(Received for publication, November 18, 1994; and in revised form, January 25, 1995)
From the
The molecular basis for the treatment of human herpesviruses
with nucleoside drugs is the phosphorylation of these drugs by the
viral-encoded thymidine kinases. In order to better understand the
structural and enzymatic mechanisms by which herpesviral thymidine
kinases recognize their substrates, photoaffinity labeling with
[-
P]5-azido-2`-deoxyuridine-5`-monophosphate
and [
-
P]8-azidoadenosine-5`-triphosphate
was used to characterize the thymidine, thymidylate, and ATP active
sites of the herpes simplex virus-1 (HSV-1) thymidine kinase. For this
study, HSV-1 thymidine kinase and a site-specific mutant enzyme (C336Y,
known to confer acyclovir resistance) were expressed in bacteria and
purified by a rapid, two-step protocol. The specificity of
photoaffinity labeling of these HSV-1 thymidine kinases was
demonstrated by the ability of site-directed substrates such as
thymidine, thymidylate, acyclovir, 5-bromovinyl-2`-deoxyuridine, and
ATP to inhibit photoinsertion. Differences in inhibition patterns of
photoaffinity labeling correlated with kinetic differences between the
wild-type and C336Y HSV-1 thymidine kinases. Cumulative results suggest
that the acyclovir-resistant cysteine 336 mutation primarily affects
the ATP binding site; yet it also leads to alteration in the binding
affinity of nucleoside drugs in the thymidine site. In this study,
azidonucleotide photoaffinity analogs are shown to be effective tools
for studying the active-site environment of HSV-1 thymidine kinase and
related site-specific mutants.
The herpes simplex virus thymidine kinases (HSV-TKs) ()are the pharmacological targets of most herpesvirus
treatments, because these kinases catalyze the initial phosphorylation
of many anti-herpesvirus nucleoside drugs, such as acyclovir (ACV) and
5-bromovinyldeoxyuridine (BVDU)(1, 2, 3) .
This targeting is based primarily on the differences in substrate
specificity compared to the cellular TKs. As a highly regulated enzyme
of the pyrimidine salvage pathway, the cytosolic cellular TK
specifically phosphorylates thymidine with ATP as the phosphoryl donor (4) . The HSV-1 TK has a much broader range of substrates which
include most pyrimidine nucleosides, many guanosine derivatives (e.g. ACV), and most purine and pyrimidine nucleoside
triphosphates(5) . HSV-TK also possesses a thymidylate kinase
activity, and it has been suggested that the thymidine and TMP sites
are shared or overlapping(6, 7, 8) . Due to
significant amino acid sequence homology between the herpesviral TKs,
six putative regions involved in active-site formation have been
identified(9, 10) . In addition, drug-resistant and
site-directed mutants have been helpful in identifying some regions
involved in substrate
binding(11, 12, 13, 14) . However,
the structural basis for this broad substrate recognition is not known.
Because clinical isolates of herpesviruses resistant to ACV are
increasing in frequency(15, 16) , this structural
information is critical for designing TK molecular models and improving
nucleoside drugs. Additionally, HSV-1 TK is currently being delivered
to tumor cells as a toxin gene, via retroviral vectors, whereby these
cells are killed after administration of
ganciclovir(17, 18) . Therefore, a better
understanding of the molecular mechanism of HSV-TK could lead to future
customized, drug-specific TKs for gene therapy.
Toward these goals, we have expressed HSV-1 TK and a site-specific mutant in Escherichia coli and purified them for use in photoaffinity labeling analysis with base-substituted, azidonucleotide analogs. These analogs have found many applications in the characterization, identification, and purification of nucleotide-binding proteins(19, 20) . Upon UV irradiation, a highly reactive nitrene intermediate of short half-life is generated which allows indiscriminant insertion into active-site amino acids in a nucleotide-binding protein(19) . The most commonly used photoaffinity analogs are the 8-azidopurine nucleotides(19) , and more recently 5-azidouridine derivatives(20) . Both classes of analogs are currently finding wide usage for the identification of active-site (or regulatory-site) peptides and amino acids involved in nucleotide binding(21, 22) . Prior to this report, base substituted azidonucleosides have not been reported in the literature for use as inhibitors of viral replication or in the study of herpesviral-encoded proteins.
In this study, we report the
bacterial expression and a rapid, two-step purification of HSV-1 TK and
a site-specific mutant TK containing a tyrosine at position 336 in
place of cysteine (C336Y). A similar mutant isolated from an
acyclovir-resistant HSV-1 strain, in which C336Y was proposed to
constitute part of the ATP and nucleoside-binding sites, was shown to
have higher K values for thymidine, ATP,
and ACV relative to wild-type TK(11) . We have used two
photoaffinity analogs,
[
-
P]8-N
ATP and
[
-
P]5-N
dUMP, for comparative
analysis of the wild-type and mutant HSV-1 TKs. This active-site
directed technique is shown to be an effective tool for studying HSV-1
TK and related site-specific mutants.
The C336Y mutation was first created by site-directed mutagenesis using a modified version of Kunkel (24) as described in Black and Hruby(25) . The HSV-1 TK gene was cloned into the pET-8c at the NcoI site using the NcoI site at the initiating methionine codon and an NcoI site 3` to the HSV-TK open reading frame(26) . The orientation, site of insertion, and C336Y mutation were confirmed by sequencing.
Figure 1: SDS-polyacrylamide gel electrophoresis of proteins at different stages of HSV-1 TK purification. Protein was taken from the pooled fractions containing HSV-1 TK activity from crude supernatant (25 µg), DEAE-cellulose column (10 µg), and Bio-Scale Q2 FPLC (2 µg) as described under ``Experimental Procedures'' and separated on a 10% SDS-polyacrylamide gel, and stained with Coomassie Blue.
Figure 2: Purification of HSV-1 TK by Bio-Scale Q2 FPLC. Pooled DEAE-cellulose fraction (1 mg) was applied to a Bio-Scale Q2 column in a Pharmacia FPLC system previously equilibrated with Buffer A. HSV-1 TK was eluted in Buffer A by a 40-200 mM NaCl gradient as indicated by protein absorbance at 280 nm (-). Thymidine kinase activity(- - -) was determined by assaying 15 µl of each fraction as described under ``Experimental Procedures.'' Fractions 40-45 were pooled, concentrated, and analyzed by SDS-PAGE as shown in Fig. 1.
By use of the TK assay buffer minus bovine serum
albumin and standard procedures(19, 20) ,
photolabeling of WT and the C336Y HSV-1 TKs by
[-
P]5-N
dUMP was pursued.
Although not shown, photoincorporation of the photoprobes with the
HSV-1 TKs was always dependent on UV irradiation. As shown in Fig. 3, half-maximal saturation of photoinsertion of
[
P]5-N
dUMP was 6 (WT) and 8 (C336Y)
µM. These values are consistent with the reported K
values of TMP for both of these HSV-1
TKs(8, 28) , and are indicative of specific
active-site incorporation. To further test the active-site specificity
of photolabeling, known nucleoside and nucleotide substrates at varying
concentrations were included in the photolabeling reactions with
[
P]5-N
dUMP and the two TKs. As shown
in Fig. 4, A-C, photolabeling of WT HSV-1 TK was
competitively inhibited in a concentration dependent manner by
thymidine, TMP, ACV, BVDU, 5-N
dU, and 5-N
dUMP.
The concentrations of inhibitors that reduce photoincorporation by 50%
(ICP
) were derived from densitometric scanning of the
autoradiographs in Fig. 4and are are listed in Table 2.
Figure 3:
Saturation of
[-
P]5-N
dUMP
photoinsertion into WT and C336Y HSV-1 TKs. HSV-1 TK (10 µg) in a
40-µl reaction buffer was photolyzed with the indicated amount of
[
-
P]5-N
dUMP and subjected to
SDS-PAGE. Photoincorporation was detected by autoradiography and
quantified by laser densitometry.
, WT;
,
C336Y.
Figure 4:
Effect of various substrates on
photolabeling of WT and C336Y HSV-1 TK with
[-
P]5-N
dUMP.
Either wild-type or C336Y HSV-1 TKs (10 µg) were incubated in the
presence of the indicated concentrations of HSV-1 TK substrates,
photolyzed with 15 µM [
-
P]5-N
dUMP, and subjected
to SDS-PAGE. A, thymidine and TMP; B,
5-N
dU and 5-N
dUMP; C, ACV and BVDU; D, ATP. Photoincorporation was detected by autoradiography and
quantified by laser densitometry.
Comparisons between the
[P]5-N
dUMP photolabeling of the
C336Y and WT HSV-1 TKs are shown in Fig. 4and Table 2.
The C336Y HSV-1 TK consistently resulted in 1.3-fold higher levels of
photoincorporation of 15 µM [
P]5-N
dUMP relative to WT HSV-1
TK (data not shown). Of the compounds tested, ICP
values
for TMP did not differ between C336Y and WT HSV-1 TKs, and were over
2-fold higher for 5-N
dUMP. However, these values
significantly differed for the nucleosides tested: thymidine 5.7-fold,
5-N
dU 16.7-fold, ACV >6-fold, and BVDU >16.7-fold (Table 2). Addition of ATP (Fig. 4D) to inhibit
[
P]5-N
dUMP photolabeling resulted in
ICP
values of 150 µM for WT and >1 mM for C336Y HSV-1 TKs.
Figure 5:
Saturation of
[-
P]8-N
ATP
photoinsertion into WT and C336Y HSV-1 TKs. Wild-type HSV-1 TK (10
µg) (A) or C336Y TK (B) were photolyzed with the
indicated concentration of
[
-
P]8-N
ATP. Proteins were
separated by SDS-PAGE followed by autoradiography and laser
densitometry quantitation.
Figure 6:
Inhibition of
[-
P]8-N
ATP
photoinsertion into WT and C336Y HSV-1 TKs by nucleoside triphosphates.
Wild-type HSV-1 TK (A,
) or C336Y TK (B,
) were preincubated with the indicated concentrations of
nucleotide and photolyzed with 15 µM [
-
P]8-N
ATP. The reaction
mixture was subjected to SDS-PAGE followed by autoradiography and laser
densitometry quantitation.
, CTP;
, GTP;
,
TTP.
In order to facilitate rapid and high-yield purification of
HSV-1 TKs expressed in bacteria, a novel purification protocol was
developed independent of the conventional thymidine-affinity
chromatography resins routinely employed with this enzyme. Because we
intend to apply this purification protocol to other HSV-1 TKs mutated
in the thymidine or TMP active-sites, the thymidine affinity resins
would have had limited utility for these purposes. The rapid
purification technique consists of only two steps following sonication
of the isopropyl--D-thiogalactopyranoside-induced E.
coli cells: a selective, modified DEAE-cellulose chromatography
procedure and Q2 FPLC. This purification reproducibly results in
greater than 95% purity of HSV-1 TK. Comparison of specific activities
obtained with this protocol and thymidine-affinity chromatography
indicates little differences(28) .
Site-directed mutagenesis
studies of HSV-1 TK have been useful in correlating conserved sites
with activity(12, 13, 14) , but the basis of
the molecular differences in substrate and drug specificities of the
HSV-1 TK remains unclear. In combination with site-specific mutants of
HSV-1 TK, the active-site directed photoaffinity analogs described here
should aid in elucidating the molecular mechanism underlying HSV-1 TK
activity. As a model, we have compared the photoaffinity labeling of
the WT and an ACV-resistant TK, C336Y, with 5-NdUMP and
8-N
ATP. The experiments presented in Fig. 3Fig. 4Fig. 5Fig. 6indicate specific
active-site photoincorporation of these analogs and highlight several
important observations regarding the nature of the C336Y mutation and
ACV resistance. For WT HSV-1 TK, the most effective inhibitors of
[
P]5-N
dUMP photolabeling are
nucleosides, not TMP or 5-N
dUMP (Table 2). This
observation is consistent with previous kinetic studies which have
shown the K
values for thymidine and TMP differ by
12-25-fold, and both compounds to be competitive inhibitors of
each other, indicating that their sites overlap or are partially shared (6, 7, 8) . However, as shown in Fig. 3and Fig. 4A, the TMP site in the C336Y
mutant remains unaffected in contrast to the thymidine and ATP sites (Fig. 4Fig. 5Fig. 6). This suggests that although
nucleosides (and nucleoside derived drugs) are preferred in the T/TMP
site(s) compared with nucleoside monophosphates, the thymidine kinase
and thymidylate kinase activities can be uncoupled and are independent.
The data also suggests that a mutation at either the
nucleoside/drug-binding site or in the ATP site will lead to increases
in K
values for both substrates and potentially
lead to a drug-resistant enzyme. This is currently being tested with
the photoaffinity analogs and other HSV-1 TK site-specific mutants
containing amino acid substitutions thought to be involved in either TK
or TMP kinase activity.
The photolabeling data with
[P]8-N
ATP and the C336Y mutant ( Fig. 5and Fig. 6) indicate that the primary effect of
this amino acid substitution is in the ATP site. Therefore, an apparent
structural conformation change as a result of the mutation somehow
alters ATP/nucleoside binding, yet still retains a WT-like ATP/TMP
binding. The cysteine 336 of HSV-1 TK is believed to comprise part of
the ATP-binding site, as suggested by comparison of homologous domains
with other viral TKs and a related motif conserved in porcine adenylate
kinase(8, 11) . Interestingly, 8-N
ATP was
used to identify an ATP active-site peptide 5 amino acids N-terminal to
this conserved site in adenylate kinase(21) . Thus, if this
homology is reflective of HSV-1 TK function, Cys-336 is likely to be
near the adenine base in the ATP site. An interesting question to
address in the C336Y mutant is why the altered affinity for ATP binding
effects phosphorylation of nucleosides, but does not apparently affect
thymidylate kinase activity. The lack of competition by CTP of
[
P]8-N
ATP photolabeling for either
HSV-1 TK was not expected. Consistent with previous
studies(5) , the purified HSV-1 TK utilizes CTP as a phosphoryl
donor very efficiently (data not shown). This lack of CTP inhibition of
photolabeling may indicate a distinct binding site for CTP and is
currently being studied.
BVDU is one of the most potent inhibitors
of HSV-1 replication in cell culture, yet has little effect on the
replication of HSV-2(3) . Of the azidonucleosides tested, the
best inhibitor of HSV-1 replication was 5-NdU (Table 3). This compound would be the closest structural mimic of
BVDU, so it is not surprising that it had anti-HSV-1 activity and
little activity against HSV-2. The 5-azido moiety is consistent with
the criteria established for determining the effectiveness of a
5-substituted deoxyuridine as an anti-HSV-1 compound (35) : it
is an unsaturated, hydrophobic, electronegative 5-substituent of no
more than 4 atoms in length. Metabolic labeling experiments indicate
that [
H]5-N
dU can be phosphorylated
by HSV-1 TK in HSV-1 infected cells. Even though 5-N
dU is a
potent inhibitor of HSV-1 replication in cell culture, the poor
half-maximal cytoxicity doses of 10-20 µM preclude
any potential clinical applications. These results, however, do
validate the hypothesis that azidonucleosides can act as biological
mimics of anti-HSV nucleoside drugs. Therefore, if they are used as
photoaffinity analogs and UV-cross-linked to HSV-TK active-site
peptides, these are likely to be the same peptide domains that interact
with the known antiviral drug. Cumulatively, this study has shown that
5-N
dUMP and 8-N
ATP are effective tools for
determining the active-site environment of HSV-1 TK.