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

Structural studies of nucleoside analog and feedback inhibitor binding to Drosophila melanogaster multisubstrate deoxyribonucleoside kinase Nils E. Mikkelsen1, Birgitte Munch-Petersen2 and Hans Eklund1

1 Department of Molecular Biology, Swedish University of Agricultural Sciences, Biomedical Center, Uppsala, Sweden 2 Department of Science, Systems and Models, Roskilde University, Denmark

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.

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]. 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.

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.

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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 In addition, many pharmacological nucleoside ana- logs (NAs) that are used in both antiviral therapy and cancer therapy need activation by deoxynucleoside

N. E. Mikkelsen et al.

Nucleoside analog deoxynucleoside kinase complexes

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).

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 important the deoxynucleoside kinases and makes 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.

α3

is two- 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 kcat to threefold higher, it is even faster [15].

LID

α7

α8

α5

P-loop

α2

α6

α4

β5

β1

ERS

β4

β3

α1

Table 1. Kinetic parameters for dNK with natural substrates and NA from the crystal structures.

β2

kcat (s)1)

Vmax (lmolÆmin)1Æmg)1)

kcat ⁄ Km (lM

)1Æs)1)

Km (lM)

12

1.2 2.3

225 665

7.2 0.092 0.029

14

29.5 34.2 42.7 31.3 29.8 13.2

1.0 2.2 8.3

1124

0.073 8.6

14.2 16.5 20.6 19 14.2 5.9 0.036 4.2

2.7 0.0043 0.0037

dTa dCa dAa dGa 5-FdU BVDUb AZTc ddCc

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.

a Data are from [15]. b Data are from [16]. c Data are from [6].

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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.

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Nucleoside analog deoxynucleoside kinase complexes

Results and Discussion

zalcitabine [BVDU, Quality of the structures

uridine), zidovudine (AZT, 3¢-azido-2¢,3¢-dideoxythymi- (ddC, 2¢,3¢-dideoxycytidine) and dine), brivudin (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 Km reflects the nucleoside binding affinity. All refinement statistics can be found in Table 2.

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).

in turn,

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, inhibits DNA synthesis [17]. 5FdU is phosphorylated efficiently by dNK with the same high kcat ⁄ Km of 2 · 107 m)1Æs)1 as with thymidine, and 10-fold higher than with TK1 [14].

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

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 Cell dimensions

P21 70.5 70.8 226.0 4 dimers 50–2.9 97.3 (97.1)

P21 70.4 70.7 225.4 4 dimers 30–3.0 99.3 (99.3)

P21212 140.0 111.9 71.1 2 dimers 20–2.8 99.4 (99.8)

P21212 137.5 112.8 69.7 2 dimers 30–2.9 83.9 (87.4)

P21212 119 64.9 69.1 1 dimer 45–2.2 99.6 (99.6)

P21 67.9 119 70.5 2 dimers 50–2.2 99.5 (99.5)

P21212 119.7 65.1 69.2 1 dimer 50–2.5 99.8 (99.7)

P21212 120.6 62.5 68.2 1 dimer 50–2.3 98.5 (91.2) 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) 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) 13.1 (2.1) 3.4 (2.7) 22045 23.4 27.3

11.3 (2.0) 2.9 (3.0) 46314 25.6 28.1 0.013 1.326 63.1 ID23-1 2vp6

13.0 (2.1) 3.7 (3.8) 50991 24.8 28.7 0.015 1.471 45.6 ID14-2 2vp9

9.7 (2.3) 2.8 (2.7) 19428 24.2 28.6 0.013 1.398 54.6 ID14-1 2vqs

9.3 (3.1) 3.6 (3.6) 26596 23.5 27.1 0.012 1.836 39.7 ID-14-1 2jj8

11.3 (3.1) 2.8 (2.9) 53215 19.5 24.9 0.012 1.403 31.8 ID-29 2vp4

17.4 (4.0) 7.1 (7.3) 18113 20.7 26.7 0.011 1.421 34.4 ID-29 2vp2

16 (3.2) 3.4 (3.5) 26365 21.4 25.9 0.010 1.183 36.1 ID-29 2vp0

Content au Resolution (A˚ ) Completness (%) Rsym Rmeas Mn(I) ⁄ sd Redundancy Reflections R factor (%) Rfree (%) rmsd bond lengths 0.009 rmsd bond angles 1.151 Mean B value (A˚ 2) 39.2 Beamline PDB-code

ID14-4 2vp5

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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, We determined the structures of dNK with four pyrimidine NAs: floxuridine (5FdU, 5-fluoro-2¢-deoxy-

N. E. Mikkelsen et al.

Nucleoside analog deoxynucleoside kinase complexes

A

Y70

A

Y70

M69

M69

E172

M118

R169

M118

R167

E172

Q81

Q81

A110

A110

M88

T34

K33

M88

E52

E52

R105

R105

B

Y70

E172

M69

R169

B

Y70

M118

M69

R167

M118

Q81

E172

Q81

A110

A110

T34

M88

K33

E52

R105

M88

E52

R105

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.

C

Y70

M69

M118

Q81

expelling the two water molecules in a manner similar to that previously reported for dT and its methyl group [13].

A110

E52

S106

R105

M88

5FdU is phosphorylated efficiently by dNK with the same Km and kcat values as with thymidine (Table 1). This is in agreement with the high similarity observed between the crystal structures obtained with dT and 5FdU.

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.

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.

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The Km for ddC is almost 500-fold higher than for is decreased only by 3.3-fold. the catalytic step should be expected to be dC, whereas the kcat Thus,

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Nucleoside analog deoxynucleoside kinase complexes

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. was no density for the N3 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 N3 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 N3 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.

and Spodoptera frugiperda embryonic 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 cells showed high sensitivity towards BVDU.

According to a kcat for AZT that is more than 400- fold lower than with thymidine, and a Km that is increased by eightfold, the catalytic step should be effected considerably more than the binding. This is in agreement with the N3 group being somewhat of a hindrance for proper binding but the LID being completely distorted, making P transfer very difficult.

complex was (dTMP) In yeast thymidylate kinase a similar shift in the P loop was observed when the deoxythymidine mono- phosphate compared with the AZT-monophosphate (AZTMP) complex. It was

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 and the [19] VZV + BVDUMP and ADP complex [20].

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.

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 kcat ⁄ Km that is about 2800-fold lower than the kcat ⁄ Km for dT (Table 1).

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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 located substrate complexes, and the other between the first sulfate ion and the substrate. There

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Nucleoside analog deoxynucleoside kinase complexes

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.

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.

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

E172

Y70

R169

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.

A

M69

R167

M118

Q81

A110

T34

K33

R105

R169

Deoxynucleoside triphosphate complex structures

B

E172

Y70

R167

M69

M118

Q81

T34

A110

K33

R105

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.

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).

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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-

N. E. Mikkelsen et al.

Nucleoside analog deoxynucleoside kinase complexes

E52

Mg

dGTP/dCTP/dTTP

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.

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 AP4dT and AP5dT 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

same

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.

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 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

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.

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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 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

N. E. Mikkelsen et al.

Nucleoside analog deoxynucleoside kinase complexes

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.

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. 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.

larger to the

AZT

containing

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 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 Km values with these substrates.

Experimental procedures

Hanging drops consisted of 2 lL of crystallization solution containing 0.1 m MES, pH 6.5, 0.2 m Li2SO4 and 26% polyethylene glycol 2000 monomethylether added to 2L of enzyme solution containing 30 mgÆmL)1 of protein and 5 mm AZT. The crystals were cryo-protected by a quick wash through crystallization liquid 26% mPEG2000.

Nucleosides and nucleotides were from Sigma (St Louis, MO, USA).

Materials ddC

Hanging drops consisted of 2 lL of crystallization solution containing 0.1 m MES, pH 6.5, 0.2 m Li2SO4 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.

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].

BVDU

Hanging drops consisted of 2 lL of crystallization solution containing 0.1 m MES, pH 6.5, and 2.5 m Am2SO4 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.

Crystallization

Hanging drops consisted of 2 lL of crystallization solution containing 0.1 m MES, pH 6.5, 0.2 m Li2SO4 and 22% mPEG2000 M added to 2 lL of enzyme solution contain- ing 10 mgÆmL)1 of protein and 5 mm 5FdU. The well

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 (cid:2)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.

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5FdU

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Nucleoside analog deoxynucleoside kinase complexes

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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].

Structure determination and refinement

Acknowledgements

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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.).

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