From the Cancer Research Campaign Institute for
Cancer Studies, University of Birmingham, Edgbaston,
Birmingham B15 2TA, United Kingdom and the ¶ Institute of
Molecular Virology, St. Louis University Medical Center, St. Louis,
Missouri 63110
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ABSTRACT |
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The C-terminal binding protein (CtBP) has
previously been shown to bind to a highly conserved six-amino acid
motif very close to the C terminus of adenovirus early region 1A (Ad
E1A) proteins. We have developed an enzyme-linked immunosorbent assay
that has facilitated the screening of synthetic peptides identical or
similar to the binding site on Ad E1A for their ability to bind CtBP
and thus inhibit its interaction with Ad12 E1A. It has been shown that
amino acids both C-terminal and N-terminal to the original proposed
binding site contribute to the interaction of peptides with CtBP.
Single amino acid substitutions across the binding site appreciably
alter the Kd of the peptide for CtBP, indicative of
a marked reduction in the affinity of the peptide for CtBP. The
solution structures of synthetic peptides equivalent to the C termini
of both Ad5 and Ad12 E1A and two substituted forms of these have been
determined by proton NMR spectroscopy. Both the Ad12 and Ad5 peptides
dissolved in trifluoroethanol/water mixtures were found to adopt
regular secondary structural conformations seen as a series of
-turns. An Ad12 peptide bearing a substitution that resulted in only
very weak binding to CtBP (Ad12 L258G) was found to be random coil in
solution. However, a second mutant (Ad12 V256K), which bound to CtBP
rather more strongly (although not as well as the wild type), adopted a
conformation similar to that of the wild type. We conclude that
secondary structure (
-turns) and an appropriate series of amino acid
side chains are necessary for recognition by CtBP.
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INTRODUCTION |
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Adenovirus E1A DNA encodes two major proteins that are expressed from differentially spliced mRNAs of sedimentation coefficients 13 and 12 S. These two proteins are co-terminal and only differ by the presence of an extra peptide located toward the C terminus of the larger molecule. Adenovirus E1A proteins (Ad E1A)1 are multifunctional, being able to regulate transcription of viral and cellular genes, initiate cell cycle progression and DNA synthesis (1, 2), inhibit differentiation (3, 4), and, in some cases, transform cells in culture (reviewed in Refs. 5 and 6).
It is now clear that Ad E1A proteins have no enzymic properties of their own but exert their influence on the host cell through a series of protein-protein interactions with important cellular regulatory molecules. Thus, E1A has been shown to bind to pRb105 and the related p107 and p130 proteins, p300/CBP, ATF2, and TBP (reviewed in Ref. 6). The interaction sites for all of these proteins on E1A have been established by mutational and deletional analysis, and most have been shown to occur in regions conserved in the E1As from different virus serotypes. For example, the pRb family of proteins binds in conserved regions (CRs) 1 and 2 (7, 8), whereas TBP (9-12) and ATF2 (13-16) interact with the N- and C-terminal halves of CR3, respectively.
A further region conserved between different E1As, although not generally included in the group of E1A conserved regions, occurs at the C terminus of the protein (17, 18). This amino acid sequence is involved in binding to the C-terminal binding protein (CtBP), which appears to be involved in the regulation of E1A CR1-dependent control of transcription (19). This, in turn, plays a role in E1A-mediated transformation. Thus, deletion of the C-terminal portions of exon 2 enhances transformation by E1A and activated ras and the tumorigenesis of the resulting cell lines (20).
Schaeper et al. (21) have mapped the binding site for CtBP on Ad5 289-a.a. E1A to amino acids 279-284 by fine deletional analysis. We have enlarged on this previous study and examined the effects of deletions on either side of the six-amino acid sequence as well as the effect on binding of each amino acid in turn. Thus, we have used an ELISA to determine the contribution of each amino acid to the E1A binding site for CtBP. In addition, using NMR spectroscopy the structure of the C terminus of Ad E1A, which includes both the CtBP binding and the nuclear localization signal (22), has been determined. The effects of mutations that disrupt binding to CtBP on the structure of the C terminus of E1A has also been examined.
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EXPERIMENTAL PROCEDURES |
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Preparation of Peptides and Proteins-- Twenty-nine synthetic peptides were prepared (Table I), using standard f-moc procedures and purified by high performance liquid chromatography on a Vydac C18 column eluted with a gradient of acetonitrile (0-100%) containing 0.1% trifluoroacetic acid. Both 235-a.a. and 266-a.a. Ad12 E1A proteins were expressed in Escherichia coli and purified by protocols described elsewhere (23). GST-CtBP was expressed in E. coli and purified using glutathione-agarose beads (Sigma).
Interaction of Ad12 E1A Peptides with CtBP-- The interaction of peptides identical to the C-terminal region of Ad12 E1A or mutant peptides (peptides in which substitutions to the wild type sequence have been made are termed "mutants") with CtBP was examined using ELISA techniques. Ninety-six-well plates were coated with purified Ad12 E1A (0.1 µg/well) by incubation overnight at 4 °C. E1A peptides, serially diluted from 1.5-400 µg/ml, were mixed with GST-CtBP (12 µg/ml) for 30 min at room temperature prior to adding to each well. After incubation for 1 h at 37 °C, the plates were washed six times with phosphate-buffered saline containing 0.1% Tween-80. CtBP bound to E1A on the plate was determined by incubation with an antibody against GST (antibody raised in goats, diluted 1:5000; Amersham Pharmacia Biotech) followed by horseradish peroxidase-linked antibody against goat IgG (1:1000 Santa Cruz). After the addition of substrate, absorbance was determined at 405 nm using a Bio-Tek plate reader. Determinations were carried out in quadruplicate, and kinetic analysis was performed (see below).
Kinetic Analysis-- The interactions between CtBP and full-length Ad E1A and between CtBP and synthetic peptides identical to portions of the C terminus of Ad E1A (Table I) were analyzed using a nonlinear least-squares approach. The standard binding function,
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(Eq. 1) |
NMR Spectroscopy -- 1H NMR spectra were recorded on a Bruker AMX500 MHz spectrometer using samples of peptides of between 5 and 10 mM concentration in 50% (v/v) d3 TFE/40% H2O/10% 2H2O, pH 5.5, at 285 K. Trifluoroethanol (TFE) was used as a co-solvent with water because TFE-water mixtures are known to promote hydrogen bond formation within peptides that already possess an intrinsic folding capability (25). Where appropriate (for the Ad12 wild type and Ad5 E*8 peptides), deuterated dithiothreitol was added to a final concentration of 10 mM to prevent intermolecular disulfide bridge formation.
Two-dimensional experiments were acquired with 2000 data points in F2 with a sweep width of 11ppm and with 480-608 rows in F1. The water resonance was suppressed by either a weak presaturation applied during the relaxation delay (1.5 s) or by using a WATERGATE sequence (26). Solvent impurities were removed using pulsed field gradients. Standard pulse sequences were employed for one-dimensional (simple pulse-collect and two-pulse Carr-Purcell spin-echo) experiments and two-dimensional (TOCSY, NOESY, and COSY) experiments. Total correlation spectroscopy (TOCSY) experiments (32-64 transients) used an MLEV-17 mixing pulse of 60 ms duration (10 kHz spin locking field). Thirty-two transients were acquired for double quantum filtered correlation spectroscopy (COSY) experiments. Nuclear Overhauser effect (NOESY) experiments used mixing times of 100, 200, and 400 ms duration with between 96 and 128 transients. TOCSY and double quantum filtered COSY experiments were used to identify spin systems within residues that were linked to sequential NOE assignments (d ![]() |
RESULTS |
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Interactions between Ad12 E1A and CtBP-- Previous studies (21) have shown that a 6-a.a. sequence (279PLDLSC284), located close to the C terminus of Ad5 289-a.a. E1A, is responsible for binding to the 48-kDa cellular phosphoprotein, CtBP. An homologous sequence in the Ad12 protein (255PVDLSV260) was also suggested as a binding site.
Western blotting was used to confirm that bacterially expressed purified Ad12 E1A protein interacts with GST-CtBP (data not shown). To quantify the interaction between E1A and CtBP, an ELISA was developed. This also had the advantage of allowing relatively rapid processing of a large number of samples so that the effects of substitutions within, and deletions of, amino acids adjacent to the binding site on E1A could be determined. The data presented in Fig. 1A show that on addition of increasing concentrations of GST-CtBP to either 266-a.a. or 235-a.a. Ad12 E1A (bound to the ELISA plate), binding occurred. Analysis of these data using Equation 1 (see under "Experimental Procedures") gave values of 4.5 × 10
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Amino Acid Determinants of the CtBP Binding Site on Ad 12 E1A-- In order to define structural elements within the C terminus of Ad12 E1A involved in the interaction with CtBP, four sets of synthetic peptides were prepared (Table I), and the ability of each to inhibit the binding of GST-CtBP to Ad12 E1A was determined. Examples of some competitive binding experiments are presented in Fig. 1B. Initially, peptides equivalent to various lengths of the Ad12 wild type sequence were examined (Table I, set 1). It can be seen that deletion of the amino acids from either end of peptide A4 (Glu249-Val254 and Arg262-Asn266) increased the Kd seen in peptides A5, A6, A7, B5, and B6, indicating that amino acids outside the suggested binding site of E1A (21) contribute to structural determinants required for optimal recognition by CtBP. This is particularly evident with peptides A6 and A7, which appear to bind CtBP much more weakly than A4. The absence of amino acids at either the N or C terminus will have structural implications for the binding site itself. However, these findings clearly demonstrate that the presence of the amino acid sequence 255PVDLSV260 alone is not sufficient for recognition and strong binding by CtBP and that these residues must adopt an appropriate configuration. It is also possible that the deleted N- or C-terminal residues participate directly in the interaction. The increased Kd values determined for B5 and B6 and the peptides in set 2 (see below) certainly supports the proposition that amino acids on both sides of the proposed site make sufficient contribution to binding to change the Kd severalfold. It should be noted, however, that although these truncations reduce the affinity (i.e. increase Kd), they do not totally abolish interactions. Thus, there is not an absolute requirement for a structured binding site, although its presence certainly strengthens binding.
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Amino Acid Determinants of the CtBP Binding Site on Ad5 E1A-- Because CtBP is able to interact with C-terminal regions of both Ad12 and Ad5 E1A, we investigated whether a synthetic peptide equivalent to the Ad5 protein would compete with the Ad12 E1A for CtBP. It was found that a peptide equivalent to Ad5 E1A (Table I, set 4, F*1) binds considerably more weakly to CtBP than peptide A4 (set 1), suggesting that the CtBP·Ad12 E1A complex may be inherently more stable than the CtBP·Ad5 E1A complex. However, a peptide, E*8 carrying the N-terminal Ad12 sequence and spanning the C terminus of Ad5, and thereby carrying Val254 (see set 1, above) was found to have a Kd similar to the wild type Ad12 peptide. These findings suggest again that Val254 contributes to the interaction with CtBP.
Structure of the C Terminus of Ad12 E1A--
Having established
that there are considerable conformational restraints imposed on the
binding site for CtBP, we considered that it would be of appreciable
interest to investigate the structure adopted by this region of Ad E1A.
To obtain as much information as possible, a peptide identical to
residues Glu247-Asn266 of Ad12 E1A was
examined by 1H NMR spectroscopy (Fig.
2). Although this peptide bound to CtBP with affinity equivalent to that for A4, it was anticipated that the
extra 2 a.a. at the N terminus would contribute to the stability of the solution conformation. The NOE cross-peaks observed in the
amide- region of a NOESY spectrum (mixing time, 200 ms) along with a
summary of the observed NOEs are shown in Fig. 2. Whereas weak
connectivities of the type d
N(i,
i+2) Glu247-Glu249,
Glu251-Thr253, and
Gln252-Val254 were observed, the conformation
over residues Glu247-Glu251 is poorly defined,
and in the absence of medium range NOEs, residues Pro263-Asn266 show evidence of pronounced
fraying (Fig. 3). However, NOEs of the
type d
N(i, i+2)
Val256-Leu258,
Asp257-Ser259,
Leu258-Val260,
Ser259-Lys261,
Val260-Arg262, dNN(i,
i+2) Val256-Leu258,
Leu258-Val260, and weak
d
N(i, i+3)
Val254-Asp257 and
Asp257-Val260 (Fig. 2B) indicate
that on average, the backbone displays a considerable conformational
preference between residues Val254 and Arg262,
illustrated by good alignment between the superimposed structures (Fig.
3).
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Effect of Mutation on the Structure of the Ad12 Peptide--
The
data presented in Table I clearly indicate that different substitutions
within, and deletions outside, the proposed binding site result in a
reduction in affinity of Ad12 E1A peptide (and by implication intact
E1A) for CtBP. We considered that it would be interesting to examine
the effects of these amino acid substitutions on the peptide structure.
First, we examined the structural consequences of the substitution
L258G for the wild type peptide. Thus, peptide C4 (L258G), with a
Kd 12-fold greater than A4 (wild type), was
examined. NOESY experiments (mixing time, 200 and 400 ms at 285 K) were
performed on a sample of the peptide (6 mM, 50% TFE, pH
5.5). Only the sequential dN(i,
i+1) connectivities and a single weak NOE of the type
d
N(i, i+2)
Val254-Val256 were observed (Fig.
4). The substitution L258G would be
predicted to result in the formation of a type II turn over the
sequence Val256-Asp257-Gly258-Ser259
because Gly is commonly found at position (i+3) in a turn of this type (35). However, the absence of medium range NOEs between the
residues spanning the C terminus indicates that this region adopts a
predominately unfolded conformation. This finding is consistent with
the high 3JNH-
coupling constants
(~7-8 Hz, Fig. 4) and indicates that the peptide looses
-turn
propensity in solution. Thus, the increase in Kd
most probably reflects the loss of the hydrophobic side chain of
Leu258 upon substitution with Gly or disruption of the
hydrophobic cluster formed by Val256, Leu258,
and Val260 in the wild type peptide (see above). This
finding suggests that the peptide may only adopt a more ordered
conformation in the presence of CtBP, although confirmation of this
will have to await further investigation.
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Structure of the C Terminus from Ad5 E1A--
The data presented
in Table I indicate that Ad12 E1A peptides interact more strongly with
CtBP than the Ad5 peptide (F*1), suggesting that Ad12 sequence
Gln252-Val254, which is absent in Ad5,
contributes to the interaction between Ad12 E1A and CtBP. Thus, it was
considered important to examine the structure of peptide E*8. For
simplicity, the N-terminal Ad12 residues Glu251,
Gln252, Thr253, and Val254 of
peptide E*8 are numbered using the Ad5 13SE1A nomenclature, and a
summary of the observed NOEs, together with the calculated structure of
this peptide, are illustrated in Fig. 6.
Owing to overlap of some NOESY spectral signals some NOEs
(dN(i, i+2) Val278-Leu280) are unassignable, and as a
consequence of shorter sequence length (compared with wild type Ad12),
some NOEs are weak (d
N(i, i+2)
Leu280-Leu282 and d
N
(i, i+2) Pro279-Asp281.
Thus, there is some variation in the precise backbone conformation over
residues Glu275-Val278, which forms a
"frayed" end, and consequently, the implications of the presence of
Val278 (Val254 in Ad12) for the structure
cannot be fully assessed. However, over the sequence
Pro279-Cys284, cross-peaks of the type
d
N(i, i+2)
Leu280-Leu282,
Asp281-Ser283,
Leu282-Cys284,
Ser283-Lys285; d
N
(i, i+2) Pro279-Asp281,
Asp281-Ser283, and
Cys284-Arg286 and
3JNH-
coupling constants in the
range 5.8-8.0 Hz (Fig. 6A) define a series of overlapping
-turns, again illustrated by good alignment between the superimposed
C-
traces over the region Pro279-Cys284
(Fig. 6B). Interestingly, the averaged conformational
preference calculated for the Ad5 peptide (Fig. 6A) is
comparable to the wild type Ad12 peptide as the backbone conformation
(i.e.
-turn) is maintained by Leu280 and
Cys284 (Ad5) and Val256 and Val260
(Ad12) (see above). Thus, the increase in the Kd for F*1 compared with A4 (Table I) implies that the global interactions between CtBP and Ad5 E1A compared with Ad12 E1A are different although,
this would not be identified in these NMR studies.
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DISCUSSION |
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It is now well established that Ad E1A exerts its influence on adenovirus-infected and E1 transformed cells through a complex series of protein-protein interactions (reviewed, for example, in Refs. 6 and 36). The main purpose of these interactions is to allow the virus to usurp control of the cell's machinery for DNA synthesis. One of the more recently characterized proteins which is bound by Ad E1A is CtBP. It seems to play a role in the regulation of transcription mediated through the CR1 region of E1A (19).
The binding site for CtBP on Ad E1A has previously been mapped, by deletional analysis, to a highly conserved region very close to the C terminus (21). In that original study, pairs of amino acids were deleted in turn, and the ability of the mutant proteins to bind CtBP was assessed by co-precipitation. It has been our intention in the work presented here to enlarge upon these initial studies and to investigate the structural determinants on Ad E1A which are required for binding of CtBP. To this end, an ELISA was developed that allowed us to assess the ability of synthetic peptides, equivalent to the C-terminal region of Ad12 E1A to compete with full-length Ad12 E1A for the binding site on CtBP. The binding constants, Kd values, for these peptides (summarized in Table I) have allowed us to draw two major conclusions about the binding site for CtBP on Ad12 E1A (and by implication on Ad5 E1A as well).
First and foremost, structural integrity of the binding site is essential for the interaction with CtBP to occur. Peptides carrying deletions of amino acids at the N or C terminus of the peptide (A6 and A7), bind to CtBP only weakly (Table I). Thus, the binding site for CtBP extends over a slightly larger sequence than that originally suggested, (Pro279-Cys284 on Ad5 E1A) by Scheaper et al. (21). The data summarized in Table I indicate that Val254, which resides N-terminal to the original proposed site of interaction in Ad12 E1A, also makes a contribution to binding. This is supported by the finding that inclusion of residues Glu251-Val254 (from Ad12) at the N terminus of the Pro280-Cys284 motif from Ad5 enhances the binding of peptide E*8 to CtBP compared with the wild type Ad5 peptide F*1 (Table I). On the whole, however, the data presented in Table I are, apart from this, consistent with amino acids Pro255-Val260 comprising the major portion of the CtBP binding site in Ad12 E1A, as suggested (21).
The second conclusion, drawn on the basis of the NMR studies, is that
the region of the wild type Ad12 synthetic peptide equivalent to the
binding site displays a conformational preference for -turns stabilized (for at least part of the time) by intramolecular hydrogen bonds. A similar structure was observed for the peptide equivalent to
the binding site of Ad5 E1A (Fig. 6). Thus, the differences between the
amino acid sequences of the two E1A proteins from different virus
serotypes (in particular the Cys284-Val260
substitution in Ad5 compared with Ad12) do not alter the overall solution structure of the binding site. It appears that substitutions in the peptides which radically reduce the affinity for CtBP
(i.e. increase the Kd) either disrupt the
solution structure (for example, peptide C4; (L258G) and most likely
peptide D*2 (P255A)) or alter some side chain contribution required for
recognition by CtBP (peptide A*7, V256K). Although overall, the
-turn conformation remains intact, it is clear that this particular
substitution in A*7 affects the integrity of the turn adopted over
residues Val254-Asp257 (Fig. 5) and yet
increases the Kd (Table I). It is possible that this
is attributable to the positively charged Lys side chain, which
disrupts important hydrophobic interactions formed between E1A and
CtBP, thereby reducing the affinity. Thus, it seems that the overall
-turn conformation in conjunction with the appropriate amino acid
side chains is required for optimum binding. Changes in either of these
parameters will result in an increase in Kd. It
should be noted, however, that some peptides (for example, A7, C5, and
C6) have Kd values considerably in excess of that
for C4. It appears that the single d
N(i, i+2) NOE
Val254-Val256 defines a
-turn over residues
Thr253-Val256, whereas the C-terminal 8 residues are ill-defined. These findings imply that peptide C4 binds
weakly to CtBP, even though few elements of the wild type structure
were detected in the absence of the target protein. A similar situation
is likely to occur for some other mutations (e.g. in
peptides C5 and C6). It is likely that structural integrity of the
binding site is required for strong binding of E1A peptides to CtBP,
although predominately unstructured peptides bind weakly.
The data presented define a conformational preference in solution over
residues Glu249-Asn266 that form the binding
site for CtBP on Ad12 E1A. Within the environment of a rigid protein,
-helices and
-sheets are commonly connected by loops (see, for
example, Ref. 37). Interestingly,
-turns have been observed in
Aspergillus oryzae
-amylase (38), CoA-binding protein
aconitase (39), galactose oxidase (40), wheat germ agglutinin (41),
cytochrome c peroxidase (42), and EcoRV
endonuclease (43). It is well known that trifluoroethanol stabilizes
-helical conformations in small peptides that posses a high
intrinsic helical capability (25), whereas
-turns can remain
unaffected (for example, whale myoglobin G-H hairpin peptide (25)), or
are occasionally enhanced in TFE (see, for example, Ref. 44).
Therefore, it was of interest to note that the latter peptide is
equivalent to a sequence that forms part of the hydrophobic core of
-lactalbumin and adopts a helical conformation in the crystal
structure of the native form of the protein (45).
The above discussion implies that -turns adopted by the backbone of
small flexible peptides may not accurately reflect the structure
adopted over the corresponding region within the native protein. Thus,
protein folding patterns are dependent on global tertiary interactions,
which play a role in determining the propensity of individual amino
acids to form secondary structure, most notably
-strands (46-48).
Such interactions would not be observed in the study described here.
However, the E1A peptides used in this study are equivalent to
sequences at the extreme C terminus and potentially provide information
concerning the structure on the surface of the molecule. This
observation is consistent with the view that short isolated peptides
can form significant amounts of secondary structure in solution (49,
50). It was therefore of considerable interest to note that a 13-a.a.
peptide equivalent to the C terminus of yeast
-MF adopts a
-turn
conformation, which is deemed to be essential for biological activity
of the protein (51). Similarly, the C terminus of Tax has been shown to
be responsible for interactions with the PDZ domain of post synaptic
density protein PSD-95 (52, 53). Furthermore, the C terminus of
-amylase forms two
-turns (38), whereas the
-turn formed by
residues PPQEE at the C terminus of integrin
V subunit is required
for transmembrane signaling (54). In view of these observations, it is
probable that the structures determined for the Ad wild type synthetic
peptides are the same as those present at the C terminus of the intact protein.
Interestingly, the nuclear localization signal for Ad E1A has been
mapped to the basic amino acids immediately C-terminal to the CtBP
binding site. It can be seen from the structures shown in Figs. 3, 5,
and 6 that these amino acids form the C terminus of the series of
-turns discussed above. The fact that these two binding sites occur
within a 10-amino acid sequence at the C terminus of E1A suggests that
this highly conserved domain is likely to be on the surface of the
protein making it available for binding. The juxtaposition of binding
sites involved in CtBP interaction and nuclear localization implies
that transport of E1A to the nucleus precedes complex formation with
CtBP as simultaneous interaction is likely to be spatially restricted.
Confirmation of this will, however, have to await more detailed
study.
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ACKNOWLEDGEMENTS |
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We are most grateful to The Wellcome Trust and The University of Birmingham Biomedical NMR unit for the provision of facilities maintained by A. J. Perberton and to Dr. K. John Smith (School of Biochemistry) for helpful discussions. We thank Drs. Helen Mott and Darerca Owen (University of Cambridge) and Dr. Noeleen Keane (University of Sussex) for critically reading the manuscript.
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FOOTNOTES |
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* Financial support was provided by the Cancer Research Campaign.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.
§ To whom correspondence should be addressed: Tel.: 0121-414-4483; Fax: 0121-414-4486; E-mail: D.P.Molloy{at}cancer.bham.ac.uk.
A Cancer Research Campaign Gibb Fellow.
The abbreviations used are: Ad E1A, adenovirus early region 1A; ELISA, enzyme-linked immunosorbent assay; CtBP, C-terminal binding protein; TFE, trifluoroethanol; CR, conserved region; TOCSY, total correlation spectroscopy; NOESY, nuclear Overhauser effect spectroscopy; a.a., amino acid(s); GST, glutathione S-transferase.
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REFERENCES |
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