From the Departments of Chemistry and Biochemistry
and § Basic Pharmaceutical Sciences, University of South
Carolina, Columbia, South Carolina 92908 and the ¶ Department of
Molecular Physiology and Biological Physics, University of Virginia,
Charlottesville, Virginia 22901
Received for publication, October 18, 2000, and in revised form, January 11, 2000
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ABSTRACT |
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Thymidylate synthase (TS) is a major target in
the chemotherapy of colorectal cancer and some other neoplasms. The
emergence of resistance to the treatment is often related to the
increased levels of TS in cancer cells, which have been linked to the
elimination of TS binding to its own mRNA upon drug binding, a
feedback regulatory mechanism, and/or to the increased stability to
intracellular degradation of TS·drug complexes
(versus unliganded TS). The active site loop of human TS
(hTS) has a unique conformation resulted from a rotation by 180°
relative to its orientation in bacterial TSs. In this conformation, the
enzyme must be inactive, because the catalytic cysteine is no longer
positioned in the ligand-binding pocket. The ordered solvent structure
obtained from high resolution crystallographic data (2.0 Å) suggests
that the inactive loop conformation promotes mRNA binding and
intracellular degradation of the enzyme. This hypothesis is supported
by fluorescence studies, which indicate that in solution both active
and inactive forms of hTS are present. The binding of phosphate ion
shifts the equilibrium toward the inactive conformation; subsequent
dUMP binding reverses the equilibrium toward the active form. Thus, TS
inhibition via stabilization of the inactive conformation should lead
to less resistance than is observed with presently used drugs, which
are analogs of its substrates, dUMP and
CH2H4folate, and bind in the active site,
promoting the active conformation. The presence of an extension at the
N terminus of native hTS has no significant effect on kinetic
properties or crystal structure.
Thymidylate synthase
(TS)1 catalyzes the reductive
methylation of 2'-deoxyuridine 5'-monophosphate (dUMP) to thymidine
5'-monophosphate (dTMP), using the co-substrate,
5,10-methylenetetrahydrofolate (CH2H4folate) as
a 1-carbon donor and reductant. The physical structures of bacterial
TSs have been relatively well defined, and crystallographic data, in
concert with data derived from kinetic, spectroscopic, and
site-directed mutagenesis studies, have led to a detailed understanding
of the catalytic mechanism of these enzymes (1). In contrast,
relatively few investigations of mammalian TS structure and catalysis
have been conducted. The three-dimensional structure of the native
human TS (hTS) has been reported previously (2). The data showed a
surprising feature not observed in TSs from other sources: loop
181-197 containing the catalytic cysteine, Cys-195, was in an
inactive conformation, rotated ~180° with respect to its
orientation in bacterial TSs, with the sulfhydryl of Cys-195 over 10 Å from the location of sulfhydryls of corresponding cysteine residues in
bacterial enzymes. Subsequent determination of the structure of a
ternary inhibitory complex between closely related ratTS (rTS) and dUMP
and Tomudex (3) has shown that the ligands bind to the enzyme in the
active conformation. Recently, it was found that also in the
hTS·dUMP·Tomudex complex hTS is in the active conformation (4). The
inactive conformation has not been observed in TSs other than human.
TS has been a primary target for chemotherapy aimed at cancers of the
gastrointestinal tract and head and neck (5) despite moderate response
in 30-40% of patients. A major problem affecting TS-directed
treatments is that tumor cells often react to an exposure to TS
inhibitors by raising levels of intracellular TS activity about 2- to
4-fold, which may lead to resistance. The levels of TS mRNA do not
change significantly; the increase is predominantly at the protein
level. Two effects have been found that can explain the observed
increased TS levels. TS was observed to bind to its own mRNA, which
is proposed to act as a feedback inhibition mechanism regulating TS
levels (6). When TS is bound by its physiological substrates, dUMP
and/or CH2H4folate, or inhibitors, FdUMP or
antifolates, it is unable to interact with its own mRNA. The
end-result of this disruption in RNA binding is relief of translational
repression with resultant synthesis of new TS. Other researchers (7), using different cell lines, did not observe changes in the extent of
ribosome binding to TS mRNA in the presence of TS inhibitors but
have shown in an elegant way that ligand-mediated induction of TS
occurs by lowering protein turnover.
The primary structure of hTS differs from that of bacterial TSs in
three regions: the N terminus of hTS is extended (by 29 residues with
respect to the most studied Escherichia coli TS, ecTS) and
two insertions of 12 and 8 residues are present at positions 117 and
146, respectively, in the human protein (1). Recent studies of hTS
isolated from bacteria as a recombinant protein and TS isolated from
human cells suggested that catalytic and some RNA binding activities of
these enzymes are dependent upon the N terminus (8). The sole
difference between the proteins was the presence of a blocked N
terminus, probably acetylation, in the enzyme isolated from human
cells. In addition, a recombinant hTS in which the N terminus was
blocked with a polyhistidine peptide exhibited catalytic and RNA
binding activity similar to that of the native, blocked protein (8).
These data suggested that the N terminus influences enzyme function.
Interestingly, the recombinant human enzyme that was utilized as the
source for the originally studied crystals was heterogeneous; ~80%
of the protein was a fusion protein containing an additional 13 amino
acids at the N terminus (see Fig. 1) that are not present in human TSs (2). The original structure determination of the native hTS was at
medium (3.0 Å) resolution, and the solvent structure could not have
been analyzed. We report here the first determination of high
resolution, 2.0 Å, structures of recombinant unblocked hTS and
NTE-hTS, both with well-defined solvent structures, and kinetic and
ligand binding studies for NTE-hTS. Analysis of the solvent structure
suggested that phosphate ions stabilize the inactive conformation. We
have conducted fluorescence studies that support this hypothesis.
Materials--
Nucleotides, salts,
isopropyl- Expression and Purification of Human Thymidylate
Synthase--
Recombinant hTS was expressed and purified as described
previously (10). The plasmid pCD1 used to express N-terminally extended hTS (NTE-hTS) with an additional 42 amino acids at the N terminus (Fig.
1) was created by subcloning the human
thyA gene from the plasmid pDHTS-1 (11) into the expression
vector pProEX-1 (Life Technologies, Inc., Gaithersburg, MD). The
bacterial strain X2913, which has a deletion of the Escherichia
coli thyA gene, was transformed with plasmid pCD1 using the
CaCl2/heat shock method (12). Transformants were grown in
Luria Bertani (LB) medium containing 100 µg/ml ampicillin. At
A600 of 1.0, isopropyl
Steady-state Kinetic Measurements--
Enzyme activity was
measured spectrophotometrically by monitoring the absorbance change
accompanying the conversion of CH2H4folate to
H2folate (13) using either a Shimadzu UV 1601 spectrophotometer equipped with a TCC 240A temperature-controlled cell
holder or a Shimadzu UV 2101 PC spectrophotometer (Shimadzu Corp.,
Columbia, MD). Measurements were carried out at 25 °C and pH 7.4 in
Morrison buffer (14). One unit of enzyme activity is defined as the
amount of enzyme required to synthesize 1 µmol of dTMP per minute.
Enzyme concentration was determined by measurement of absorbance at 280 nm, as described previously (15).
Transient-state Kinetic Measurements--
Kinetic data
describing the binding of ligands with hTS were obtained using a
stopped-flow fluorimeter/spectrometer (Hi-Tech SX.18MV, Applied
Photophysics, UK) as described previously (10, 16). Kinetic and
thermodynamic constants for nucleotide binding to NTE-hTS were
determined by titrating the enzyme with various concentrations of dUMP,
FdUMP, or dTMP. Data were collected over 20-ms or 50-ms time periods.
Final concentrations of dUMP, FdUMP, and dTMP were 5-200, 10-250, and
5-250 µM, respectively. Transient-state kinetic
constants for CH2H4folate binding to the binary
complex of NTE-hTS and dUMP were measured by titration of the
binary complex with various concentrations of
CH2H4folate. Data were collected over a 1-s
time period. The concentration range of
CH2H4folate was 5-250 µM. The
final concentration of dUMP was 0.50 mM.
Crystallization of Human Thymidylate Synthase--
Crystals of
unblocked hTS and NTE-hTS were grown by the vapor diffusion method in
the hanging-drop setup at conditions similar to those reported
previously (2). Typically, 5 µl of enzyme solution (18 mg/ml)
containing 1 mM EDTA, 10 mM 2-ME, 0.1-1
M potassium chloride, 100 mM imidazole
(NTE-hTS), 50 mM Tris-HCl (pH 7.5-8.5) was mixed with an
equal volume of precipitant solution containing 38-50% saturated
ammonium sulfate, 40 mM potassium phosphate, 20 mM 2-ME, and 100 mM Tris-HCl (pH 7.8), and
allowed to equilibrate with 0.6 ml of precipitant solution present in the well. The culture plates were stored undisturbed at room
temperature, and crystals appeared after 3 weeks and grew to the size
of 0.1-0.7 mm within 1 week.
X-ray Diffraction Data Measurement and Processing--
Data were
collected on a single crystal of wild type hTS and NTE-hTS with
approximate dimensions of 0.45 × 0.50 × 0.50 and 0.15 × 0.15 × 0.15 mm, respectively. The crystals were transferred to
a cryoprotectant solution containing 45% saturated ammonium sulfate,
30% concentrated ethylene glycol, and 100 mM Tris-HCl (pH
7.8), and flash-cooled in N2 vapors at Fluorescence Studies--
Evidence for the existence of the
inactive loop conformation in solution was obtained utilizing a
spectrofluorometer (Model 8100, SLM-Aminco Inc., Urbana, IL). The
excitation wavelength was set at 295 nm, and the emission was scanned
from 305 to 450 nm at a rate of 5 nm/s. An Ultra-Vu polystyrene cuvette
(Baxter Diagnostics Inc., Deerfield, IL) was used to hold a 3-ml sample containing 4 µM protein in 50 mM Tris-base
(pH 7.4). The purity of the protein (recombinant wild type TS) was
better than 95%. The titration was carried out by addition of
potassium phosphate (pH 7.4) or dUMP in increments up to 10% of the
sample volume. No fluorescence signal was observed from the buffer
(Tris-HCl), phosphate, or dUMP in the experiments. All measurements
were done in triplicate to eliminate errors introduced by
non-homogeneity of the sample and instrumental drift.
Determination of Steady-state Kinetic Constants for NTE-hTS and
hTS--
The specific activities, determined at 25 °C, for NTE-hTS
and hTS were 1.1 and 1.2 units/mg, respectively, whereas catalytic rates (kcat) were 1.5 and 1.6 s Determination of Kinetic and Thermodynamic Constants for Nucleotide
Binding to NTE-hTS--
The time courses of fluorescence quenching
upon nucleotide binding were analyzed using single-exponential curve
fits (23). Kinetic and thermodynamic constants for nucleotide binding
were determined as previously described (16, 23). A linear relationship was observed between kobs and nucleotide
concentration. The kinetic and thermodynamic constants for nucleotide
binding to NTE-hTS are listed in Table I.
The kinetic and thermodynamic constants describing nucleotide binding
to NTE-hTS are nearly identical to those determined for hTS by
transient-state kinetic analysis (16).
Determination of Kinetic and Thermodynamic Constants for
CH2H4folate Binding to the Binary Complex of
NTE-hTS·dUMP--
Fluorescence quenching due to
CH2H4folate binding to the binary complex of
NTE-hTS·dUMP was analyzed using a single-exponential curve with a
steady-state parameter to account for product formation as previously
described (23). Mixing NTE-hTS with substrates resulted in a
fluorescence burst (kburst) at 340 nm. The
dependence of kburst on
CH2H4folate concentration was hyperbolic as
previously observed for hTS (13), and the kinetic and thermodynamic
constants for CH2H4folate binding to
NTE-hTS·dUMP were determined using the expression:
kburst = kiso[L]/(L + Kd) + kr, iso + kchem (23). The kinetic and thermodynamic
constants determined for NTE-hTS are shown in Table
II and are similar to those determined for hTS by transient-state kinetic analysis (16).
Crystallization--
The typical high salt conditions used by
Schiffer et al. (2) and in the present report exclusively
support the growth of hTS crystals in which the active-site loop adopts
the inactive conformation. We have investigated other crystallization
conditions, especially those with low ionic strength, and obtained
several systems that were able to grow hTS crystals. A semi-factorial approach was employed to screen a wide variety of conditions arranged into matrices containing polyethylene glycols, different buffers, and a
multitude of salts at varying concentrations (data not shown). At 38%
(weight/volume) PEG 4000, 100 mM Tris-HCl (pH 7.6), 20 mM 2-ME, and 2% saturated ammonium sulfate, thin and
irregularly shaped crystals were observed after 3 weeks measuring
~0.1 × 0.2 × 0.2 mm. Despite their fair size, these
crystals did not scatter x-rays at all. However, we succeeded in
growing trigonal crystals, similar to those reported here and by
Schiffer et al. (2) with the PEG/ammonium sulfate biphasic
system (24). The optimum conditions were at 15-30% (weight/volume)
PEG 4000, 42-50% saturated ammonium sulfate, and 100 mM
Tris-HCl (pH 8.5). The results were similar for hTS and NTE-hTS.
Despite the extra 42 residues at the N terminus, the crystals of hTS
and NTE-hTS are isomorphous and isomorphous to those reported by
Schiffer et al. (2). The crystals belong to the space group
P3121 with one subunit in the asymmetric part of
the unit cell. The parameters and statistics of crystallographic
refinements are summarized in Table
III.
Folding of hTS and NTE-hTS--
The protein model reported
by the Stroud group (2) was, in general, excellent; however, stretches
of additional residues were fit and some side chains were rebuilt to
fit to the more resolved density. The hTS data, which were refined
first, having a mosaicity of 0.22 and an overall
Rmerge of 0.033, yielded excellent electron
density maps. The structural differences between hTS and NTE-hTS in
unliganded form appear to be insignificant and limited to occasional
shifts in the solvent structure. The root-mean-square deviations of the
positions of Ca between hTS and NTE-hTS, was 0.16 Å. A
significantly smaller number of structured water molecules observed in
the NTE-hTS structure is likely to be related to the smaller crystal
used and resulting quality of the data. The discussion below refers to
the wild type enzyme but is valid as well for the NTE-hTS.
The N terminus is largely disordered; good density starts at Pro-26,
and this region does not change between hTS, NTE-hTS, and an inhibitory
ternary complex of hTS with dUMP and Tomudex (ZD1694 or
raltitrexed) (4) or analogous rat TS complex (PDB entry 2tsr
(3)). A least-squares superposition of the C
The active site loop 181-197 is in the inactive conformation that was
reported in previous investigations (2). The loop forms only limited
intermolecular interactions: hydrophobic contacts between the side
chain of Leu-189 and Pro-305 and a hydrogen bond between Pro-188 O and
Arg-274 NH1. A major intramolecular interaction stabilizing the
inactive conformation is hydrogen bonding between the guanidinium group
of Arg-163' from the other subunit and peptide carbonyls of Ala-191 and
Leu-192. These interactions must stabilize the inactive conformation to
the extent that in this region we do not observe any disorder in
electron density maps (Fig. 2, bottom) nor high temperature
factors, which would be indicative of a significant population of the
active conformer. In the inactive conformation, the catalytic Cys-195
is directed toward the dimer interface and, together with Cys-180 and
symmetry equivalent Cys-195' and Cys-180' from the other subunit of the
dimer, forms a cluster of four thiols, with S-S distances of 5-7 Å.
The thiols do not form disulfide bonds between themselves and are
accessible through a short channel. Indeed, they react with exogenous
thiols. During the investigation, it became apparent that the enzyme is
prone to oxidation despite the presence of 20 mM 2-ME in
the crystallization medium. That oxidation is occurring was suggested
by the observation that some cysteine residues in the crystals were
modified to form S,S-(2-hydroxyethyl)thiocysteine, presumably by
reacting with 2-hydroxyethyl disulfide, the product of oxidation of
mercaptoethanol (2-ME). Although disorder may contribute to the spread
of electron density, leading to difficulty in assessment of cysteine
derivatization, density maps were analyzed at very low contouring
levels to lower the probability of misinterpretation.
Solvent Structure and Ligand Binding--
The high resolution and
low temperature of the data collection resulted in well-resolved
solvent structure. The positions of 211 water molecules, 4 phosphate/sulfate ions, and 1 glycol molecule were identified per
asymmetric part of the unit cell, or one hTS subunit. The ligand, which
we tentatively identified as a glycol molecule originating from the
cryosolvent, is located together with a water molecule in a large
cavity (Fig. 3, top). This
cavity is not present in ecTS or lcTS and is created by the replacement
of ecTS Met-34 and Trp-201 with hTS Ala-63 and His-250, respectively.
Alanine in this position is unique for mammalian TSs; other TSs, with
the exception of the viral enzymes, have a methionine or a large
hydrophobic residue in this position. The cavity is lined mostly with
hydrophobic residues, although some hydrogen-bonding functions are
present. The cavity is elongated and connects to the molecular surface
on one side, the other end being blocked by the side chain of Gln-36,
preventing the cavity from becoming a channel (Fig. 3,
bottom). The occurrence of such cavity must destabilize the
folded state, so it is likely that it has some function compensating
for the reduced stability. It appears that its presence is linked to
the inactive conformation; in the ternary inhibitory complex
hTS·dUMP·Tomudex the cavity does not exist. It may be speculated
that the cavity facilitates loop 181-197 flipping. The glycol binding
energy must stabilize the inactive conformation and thus it was
possible that glycol might be a noncompetitive inhibitor. However,
measurements of catalytic activity of hTS in the presence and absence
of up to 200 mM glycol did not show significant
differences. The cryoprotectant, from which the bound glycol
originated, was about 5 M and moreover synergism between
phosphate and glycol binding is likely.
There are four tetrahedrally shaped peaks in the ordered solvent region
with environment suitable for bound phosphate/sulfate ions. The mother
liquor of the crystal used to collect data was 40 mM in
phosphate and ~2.0 M in sulfate. Because sulfate ions often occupy phosphate-binding sites in crystals obtained with ammonium
sulfate due to the similarity of their shape and charge, it is likely
that the bound ions are some mixture of the two species. Three of them
are located within or close to each other and in vicinity of the active
site (Fig. 4). It is not unlikely that the bound phosphate/sulfate ions map the contacts between the enzyme
molecule and the phosphate moieties of a nucleic acid. Indeed, in the
structure of a complex between mRNA and a sequence-nonspecific mRNA binding protein, all protein·mRNA interactions are
through the sugar phosphate backbone and most of them through
arginine-phosphate ion pairs (26). In the structure of a complex
between the Sex-lethal protein isolated from Drosophila
melanogaster and 12-nucleotide, single-stranded RNA, which is an
example of mRNA sequence-specific binding, many arginine-phosphate
ion pairs are observed (27). In hTS, one distance between two
phosphate/sulfate sites, 6.5 Å, is the same as phosphate-phosphate
distances in single-stranded RNA helices (26). The other distance is
10.0 Å and may correspond to a second neighbor phosphate-phosphate.
Indeed, in the Sex-lethal protein·RNA complex, several second
neighboring phosphates are separated by distances of ~10.4 Å (27).
Fluorescence Measurements--
The observed involvement of
the residues of loop 181-193 in phosphate ion binding sites suggested
that phosphate concentration might affect the loop conformation. The
loop has a tryptophan at position 182 and our modeling, based on the
structure of the hTS·dUMP·Tomudex complex, showed that the position
of the indole moiety differs between the active and inactive
conformations by about 5 Å. It appeared likely that even with five
tryptophans per subunit such change would be reflected in the enzyme
fluorescence. This hypothesis turned out to be true, because there
is a strong phosphate ion concentration-dependent increase
in the intensity of hTS fluorescence (Fig.
5, top). Subsequent dUMP
binding effectively reverses this signal enhancement as fluorescence
falls below the intensity level observed for the unliganded hTS (Fig.
5, center). This indicates that even without phosphate ions
present a significant fraction of the enzyme is in the inactive
conformation and eliminates the possibility that the inactive
conformation is an "artifact of the crystal field." Such effects
were not observed in ecTS (Fig. 5, bottom) for which the
inactive conformation has not been observed.
Previous studies indicated that modifications at the N terminus of
hTS alter catalytic activity and RNA binding activity, presumably
through effects on the enzyme conformation (8). In the present
investigation, the reaction mechanisms and crystal structures of
recombinant hTS with an unmodified N terminus and hTS with an extended
N terminus have been determined. Our data indicate that the presence of
an additional 42 amino acids at the N terminus of hTS exerts a minimal
effect on ligand binding and on the kinetically measurable steps in the
reaction pathway. These results are supported by crystallographic data,
which indicate no significant differences in the arrangement of atoms
that were ordered in crystals. It appears that the modification of the
N terminus induces only local differences that do not propagate to the
rest of the molecule. The poor conservation of the amino acid sequence
at the N terminus strongly suggests that the physiological function of
the N-terminal extension, if such indeed exists, is strongly
species-specific and thus is unlikely to be related to catalysis, in
agreement with our experimental data.
Several new structural features of hTS have been deduced from the high
resolution model reported here. Of interest is the possibility that
they may represent sites for interactions of TS with macromolecules.
Evidence that TS is involved in protein·protein interactions was
derived from studies indicating that it is a component of a multienzyme
DNA-biosynthesizing complex termed the replitase (29). The
identification of two cavities specific for mammalian TSs suggests that
they are related to interaction between hTS and other macromolecules
and opens a new field for studies of this amazingly complex enzyme.
A large body of evidence indicates that hTS interacts with ribonucleic
acids (6, 30-32). At least 11 ribonucleoprotein complexes involving
hTS have been identified in vivo, including complexes with
TS mRNA and mRNA encoding the tumor suppressor gene product, p53 (30-32). No consensus sequences or structures have been identified among the RNAs interacting with hTS. The binding of hTS to TS mRNA
and to p53 mRNA inhibits translation in vitro (6, 32). That translational regulation by TS occurs in vivo is
supported by recent studies of interactions between TS and p53 mRNA
(33). Binding motifs of 36 and 70 nucleotides have been identified for the interaction between TS and TS mRNA (30, 34); however, the
site(s) of interaction on the human protein have not been identified.
The crystal structures analyzed in the present investigation provide
the first suggestion of the location of a region in which multiple
phosphate substituents are predicted to bind whereas fluorescence
studies correlate it with the inactive conformation of loop 181-197.
It is possible that the inactive conformation makes hTS less vulnerable
to an oxidative stress or other factors that derivatize thiols,
however, it appears more probable that the inactive conformation
facilitates the binding of nucleic acids, because Arg-185 of the loop
binds one of the closely positioned phosphate/sulfate ions.
The existence of the inactive conformation offers a novel mechanism for
inhibition of hTS. Its stabilization should be an effective manner of
inhibiting hTS and providing an alternative to active site-directed
inhibitors that are currently used in cancer therapy. Inhibitors
stabilizing the inactive conformation and binding away from the active
site may be of significant advantage, because they would be expected
not to interfere with mRNA binding to hTS. Active site-directed
inhibitors, such as FdUMP, inhibit the binding of hTS to TS mRNA
in vitro, reducing the repression by TS of TS mRNA
translation (6). In addition, exposure of tumor cells to active
site-directed inhibitors results in a significant increase in
steady-state levels of TS protein (35-37). Two mechanisms underlying the elevation in protein have been documented in different cell lines. The first emphasizes translational derepression by TS
binding to TS mRNA, the second the increased stabilization of the
protein upon ligand binding (6, 7). Our structural data strongly
support both mechanisms. The putative mRNA binding site involves
the inactive conformation of loop 181-197 and overlaps the active
site. Thus, the formation of inhibitory complexes must eliminate
mRNA binding. The ternary inhibitory complex has a much more
compact structure than the native TS (4). Disordered loop 107-128 is
likely to be more prone to degradation than the same residues in the
compact conformation observed in the inhibitory complex.
Therapeutic approaches aimed at the stabilization of the inactive
conformer should not activate those mechanisms that lead to TS
accumulation and may improve the outcome of cancer chemotherapy in
which TS is the target. Such approaches may utilize more powerful inhibitors binding in the glycol cavity or cross-linking of the cluster
of four thiols from Cys-195, Cys-180, and their equivalents from the
other subunit.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-thiogalactopyranoside,
2-mercaptoethanol (2-ME), phenylmethylsulfonyl fluoride, ethanediol, polyethylene glycols (PEGs), and folic acid were obtained from Sigma
Chemical Co. (St. Louis, MO). Ultra-pure ammonium sulfate was from ICN
Biomedicals, Inc. (Aurora, OH). Restriction enzymes and T4 DNA ligase
were from New England BioLabs (Beverly, MA). (6S)-5,6,7,8-Tetrahydrofolic acid (H4folate) was
prepared from folic acid and converted to
(6R)-CH2H4folate as described
previously (9).
-D-thiogalactopyranoside was added to a final
concentration of 0.6 mM. The culture was incubated for
4 h, cells were harvested by centrifugation at 1000 × g at 4 °C, and frozen at
70 °C. Cell pellets (2-3
g) were thawed on ice and suspended in buffer X (20 mM
Tris, 100 mM KCl, 20 mM imidazole, and 14 mM 2-ME at pH 8.5 and 4 °C) containing 0.1 mM phenylmethylsulfonyl fluoride. Cells were lysed at
4 °C by sonication using a Branson Sonifier 450 (Branson
Ultrasonics, Danbury, CT). Cell debris was removed by centrifugation at
18,000 × g at 4 °C for 30 min. NTE-hTS was purified
from the cell-free extract at 4 °C using a nickel-nitrilotriacetic acid column (Qiagen, Chatsworth, CA). The cell
extract was loaded onto the column and washed with buffer X. The column
was then washed with buffer Y (20 mM Tris, 1 M
KCl, and 14 mM 2-ME, pH 8.5) and NTE-hTS was eluted with
buffer Z (20 mM Tris, 100 mM KCl, 100 mM imidazole, and 14 mM 2-ME, pH 8.5).
Fractions containing NTE-hTS were pooled and dialyzed overnight against
buffer A (10). Purified NTE-hTS was analyzed by 12% SDS-polyacrylamide
gel electrophoresis for purity and stored in buffer A containing 15%
glycerol at
20 °C.
View larger version (11K):
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Fig. 1.
Sequences of amino acid extensions at the N
terminus of hTSs.
178 °C.
Crystallographic diffraction experiments were carried out at the SBC
insertion device beamline of the advanced photon source at Argonne
National Laboratory using x-rays of 0.9793-Å wavelength. The data for
unblocked hTS and NTE-hTS were collected at a crystal to detector
distance of 225 and 180 mm, respectively, and processed with the HKL
2000 suit of programs (17). At the initial stage of our analysis the
coordinates of hTS were not available; therefore, a structure of ecTS
(Protein Data Bank code 1qqq (18)) with appropriate modifications was
used as the search model in the molecular replacement method carried
out with the CNS software (19). The final structure of unblocked hTS
replaced ecTS as the initial model for NTE-hTS. The data were refined
and optimized with the CNS software (19) using simulated annealing with
torsional dynamics and positional and temperature refinements. Electron
density maps calculated with 2Fo
Fc, and Fo
Fc coefficients were utilized to introduce manual
corrections to the model with the interactive graphics program CHAIN
(20). Ribbon diagrams and wire-and-basket models were prepared with the
programs MOLSCRIPT (21) and CHAIN (20), respectively.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.
These values were very similar to previously published values of 1.2 units/mg for the specific activity and 1.5 s
1 for
kcat of hTS at 25 °C (10, 22).
Comparison of kinetic and thermodynamic constants for nucleotide
binding to NTE-hTS and hTS
Comparison of kinetic and thermodynamic constants for
CH2H4folate binding to NTE-hTS and hTS in the presence
of dUMP
Crystallographic and refinement statistics
plots of
hTS and the inhibitory complex is shown in Fig.
2. Apparently, the extra N-terminal
residues of NTE-hTS occupy intermolecular space in multiple positions
without affecting crystal packing. The main chain follows closely the
fold of ecTS starting from Glu-30, which is equivalent to Met-1 of
ecTS. As previously observed in a Lactobacillus casei TS
(lcTS) structure (25), the carboxylate moiety of the
N-carbamoylated ecTS occupies the same position as the
carboxylate of a glutamic acid (Glu-30 in hTS), apparently forming a
structural lock that isolates the rest of the TS molecule from the
presence and/or conformational state of the N terminus. Indeed, a
glutamic acid at this relative position is conserved in all TSs that
contain additional N-terminal sequences with one exception, which
contains aspartic acid. In the region preceding the first insert, there
is reasonable electron density for residues 99-106, which was
not observed previously. Region 107-128, which includes the insert, is
disordered in the crystals. This is in contrast to the structures of
hTS·dUMP·Tomudex and rTS·dUMP·Tomudex complexes, in which this
region has interpretable electron density (3, 4). The second insert,
residues 146-153, has a well-defined structure in our crystals. A
short loop, Thr-103 to Arg-107 in ecTS, which is solvent-exposed, is
replaced in hTS with a longer loop, Ala-144 to Gln-156, which is
directed in the opposite direction toward the hTS surface (Fig. 2,
top). The insert loop is quite polar with the exception of
Met-149, which forms hydrophobic contacts with Thr-96 and Asn-97, and
Tyr-153 interacting with Phe-137. These interactions likely anchor the
loop to the rest of the molecule and induce the observed ordered
conformation. Although Met-149 is not conserved, equivalent residues
are usually hydrophobic and probably function in a similar manner. The
presence of the loop creates a cavity in which four ordered water
molecules are present. The structure of this loop is in excellent
agreement with that observed in the hTS·dUMP·Tomudex and
rTS·dUMP·Tomudex complexes (3, 4), indicating that its function is
not related to catalysis.
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Fig. 2.
Top, superposition of the C
trace of unblocked hTS (thick line) on ecTS (Protein Data
Bank entry 1qqq (18)). The orientation of the second insert, Ala-144 to
Gln-156, is opposite to that of the corresponding shorter loop in ecTS,
and is directed toward the surface of the molecule. Bottom,
electron density for the loop 181-197 contoured at 1.2s
does not suggest disorder and thus the presence of active conformer in
the crystal.
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Fig. 3.
Top, stereo view of the binding site for
a cryogen molecule, ethanediol (EDO), is shown with
2Fo Fc density maps contoured
at the 1.2s level. This cavity is not present in
bacterial TSs in which bulky groups replace Ala-63 and His-250.
Bottom, ribbon representation (21) of the glycol-binding
cavity, which is not far from the putative RNA binding site in hTS. The
glycol molecule is in the center, between the side chains of His-250
and Gln-36. The cavity is surrounded by hydrophobic and polar residues,
which form a channel leading to the active site where three
phosphate/sulfate ions are bound.
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Fig. 4.
Stereo diagram of the phosphate/sulfate
binding sites. Electron density presented as basket contouring is
at the 1.2s level. The distances between the phosphate ions
are very similar to those between the phosphate moieties in RNAs,
possibly mapping the sites of TS-RNA binding.
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Fig. 5.
Fluorescence of hTS and ecTS.
Top, enhancement effect of phosphate ion on hTS
fluorescence. Middle, reversal of the effect by dUMP. All
samples, except hTS, have the same concentration of Pi (64 mM) and, additionally, dUMP at the concentration as shown
in the inset (the order of the curves is the same as
indicated in the legend, except for unliganded hTS). Bottom,
no effect on ecTS.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Dr. Cathy Murphy for making her fluorimeter available and Dr. Trent Spencer for many helpful discussions.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant CA76560 and the South Carolina Cancer Center. Some instrumentation used in this research was purchased with National Science Foundation Grant BIR9419866 and United States Department of Energy Grant DE-FG-95TE00058. Use of the Argonne National Laboratory Structural Biology Center beamlines at the Advanced Photon Source was supported by the United States Department of Energy, Office of Science, under contract W-31-109-ENG-38.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and the structure factors (code 1HW3 and 1HW4) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
To whom correspondence may be addressed: Dept. of Chemistry
and Biochemistry, University of South Carolina, 631 Sumter St., Columbia, SC 29208. Tel.: 803-777-2140; Fax: 803-777-9521; E-mail: lebioda@ mail.chem.sc.edu and berger@cop.sc.edu.
Published, JBC Papers in Press, January 24, 2001, DOI 10.1074/jbc.M009493200
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ABBREVIATIONS |
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The abbreviations used are: TS, thymidylate synthase; hTS, human thymidylate synthase; ecTS, Escherichia coli thymidylate synthase; lcTS, Lactobacillus casei thymidylate synthase; NTE-hTS, N-terminally extended human thymidylate synthase; dUMP, 2'-deoxyuridine 5'-monophosphate; dTMP, 2'-deoxythymidine 5'-monophosphate; CH2H4folate, 5,10-methylenetetrahydrofolate; raltitrexed, Tomudex or ZD 1694, N-(5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino]-2-thenoyl)-L-glutamic acid; FdUMP, 5-fluoro-2'-deoxyuridine 5'-monophosphate; 2-ME, 2-mercaptoethanol; H4folate, (+)-tetrahydrofolate; PEG, polyethylene glycol; r.m.s.d., root mean square deviation.
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REFERENCES |
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