dCTP and dGTP bind with the base in the substrate site, similarly to the binding of the feedback inhibitor dTTP.. They further suggested that dCTP could function as a bisubstrate analog
Trang 1inhibitor binding to Drosophila melanogaster
multisubstrate deoxyribonucleoside kinase
Nils E Mikkelsen1, Birgitte Munch-Petersen2and 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
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 informafunc-tion 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.
Trang 2kinase-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 kcat 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.
Km
(l M )
Vmax (lmolÆmin)1Æmg)1)
kcat (s)1)
kcat⁄ K m
(l M )1Æs)1)
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.
Trang 3Results 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 Km 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
kcat⁄ Km of 2· 107m)1Æs)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.
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)
Trang 4expelling 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 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
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 Km for ddC is almost 500-fold higher than for
dC, whereas the kcat 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.
Trang 5affected 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 kcat⁄ Km that is about 2800-fold lower than the
kcat⁄ Kmfor 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 N3azido 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
According to a kcatfor 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
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.
Trang 6speculated 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.
Trang 7detectable 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 phosphos-phate 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 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.
Trang 8that 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 Kmvalues 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 14C 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 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 containing 26% mPEG2000
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
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
5FdU 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
Trang 9solution 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 K2SO4, 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)1of 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.)
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