixture of 80% crystalli- zation solution, 10% ethylene glycol and 10% glycerol. Full-length dNK+dTTP Hanging drops consisted of 2 lL of crystallization solution containing 0.1 m Tris, pH 7.5, 0.2 m potassium citrate, 12% polypropylene glycol P400 and 20% poly(ethylene gly- col) 3350 added to 2 lL of enzyme solution containing 10 mgÆmL )1 of protein and 5 mm dTTP. The crystals were flash frozen without further additions. Data collection X-ray diffraction data were collected at 100 K at various beamlines at ESRF Grenoble (Table 2). The data were scaled and merged using the programs mosflm [30] and scala [31]. Data collection statistics are shown in Table 2. Structure determination and refinement Structures with the same space group and similar cell dimensions as previous complexes could often readily be determined directly by a few rounds of rigid body refine- ment. If this did not succeed, the structures were solved by molecular replacement using the program phaser [32]. The refined structure of the previously determined dNK–dC dimer was used as a search model. After rigid-body and restrained refinement in refmac5 [33], an initial electron map was calculated. From this map most of the polypep- tide chains could be built using the programs o [34] and coot [35]. Acknowledgements This work was supported by grants from the Swedish Research Council (to H.E.), the Swedish Cancer Foun- dation (to H.E.) and the Danish Research council (to B.M.P) and the Novo Nordic Research Council (to B.M.P.). References 1 Thelander L & Reichard P (1979) Reduction of ribonu- cleotides. 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Structural studies of nucleoside analog and feedback inhibitor binding to Drosophila melanogaster multisubstrate deoxyribonucleoside kinase Nils. substrate and feedback inhibitors. dCTP and dGTP bind with the base in the substrate site, similarly to the binding of the feedback inhibitor dTTP. All nucleoside