Journal of General Virology (1990), 71, 2979-2987. Printedin Great Britain
2979
Herpesviral deoxythymidine kinases contain a site analogous to the
phosphoryl-binding arginine-rich region of porcine adenylate kinase;
comparison of secondary structure predictions and conservation
Nandha Kumar Balasubramaniam,t Venkat Veerisetty and Glenn A. Gentry*
Department o f Microbiology, University o f Mississippi Medical Center, 2500 North State Street, Jackson,
Mississippi 39216, U.S.A.
Twelve herpesviral deoxythymidine kinases were examined for regions of sequence similarity by multiple
alignment. Six highly conserved sites were observed.
Site 1 corresponded to a glycine-rich loop that forms
part of the ATP-binding pocket in porcine adenylate
kinase (PAK), and site 5 corresponded to a region in
PAK, located on one lobe of the cleft, that contains
arginine residues that bind substrate phosphoryl
groups. Site 3, consisting of the m o t i f - D R H - , is
thought to be involved in thymine/deoxythymidine
recognition; site 4, which is nearby, probably participates in this function as well. The functions of sites 2
and 6 have not been identified. Secondary structure
predictions were made by the Garnier method and
averaged for each position in the multiple alignment.
The structure predicted for all six sites was typically a
short flexible region (turn or coil) at or adjacent to the
site, flanked by rigid structures (helix or sheet) on
either side.
Introduction
(Otsuka & Kit, 1984; Gentry, 1985; Darby et al., 1986;
Mittal & Field, 1989; Robertson & Whalley, 1988), and
we suggest a role for a fourth (site 5), based on its
similarity to a highly conserved arginine-rich site in
porcine adenylate kinase (PAK). As it has been
characterized by X-ray crystallography, PAK serves as a
Rosetta stone by which functions may be assigned to
analogous regions of related enzymes (the function of site
1 was so assigned, not only for herpesviral dTKs, but for
many other nucleoside and nucleotide kinases as well).
Finally, in considering the secondary structures of the
herpesviral dTKs, we have developed an algorithm for
averaging the predictions for related proteins that
introduces an element of confidence in the prediction,
the ambiguity index. With this algorithm we show that
all six conserved sites share a similar predicted structure,
which consists of a short flexible region (coil or turn) at or
adjacent to the site, flanked by rigid structures (sheet or
helix) on either side.
The herpesviral enzyme deoxythymidine kinase (dTK)
(ATP : thymidine 5-phosphotransferase; EC 2.7.1.21),
a salvage pathway enzyme, phosphorylates natural
nucleoside substrates (Jamieson et al., 1974) as well as
nucleoside analogues used in the therapy of herpesvirus
infections. Following activation these analogues block
viral replication by inhibiting DNA polymerase (Aswell
et al., 1977). Analysis of the deduced amino acid
sequences of the herpesviral dTKs is of interest in drug
design (Folkers et al., 1989a, b), and has been used
further to suggest a date for the divergence of herpes
simplex virus types 1 (HSV-1) and 2 (HSV-2) and also to
infer a time for the origin of human sexual behaviour
(Gentry et al., 1988).
In this paper we report comparative sequence analyses
of the dTKs of HSV-I and HSV-2, marmoset herpesvirus
(MHV), varicella-zoster virus (VZV), feline herpesvirus
(FHV), pseudorabies virus (PRV), equine herpesvirus
type 1 (EHV-1), bovine herpesvirus type 1 (BHV-1),
turkey herpesvirus (THV), Marek's disease virus
(MDV), herpesvirus saimiri (HVS) and Epstein-Barr
virus (EBV). These analyses have identified six relatively
well conserved regions of similarity, or 'sites'. The
functions of three of these have been reported previously
I"Present address: Pioneer Hi-Bred International,Inc., Johnston,
Iowa 50131, U.S.A.
0000-9812 © 1990 SGM
Methods
Nucleotide and/or amino acid sequences were obtained from
GenBank or from the literature as follows: HSV-1, Wagner et al.
(1981); HSV-2,Swain&Galloway(1983); MHV,Otsuka&Kit (1984),
as correctedbythe authorsin GenBank;VZV,Davison&Scott(1986);
FHV, Nunberg et al. (1989); EHV-1, Robertson & Whalley(1988);
PRV, Kit & Kit (1985), as corrected by the authors (personal
communication);BHV-1, Mittal& Field (1989); THV, Martin et al.
(1989); MDV,Scottet al. (1989);HVS,Honesset al. (1989); EBV,Baer
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2980
N. K. Balasubramaniam, V. Veerisetty and G. A. Gentry
et al. (1984). For EBV and HVS, the complete open reading frames
would encode proteins considerably longer than those shown. As the
various signals for initiation of translation of the EBV dTK are most
appropriately arranged about an internal A U G (BXLF1 6113 to 6111 ;
G. A. Gentry, unpublished observations), the translations used here for
both the EBV and the HVS dTKs reflect that possibility. It is
recognized, however, that the actual polypeptides may be much longer;
in any case, the general conclusions of the present study would be
unaffected.
Sequence manipulations were done within PROPHET (available
from Bolt, Beranek and Newman, Cambridge, Ma., U.S.A.), a
comprehensive scientific computing resource operating in a Unix
environment on a Digital Equipment Corp. MicrovaxlI and VaxStation 3200 connected in a local area network. Programming was done in
PL/PROPHET, a language originally derived from PL-1 but now
resembling C.
Nucleotide sequences were translated to their corresponding amino
acid sequences in the PROPHET system. In most cases those obtained
from the literature were entered directly as amino acid sequences.
Multiple alignments were done as follows. The two most closely related
dTKs (HSV-1 and HSV-2) were aligned using the ALIGN program in
PROPHET, which is based on the algorithm of Needleman & Wunsch
(1970). A single sequence was then created that retained the common
residues of both parents, but in which a dummy character replaced
those elements in which the parental sequences differed. This common
sequence was aligned as before with the next closest relative. Gapped
versions of the first two sequences were prepared by reintroducing
original residues in place of the dummy characters. The common
residues of the three sequences were then combined into a single
sequence and the procedure repeated until all sequences had been
incorporated. Gaps, once introduced, were retained in the final product
(Feng & Doolittle, 1987), which was checked by visual inspection and
manually revised as needed. The process of making the multiple
alignment included procedures (such as ALIGN) which are available
in PROPHET, as well as others written locally in PL/PROPHET
(principally for introducing dummy characters and for creating single
sequences retaining the common residues of two or more aligned
parental sequences).
Procedures for searching sequences, for facilitating, displaying and
editing the multiple alignments, and for averaging the secondary
structure predictions were also written in PL/PROPHET. Quantification of sequence similarity was done using the algorithm TRIPEVAL
(Gentry, 1985). A similarity density score greater than 20.9 indicated a
probability of less than 0.001 that the similarity had arisen by chance
(Gentry, 1985).
Secondary structure analysis was done using the G A R N I E R public
procedure available in PROPHET (Gamier et al., 1978). Each dTK in
its original unaligned form (i.e. without gaps) was put through
GARNIER. Each of the 12 tables thus produced contained four
predictive scores (for helix, sheet, turn and coil, respectively) for each
residue, and a prediction for that residue. The highest score determined
the prediction. These tables were further analysed by locally written
procedures. For each dTK, gaps were introduced into each of the four
strings of scores, corresponding to the gaps contained in that dTK in its
multiply aligned form. Then the 12 sets of four strings were averaged to
produce a single set of four strings containing the averaged predictive
scores (helix, sheet, turn or coil) for each position in the multiply
aligned dTKs. For each position the highest of the four scores
determined the prediction. In addition the ratio of the highest score
divided by the next highest score was calculated for each position; in
those cases where all but the high score were negative the high score
itself was used. The reciprocal of this value was designated the
ambiguity index, and varied between limits of 1 and 0, reflecting
maximum (two or more equal high scores) or minimum ambiguity
respectively in the prediction.
Results and Discussion
Comparison of herpesviral dTKs
Twelve herpesviral dTKs were aligned. The properties
and functions of herpesviral and other dTKs have been
reviewed (Gentry et al., 1983; Kit, 1985). Alignment of
the herpesviral dTKs is shown in Fig. 1. Conserved
regions were located and quantified by TRIPEVAL,
which evaluates triply aligned sequences. Two such
combinations were analysed. (i) The three least related
sequences which were any one from groups 1 and 2, and
one each from group 3 and group 4 (Table 1). HSV-1,
MDV and HVS were arbitrarily selected. (ii) The three
next least related sequences which were one each from
groups I, 2 and 3. HSV-1, EHV-1 and MDV were
arbitrarily selected.
Following analysis by TRIPEVAL, the similarity
density score was plotted against the residue number
(Fig. 2).
Identification of the conserved sites
Comparison of the primary structure of the 12 dTKs
revealed six conserved segments. We refer to them as
sites because their high degree of conservation suggests
that they have specific functions. They are numbered in
order of occurrence from the amino terminus. Mittal &
Field (1989) have examined a multiple alignment of
HSV-I, HSV-2, MHV, VZV and BHV-1 dTKs, and
identified seven conserved regions. These correspond to
our sites as follows: site 1 = region I; site 2 = region II;
sites 3 and 4 = region III; site 5 = region V; site 6 =
region VIb. Although some conservation may be
observed for regions IV and Va (see Fig. 2, between sites
4 and 5 for region IV, and just after site 5 for region Va),
the score returned by TRIPEVAL was too low for them
to be included. The segments we designate as sites also
tend to be shorter than the regions.
(i) Site 1
Site 1 was first described as part of a substrate-binding
pocket in (PAK) (Pal et al., 1977) and was initially
identified in viral dTKs (Otsuka & Kit, 1984; Gentry,
1985) by the consensus sequence - GXXGXGKT/S -.
This site is also found in most enzymes that bind ATP.
The three glycines are important components of a
flexible loop that changes conformation on substrate
binding (Fry et al., 1986; Pal et al., 1977). In the
herpesviral dTKs this site consists of residues 56 to 65 (of
the multiple alignment), and the consensus sequence is
perfectly conserved for all the herpesviral dTKs. In PAK
the consensus sequence is followed by hydrophobic
residues that together with this segment form a pocket
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Herpesviral deo xythymidine kinases
Table 1. Herpesvirus relationships
Alphaherpesviruses
Mammalian
Primate
(1) HSV-I
HSV-2
VZV
MHV
Non-primate
Avian
Gammaherpesviruses
(2) F H V
PRV
E H V- 1
BHV-I
(3) THV
MDV
(4) HVS
EBV
that binds the adenine moiety of ATP (Fry et al., 1986).
There also are scattered hydrophobic residues following
the consensus sequence in the herpesviral dTKs, but
whether they perform a similar function remains to be
seen.
Site-directed mutagenesis of HSV-1 dTK supported
the concept of an important catalytic function for site 1
(Liu & Summers, 1988). When any one of the glycines in
the consensus sequence - GXXGXGKT/S - was mutated
to valine, the enzyme was inactive. Activity was also lost
if the lysine was changed to isoleucine, and when the
threonine was changed to alanine. Activity was retained,
however, if the threonine was changed to serine (as
would have been expected; MHV dTK has a naturally
occurring serine in this position), although the Km values
for dT and ATP were different. The small sized glycine is
thought to have a secondary structure breaking activity
that is essential for the dTK to function. The positively
charged lysine could participate in neutralizing the
negatively charged phosphoryl group on ATP. The
hydroxyl group of threonine (and of serine) is thought to
form an ester intermediate by reacting with the
nucleotide phosphoryl group (Liu & Summers, 1988), as
the phosphoenzyme intermediate would otherwise be
unlikely to form (Arnold et al., 1986). The hydroxyl group
is apparently required at this position because enzyme
activity was abolished by substitution with residues other
than serine (Liu & Summers, 1988).
An amber mutation close to site 1 (codon 44) results in
the deletion of the 45 amino-terminal residues and in
turn the production of a truncated polypeptide in the
HSV-1 dTK (Coen et al., 1989; Irmiere et al., 1989). This
polypeptide is unstable, and although it retains dTK
activity it is devoid of deoxythymidylate kinase
(dTMPK) activity, normally present in the dTKs of
HSV-1, VZV and EBV ( C h e n & Prusoff, 1978; Fyfe,
1982; Ayisi et al., 1984; Veerisetty & Gentry, 1985; Coen
et al., 1989). Furthermore it has been suggested that
EHV-1 and PRV dTKs lack dTMPK activity (Veerisetty
et al., 1990). Regions for which conservation correlates
with dTMPK activity have not been found.
2981
In PAK the proline (residue 17) in site 1 is thought to
play an important role in the binding of substrates, but
not for catalytic efficiency (Tagaya et al., 1989). Such a
proline residue is absent in site 1 of the herpesviral dTKs,
although proline 86, conserved in all 12 of the dTKs, is
less than 25 residues to the right.
In summary the glycine-rich loop is thought to have
multiple functions (in PAK) which include regulating
access to the substrate-binding site, modifying the
binding site affinity and relocating the catalytic groups
(Fry et al., 1986). This loop seems to be ubiquitous for all
nucleotide-binding enzymes (Walker et al., 1982). By
analogy to the other nucleotide-binding enzymes, site 1
of the herpesviral dTKs probably also participates in
binding ATP.
(ii) Site 2
The segment in the herpesviral dTKs beginning at
position 85 contains a highly conserved six residue
sequence. This is site 2, which is not recognizable in the
adenylate kinases or the poxvirus and mammalian dTKs
sequenced thus far, nor are similar segments found in the
NBRF protein database. This site has the motif
-EP(M/L/I)XYW-, and E[85], P[86], Y[89] and W[90] are
conserved in all 12 dTKs. No functional role has been
assigned to site 2, although because of its content of
hydrophobic residues it could conceivably form a part of
the ATP-binding pocket.
(iii) Sites 3 and 4
Site 3, the most conserved site in the herpesviral dTKs,
consists of the motif -DRH- and is reported to be
involved in thymine recognition (Darby et al., 1986;
Mittal & Field, 1989; Robertson & Whalley, 1988). It is
found at position 172 of the multiple alignment of the
dTKs, and D[172] may have a role analogous to the
aspartate residue found in several nucleotide-binding
proteins (Dever et al., 1987). These residues are also
found in the thymidylate synthetases of vaccinia virus
and of yeast, except that the histidine is replaced with a
tyrosine (Smith et al., 1989; Jong et al., 1984), and further
may be seen in several bacterial phosphotransferases
that use ATP as phosphate donor (Brenner, 1987). The
aspartate may interact with the phosphate groups of
ATP through a Mg z+ salt bridge. Preceding site 3 is a
hydrophobic region that may be essential for catalytic
activity (Robertson & WhaUey, 1988). Following the
histidine is a proline which is conserved in 10 of the 12
herpesviral dTKs; although it may be important for the
conformation of this site (proline can be the site of a bend
in a beta-sheet), the fact that it is not completely
conserved suggests that it is not required for activity.
Adjacent to site 3 is site 4, which consists of three
residues beginning at position 181. This site was initially
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2982
N. K. Balasubramaniam, V. Veerisetty and G. A. Gentry
i0
20
30
40
50
SITE i
70
I
I
I
I
I
*******
I
hsltk
MASYPCHQHASAFDQAARSRGHSNRRTALRPRRQQEATEVRLEQKMPTLLRVYIDGPHGMGKTTT-TQLL
MASHAGQQHAPAFGQAARASGPTDGRAASRPSHRQGASEARGDPELPTLLRWflDGPHGVGKTTTSAQLM
hs2tk
mhvtk
MSGTAGTSRILRWfLDGPHGVGKSTTAEALV
vzvtk
MSTDKTDVKMGVLRIYLDGAYGIGKTTAAEEFL
fhvtk
MASGTIPVQNE°EIIKSQVNTVRIYIDGAYGIGKSLTAKYLV
M_RILRIYLDGAYGTGKSTTARVMprvtk
ehltk
MAARVPSGEARRSASGAPVRRQVTIVRIYLDGVYGIGKSTTGRVMMAEP-ARALRVVRIYLDGAHGLGKTTTGRALbhltk
MALPRRPPTLTRVYLDGPFGIGKTSILNAMP
thvtk
MASQMTSAQLIRVYLDGSMGIGKTSMLNEIP
mdvtk
hvstk < M L E F G E S L K S K L H N D - - S K K S P D E P D G L V H V P V H L L Y P P K H Q D P V - - P A F F I F L E G S I G V G K T T L L K S M N
ebvtk
<MNVLNLDDAQDTRQAKAQRKESMRVPIVTHLTNHVPVIKPACSLFLEGAPGVGKTTMLN---
80
SITE 2
I
hsltk
hs2tk
mhvtk
vzvtk
fhvtk
prvtk
ehltk
bhltk
thvtk
mdvtk
hvstk
ebvtk
Ii0
f
160
I
I
120
130
I
I
140
i
SITE 3
i
***
SITE 4
***
190
200
I
210
I
I
DAVLAPHVGGE-A-GSSHAP---PPALTLIFDRHPIAALLCYPAARYLMGSMTPQAVLAFVAL-IPPTLP
DAVLAPHIGGE-A-VGPQAP---PPALTLVFDRHPIASLLCYPAARYLMGSMTPQAVLAFVAL-MPPTAP
EEAMRPHVGRELAEPDDNGPLPQRRDFVLVVDRHAVASMVCYPLARFMMGCVSLRSVASLISH-LPPPLP
HAKISALMDTSTSDLVQVNKE--PYKIML-SDRHPIASTICFPLSRYLVGDMSP-AALPGLLFTLPAEPP
...... H S R L S T I T G Y Q K V V C E E H P D V T L I I D R H P L A S L V C F P L A R Y F V G D M T L G S V L S L M A - T L P R E P P
...... H T R L V P L F G - - P A V E G - P P E M T W F D R H P V A A T V C F P L A R F I V G D I S A A A F V G I T k A - T L P G E P P
...... H D H T F G L F G G D S L Q R G T R P D L T W F D R H P V A S A V C F P A A R Y L I G D M S M C A L I A M V A - T L P R E P Q
VRGPVLNFARARVRAAAPPGPAPGGTVTLVFDRHPVAACLCYPFARYCLREINAEDLLMLAA-AMPPEAP
..... F H E R L S S K C R G K I E I C D T - P A I I L M L D R H P V A A I L C F P I T R Y L L G E Y S L E M L I S S I I R - L P L E S P
.... V F H E R L S S K C - H R I T G T R G N P S L I L I L D R H P I S K T V C F P I A R H L T G D C S L E M L I S M I I R - L P Q E P P
A T D R L S S P K N S L L - - - S - - - S .... D M W V M F D R H P L S A T V V F P Y - - M H F Q N G F L S F S H L I Q L W S S F K A S R
..... A S R K R S L L V T E S G A R S V A P L D C W I L H D R H L L S A S V V F P L - - M L L R S Q L L S Y S D F I Q V L A T F T A D P
220
I
hsltk
hs2tk
mhvtk
vzvtk
fhvtk
prvtk
ehltk
bhltk
thvtk
mdvtk
hvstk
ebvtk
I00
VALGSRDDI-VYVPEPMTYWQVLGASETIANIYTTQHRLDQGEISAGDAAWMTSAQITMGMPYAVT--EALGPRDNI-VY~PEPMTYWQVLGASETLTNIYNTQHRLDRGEISAGEAAVVMTSAQITMSTPYAAT--AP~CEPRRPIRSMLQEPMAYWRSTFASDAITEIYDTQHRLDSNEITAAEAGAFMTSLQ~GTPYALL--HHFAITPNRILLIGEPLSYWRNLAGEDAICGIYGTQTRRLNGDVSPEDAQRLTAHFQSLFCSPHAIM--RADENRPGYTYYFPEPMLYWRSLFETDVVGGIYAVQDR_KRRGELSAEDAAYITAHYQARFAAPYLLL-----ALGG--ALYVPEPMAYWRTLFDTDTVAGIYDAQTRKQNGSLSEEDAALVTAQHQAAFATPYLLL--A S A A S G G S P T L Y F P E P M A Y W R T L F E A D V I S G I Y D T Q N R K Q Q G D L A A D D A A S I T A H Y Q S R F T T P Y L I L --°
AAASTAGEGVLFFPEPMAYWRTMFGTDALSGILAASARCAAASHGSARARRAGAPRRIRGRGGPGCVLPGQ
D H T P D G A P I L K V Y - E P M I ~ Y W R C Q S - T D L V V A A N E T P E R R R G G A L S R F Q S D M I M A S I Q A R F A D P Y L L ....
T H S L M G V P V L K V F _ E P M K Y W R Y ~ f F _ T D L V T T V N D T C D R R R R G E F S L F Q S S M I V T A L Q S K F A D P Y L .....
--GILGGKNVLAFHEPIAYWTDVF-SNSLEEVYK-LTLPAKVGR--TSNSAKLLACQLKFASP-LLALKT
HLKAVFGDLTIVVPEPMRYWTHVY-ENAIKAMHKNVTR-ARHGREDT--SAEVLACQMKFTTPFRVL---
150
hsltk
hs2tk
mhvtk
vzvtk
fhvtk
prvtk
ehltk
bhltk
thvtk
mdvtk
hvstk
ebvtk
******
SITE 5
240
*******
I
250
I
260
I
270
I
280
I
GTNIVLGALP- E D R H I D R L A K R Q R P G E R L D L A M ~ I R R V Y G L L A N T V R Y L ..... Q G G G S W W E D W G Q L
GTNLVLGVLP - EAEHADRLARRQRPG
ERLDLAMLSA
- I R R V Y D L L A N T V R Y L ..... Q R G G R W R E D W G R L
G T N L W A S L D - FREHAAR~PGERLDLTMMAAI R N A Y A M L A N T S R Y L ..... L S G G D W R R D W G S L
G T N L W C T V S - LPSHLSRV S KRARPG ETVNLPFVMV
- LRNVYIMLINTI
I FL ...... K T N N W H A G W N T L
GGNLV-VTTUq- I EEHI]IRI~GRS RTGEQI DMKLI HA -~ L V H T K K F L
..... T K N T S W R D G W G K L
GGNLVVASLD - P D EHLRRLRARARAG
EHVDARLLTA
- L R N V Y A M L V N T SRYL ..... S S G R R W R D D W G R A
GGNIVVTTLN- V D E H V R R L R T R A R I G EQ I D M K L I A T -L R N V Y S M L A N T S N F L ..... R S G R V W R D G W G E L
G A N L W C T L P - PA EQQRRLAAR_ARPG DRADAG FLVA- V R N A Y A L L V N T C A F L - - -R A G G D G A T A G T R W S G
G C N L T V T I L P D E K E H V N R I C SRDRPG E T A D R N M L R T -LNAVYAS L V D T V K Y A N L T C PYEKE S W E M E W L G L
G C N L V I V D L H D E K E H V S R L S S R N R T G E K T D L I M L R A -LNAVYS CLVDT I MYANHI CPYS KDEWES E W L D L
GDNI ILLNUq- S Q E N L K R V K K R N R K E E K S V S IEHIRLLNNCY- -~ W f C A W L - L V Q N F T P E E I V E V C F N A
GDT IVWMKLN -VE E N M R R L K K R G R K H E S G L D A G Y L K S V N D A Y - -HAVYCAWL- L T Q Y F A P ED I V K V C A G L
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Herpesviral deoxythymidine kinases
hsltk
hs2tk
mhvtk
vzvtk
fhvtk
prvtk
ehltk
bhltk
thvtk
mdvtk
hvstk
ebvtk
hsltk
hs2tk
mhvtk
vzvtk
fhvtk
prvtk
ehltk
bhltk
thvtk
mdvtk
hvstk
ebvtk
.....
290
300
SITE 6
320
330
340
350
I
I
******
I
I
I
I
2983
SGTAVPPQGAEPQSNAGPRPHIGDTLFTLFRAPELLAPNG-DLYNVFAWALDV-LAK-RLRPMHV
. . . . . TGVAAATPRPDPEDGAGSLPRIEDTLFALFRVPELLAPNG-DLYHIFAWVLDV-LAD-RLLPM-HL
PVFKPSAFVARAAKTAYTLPLRDEPGLADTLFAALKVPEFLDARG-YPRAAHAWTLDI-I-~-RIRALRV
SFCNDVFKQKLQKSECI--KLREVPGIEDTLFAVLKLPELCGEFG-NILPLWAWGMET-LSN-CSRSMSP
KIFSHYERNRLVETTIVSDSTESD--LCDTLFSVFKARELSDQNG-DLLDNHAWVLDG-I.bIE-TLONLQI
PRFDQTTRDCL--AIAqELCRPRDDPELQDTLFGAYKAPELCDRRG-RPLEVHAWAMDA-LVA-KLLPLRV
PLSCETYKHRA--TQMDAFQERESPELSDTLFANFKTPELLDDR@-VILEVHAWALDA-IML-KLRNLSV
RTQMHWPRSQTPWletNAKCAG---AGLRDTLFAALKCRELYPGGGTGLPAVHAWALDA-LAG-RLAALEV
PWFEESLLEEFISRPRPVICSRTRMPLDRTLLAIFKRKELCSENG-ELLTQYSWILWGLLTKLHTINVEL
PWFDTSLATTFINEPR-TDYRGSRVSIA-IHTLLAIFKRRELCAEDG-SLSTTHAWILWGLLMKLRNINVER
KHITDLSSSKPSFLAK.HVSTEDMLKS---SIFNTWIEMTKAHRDSCTIME-CLLTFCKELEKVQLI . . . .
TTITTVCHQSHTPIIRSGVAEKLYKN---SIFSVLKEVIQPFRADAVLLEVCLAFTRT-LAYLQ . . . . . .
360
370
380
390
400
410
420
I
I
I
I
I
I
I
FIL-DYDQSPAGCRDALLQLTSGMVQTHVTTPGSIPTIC--DLARTFAREMGEAN*
FVL-DYDQSPVGCRDALLRLTAGMIPTRVTTAGSIAEIR--DLARTFAREVGGV*
YTL-DLTGPPEACAAAFRRLCAGLVLTEGSHPGALCELKRAAAA--YAREMSVVGSREPTTAEVESA *
FVL-SLEQTPQHAAQELKTLLPQMTPANMSS-GAWNILKELVNAVQDNTS*
FTL-NLEGTPDECAAALGALRQDMDMTFIAACDMHRISEALTIYH*
STV-DLGPSPRVCAAAVAAQARGMEVTESAYGDHIRQCVCA ..... FTSEMGV*
FCA-DLSGTPRQCAATVESLIPI~ISSTLSDSESASSLE---RAARTFNAEMGV*
FVL-DVSAAPDACAAAVLDMRPAMQAACADGAAGATLA---TLARQFALEMAGEATAGPRGL*
FDISGMSRRE--CASAIMHTMPERLSTLASWNDLCELEDDVIS---YNKGMCNEVGASR*
FNITGLSTTK--CVESFMDTMSERLVTHMSWNDAFEIEADVLA---YNKEMAM*
HVNV--SPFTDDIPGLWASIYTSIRRNSAIKPNRVNWLALEDLARTFNSQ*
FVLVDLSEFQDDLPGCWTEIYMQALKNPAIRSQFFDWAGLSKVISDFERGNRD*
Fig. 1. Multiple alignment of the amino acid sequences of 12 herpesviral deoxythymidine kinases. Sites are indicated just above the
first and below the twelfth sequence in each case. Arrows at the start of the HVS and EBV dTKs indicate the portions of the open
reading frames not shown (see text).
implicated as the nucleoside-binding site of the enzyme
(Darby et al., 1986), and the motif -C(Y/F)P- is
conserved in 10 of the dTKs. Mutational analysis has
shown that the cysteine is not essential for the activity of
the enzyme and thus not crucial to thymine recognition;
nor is a disulphide bond required at this position (Inglis
& Darby, 1987). Mutation of alanine 178 to threonine,
arginine 186 to glutamine, or cysteine 363 (not part of
sites 3 and 4) to tyrosine produced variants of HSV-1
strain SC-16 (Larder et al., 1983a, b) which showed
altered affinities for nucleoside substrates and for ATP
(Darby et al., 1986). It was further shown that the C[363]
mutant lacked dTMPK activity (Veerisetty & Gentry,
1985). Alanine 178 is between sites 3 and 4, and arginine
186 is just to the right of site 4; these two residues may be
involved in dT recognition.
(iv) Site 5
Site 5 is arginine-rich and has been recognized in
herpesviral dTKs (Gentry, 1985; Mittal & Field, 1989)
by its similarity to an arginine-rich site in PAK (Fig. 3).
This site, occurring at position 228, is highly conserved in
the herpesviral dTKs. Of the seven residues in this site
arginines 228, 232 and 234 are conserved completely in
the 12 dTKs. Two additional herpesviral dTKs not
included in the present analyses have recently been
described and deserve comment in this regard. First, the
dTK of infectious laryngotracheitis virus (Griffin &
Boursnell, 1990) contains all three of the arginines in site
5, whereas the dTK of bovine herpesvirus 2 (Sheppard &
May, 1989) replaces the third arginine (residue 234 in the
multiple alignment) with a proline. The latter is of some
interest, because it is the only wild-type herpesviral dTK
so far sequenced that does not retain all three. It should
be mentioned that the MHV dTK as shown by Scott et al.
(1989) and by Griffin & Boursnell (1990) also replaces the
third arginine with a proline, whereas Honess et al.
(1989) have shown it as an arginine. The current
GenBank entry documents Kit's correction that results
in the arginine, and probably should be considered
authoritative. Kit et al. (1987) also described an
acyclovir-resistant mutant of HSV-2 in which the third
arginine was replaced with a histidine. This mutant did
retain dTK activity, although the data shown suggested
it was somewhat less than wild-type. In PAK the third
arginine is not found, but an additional arginine (138;
Fig. 3) further to the right is essential for activity (Yan et
al., 1990). In addition to the three arginines, G[236] and
E[237] are highly conserved (10/12 and 11/12). The latter
(glutamate) is replaced in only one instance, by aspar-
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N. K. Balasubramaniam, V. Veerisetty and G. A. Gentry
2984
34-
1
34
2
30 -
5
6
! HSV-ITK EHV-IT K
L
MDV TK
P = 0 001
tate. This acidic residue may also be important for some
catalytic function of this site. In P A K the arginines in
this region are thought to bind substrate p h o s p h o r y l
groups (Dreusike et al., 1988). We assume that site 5 in
herpesviral d T K s has a similar function.
20-
(v) Site 6
Site 6 extends from residues 307 to 312. Although the
degree of conservation is as great as for sites 2 and 5 when
the d T K s of HSV-1, E H V - I and M D V are compared, it
is less well conserved in the trio HSV-1, M D V and HVS.
To the right of site 6 is a region that is still less well
conserved. N o function has been assigned to site 6.
/
10-
I I I I
0 I I
30
1
34
2
1
5
6
HSV-1 T K / M D V T K / H V S T K
r,~
P=0.001
20
10
0
,
,J
i,
, , ,
I0O
tJ,
300
400
200
Position in alignment
Fig. 2. Evaluation of similarities between the amino acid sequences of
two trios of herpesviral deoxythymidinekinases by TRIPEVAL. The
probability is equal to or less than 0.001 that scores of 21 or greater
represent similarities that arose by chance (Gentry, 1985).
0
HSV-1 TK 211 DRHIDRLAKRQRPGERLDLAMLA 233
PAK
123 ETMTKRLLKRGETSGRVDDNEET145
EBV TK 197 EENMRRI.gXRGRKItESGLDAGYL219
HSV-ITK 211 d::i:RL:KR:R::ERID:A::: 233
PAK
123 :::::::::::::::::::::: 145
EBV TK 197 E::mrRL:KRGR::E:::DA::: 219
HSV-ITK 211 W w ' W W W W W W o o v v v o ~
EBV TK 197 W W W W W W W W W W v o Y v ~
223 W W W W W W W W W o v v v o ~
SUM
233
219
245
PAK
123 W ~ o o o v v v v v W W ~
145
X-ray
123 W ~ v v v v v v v v v v v W
145
Fig. 3. Comparison of an arginine-rich site in herpesviral deoxythymidine kinases with an analogous site in PAK. Site 5 is indicated by
asterisks. The upper two panels show multiple alignments of the site
and flanks as in Fig.: 1 and 2. The lower two panels show the secondary
structure predictions. PAK, porcine adenylate kinase; SUM, the
predictions based on the averageof the 12 herpesviral dTKs; X-ray, the
actual structure of the arginine-rich site in PAK as determined by Xray crystallography. Flanking numbers refer to residues in the
individual sequences, except for suM, in which case they refer to the
residues in the multiple alignments shown in Fig. 1 and 2. Symbolsare:
W, helix; Y, sheet; o, turn; v, coil. The second panel shows matches
only.
Secondary structure predictions
Prediction of the protein secondary structure (helix,
sheet, turn or coil) by the algorithm developed by
G a m i e r et aL (1978) has a typical accuracy of 50 to 60?/0,
and is often more accurate than the C h o u - F a s m a n (1974)
method. Seventeen residues is the m i n i m u m length of the
amino acid sequence required f o r G A R N I E R and
predictions are based on data derived from globular
proteins. Examination of the secondary structure predictions for the various sites together with the associated
ambiguity indices (Fig. 4) permits several conclusions.
First, each site tends to be located at or adjacent to a
flexible region (turn or coil) that is flanked by rigid
structures on both sides (helix or sheet). This m a y range
from the single turn (site 2) to the five residue turn-coilcoil--coil-turn structure at site 5. Second, the ambiguity
index tends to be higher (prediction less certain) at
transitional residues. This is particularly striking at site
5. In fact, the two 'turns' in site 5 are more likely to be coil
or helix; a 'turn' is normally defined as four residues
flanked by sheets. Third, the lowest ambiguity indices
are found in association with rigid structures (e.g. the
helical region preceding site 5).
Because the tertiary structure of P A K has been
determined, it is possible to evaluate some of the
predictions. P A K was therefore analysed by G A R N I E R
and the results for site 5 in P A K were compared with site
5 in EBV and HSV-1 dTKs, with the average (as shown
in Fig. 4), and with the structure as determined by X-ray
crystallography (Fig. 3). The results are generally
consistent. In the helical region where the ambiguity
index (as shown in Fig. 4) was lowest there was complete
agreement with the X-ray-determined structure. The
nature of the flexible structure (loop) was fairly well
approximated by the average for the 12 herpesviral
dTKs, especially considering the high ambiguity indices
at the two turn predictions, except that the actual loop in
P A K was considerably longer (toward the carboxy end)
than the Garnier predictions either for P A K , the HSV-1
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Herpesviral deoxythymidine kinases
1
3
2
4
m
'¢Yv'~ot.x~'YY'v~tY
'O'v"t'~Yt:l
2985
m
, I . i .!. It'v',t'
0
5
6
I,l,l,l,l,l,l,l,l,l,h,t,t,rt,l,l,l,l,I
)
"
'V¥'fYYcx,o'vl
,I,I, I, 1,1,1,I,I,1,1,1J
0
W
Y
Helix
Sheet
v
h
Coil
Ambiguity
index
--
Fig. 4. Secondary structure predictions for six conserved sites in the amino acid sequences of 12 herpesviral deoxythymidine kinases.
Site locations are indicated by dark bars. The predictions are based on averaged scores for each residue (see text). The ambiguity index
is the ratio of the second highest to the highest score, unless the second highest score is negative, in which case the ratio is the inverse of
the highest score. The ratio thus varies between a maximum of one, and a minimum limit of zero.
and EBV dTKs, or the averaged version. That the
averaging procedure was this consistent was not unexpected, as Garnier et al. (1978) suggested that such an
approach would increase the reliability of the
predictions.
l
2
HSV-1
-
_~
HSV-2
-
-
MHV
-
VZV
-
PRV
Suggestions can be made about the three-dimensional
structure of the herpesviral dTKs. By analogy with PAK
it is likely that there are two substrate pockets, one
binding ATP, the other dT. The two components
identified that have analogues in PAK, sites 1 and 5,
probably have a similar structural relationship; i.e. site 1
forms part of the ATP-binding pocket and is located
within the cleft which is characteristic of nucleotide and
nucleoside kinases (Anderson et al., 1979), whereas site 5
is located near the end of one of the lobes. Sites 3 and 4
should logically be part of the dT-binding pocket.
Whether those herpesviral dTKs with dTMPK activity
have an additional pocket or whether dTMP can also
bind in the dT pocket in these enzymes remains to be
determined. In the latter case, following phospborylation
of dT, the dTMP might linger after release of the ADP
and be phosphorylated further upon the binding of a
fresh ATP. This sequence of events is consistent with the
PAK model in which the cleft narrows modestly on
binding AMP, and then closes completely with a 'huge'
movement on binding ATP (Schulz et al., 1990).
Because sites l and 5 are conserved in PAK and in the
herpesviral dTKs, it seems likely that they may be
conserved together in other related enzymes, and that
5
6
||
I
m
I
m
7
II
FHV
Conclusions
-
34
7
-
-
-
i"
II
I
a7
I
I
T
T
•
t
--
EHV-I
-
-
BHV-1
--
--
THV
=
-
|
,7
7
. . .
II
MDV
!
-
-
-
-|
HVS
~
=
EBV
-~ =
|
. .
Z"
. .
I
I
~___
-
_
II
R
•
I
!
Fig. 5. Relative linear spatial relationships among six conserved sites
of 12 herpesviral deoxythymidine kinases. Sites are indicated by solid
boxes and number. Site 1 (nearest the amino terminus) was arbitrarily
set at the same relative position for each dTK. The amino termini of the
EBV and HVS dTKs may in fact be approximately one-third longer
(see text).
site 5 should be sought whenever site 1 is observed. In
this regard the amino acid sequence of yeast guanylate
kinase (YGK) has been published recently (Berger et al.,
1989), and although the authors report substantial
similarity only with site 1 in PAK, there are two arginine
residues (R[131] and R[135]) at a location analogous to
site 5 in PAK, which leads us to suggest that site 5 is in
fact conserved in YGK.
The relative linear arrangements of the conserved sites
of the various dTKs is shown in Fig. 5. From this figure,
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2986
N. K. Balasubramaniam, V. Veerisetty and G. A. Gentry
three conclusions may be drawn. First, the distance from
the amino terminus to site I is not important. Second, the
distance from site 6 to the carboxy terminus, though
somewhat more consistent, may not be critical. Third,
the spacing of the various sites is important, with the
possible exception of the distance between sites 2 and 3,
which can vary, at least within modest limits. This region
also exhibits the least conservation among the 12
enzymes (Fig. 2).
Finally, we should be reminded that direct structural
data, such as those provided by X-ray crystallography
and other analyses, are required in order to confirm these
suggestions.
We gratefully acknowledge the technical assistance of M. Minyard,
L. Devine, A. Green and N. Lawrence. Further we appreciate the
numerous helpful suggestions of Drs Wayne Rindone and Mark Olson,
and the provision of revised versions of the PRV and MHV dTK
sequences by Dr Saul Kit. Finally we thank Drs Ursula Gompels,
Simon Scott, Dianne Aparisio, Millar Whalley and Jack Nunberg for
providing preprints of several of the dTK sequences. This work was
supported by NIH grant RR 04334.
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