Structural Determinants Present in the C-terminal Binding Protein Binding Site of Adenovirus Early Region 1A Proteins*

David P. MolloyDagger §, Anne E. MilnerDagger , Imran K. YakubDagger , G. Chinnadurai, Phillip H. GallimoreDagger parallel , and Roger J. A. GrandDagger

From the Dagger  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

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

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 beta -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 (beta -turns) and an appropriate series of amino acid side chains are necessary for recognition by CtBP.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

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.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

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,
Y=Y<SUB><UP>max</UP></SUB> · [L]/([L]+K<SUB>d</SUB>) (Eq. 1)
was applied, where Y represents the percentage of E1A bound to GST-CtBP in the presence of varying amounts of E1A peptide, [L], Kd represents the equilibrium binding constant, and Ymax represents the end point for the binding interaction (see, for example, Ref. 24). The affinity of CtBP for synthetic peptides was measured by competition with native E1A, and Kd values are quoted as the mean values from four experiments (± S.E.).

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 (dalpha N(i, i+1)) in NOESY spectra.

Inter-proton distances were calculated from NOESY spectra (200-ms mixing time) by integration of each cross-peak. Distances were then grouped into three classes between strong and very weak (strong and medium NOEs were allocated the same distance constraints in structural calculations). Backbone hydrogen bonding patterns within the peptides were assessed by following the movement of chemical shift of backbone amide protons with changes in temperature (at 285, 290, 295, 300, and 305 K) in TOCSY experiments. Temperature shift coefficients were calculated from the slope of a plot of temperature against chemical shift and were linear in every case.

The structures of the peptides were calculated using X-PLOR 3.8.5.1 (27). The CA, HA, N, HN, CB*, and CG* atoms of each residue were embedded using the distance geometry dg-sub-embed routine. The remaining atoms were ordered by template fitting, and the atomic coordinates were allowed to evolve under the applied NOE distance constraints during the dgsa and refine simulated annealing subroutines. From 100 structures calculated, approximately 30 were discarded (wrong-handed in the distance geometry routine). Of the remaining structures, only those (47 structures) with no violations of the applied NOEs greater than 0.5 Å were selected for further analysis. Donor-acceptor pairs were identified by analysis of the structures in the absence of hydrogen bond constraints. In every case the donor corresponded to a backbone amide proton signal with a temperature shift coefficient of between 1.0 and 3.0 ppb/K. Final structures were calculated by the inclusion of constraints for hydrogen bonds.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

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 × 10-9 and 10.3 × 10-9 M for the dissociation of GST-CtBP from Ad12 266-a.a. and 235-a.a. proteins, respectively, as has been reported previously (23).


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Fig. 1.   Analysis of binding interactions between EIA and CtBP using ELISA. A, kinetic analysis of Ad12 235-a.a. E1A and Ad12 266-a.a. E1A complexes with GST-CtBP. Varying concentrations of Ad12 E1A proteins were mixed with GST-CtBP and analyzed using ELISA techniques. CtBP bound to E1A was determined by incubation with an antibody against GST (goat). Following the addition of anti-goat horseradish peroxidase-linked secondary antibody and substrate, absorbance was determined at 405 nm (see under "Experimental Procedures"). The averaged data from four experiments (± S.E.) are plotted against concentration of Ad12 266-a.a. E1A (black-square) and Ad12 235-a.a. E1A (bullet ). The solid and dashed lines represent best fit of the data by Equation 1 using values for the Kd of 4.5 × 10-9 and 10.3 × 10-9 M for Ad12 266-a.a. E1A (---) and Ad12 235-a.a. E1A (- - - -), respectively. B, inhibition of the interaction between Ad12 266-a.a. E1A and CtBP in the presence of synthetic peptides. The ability of synthetic peptides equivalent to the C terminus of Ad12 E1A and Ad5 E1A to inhibit the interaction between CtBP and Ad12 266-a.a. E1A was assessed by ELISA (see under "Experimental Procedures"). The averaged data from four experiments (± S.E.) for peptides A4 (black-down-triangle ), D*1 (open circle ), A*6 (), C5 (down-triangle), and A7 (black-square) are plotted as the percentage of peptide bound to CtBP in the presence of E1A against the concentration of peptide. A 14-a.a. peptide, equivalent to residues Ser42-Phe55 of human thrombin receptor (bullet ) was included as a control and does not bind to CtBP (Kd >> 3.0 × 10-3 M). The solid lines represent best fit of the data by Equation 1 using values for Kd of 2.5 × 10-6, 7.6 × 10-6, 15.5 × 10-6, 120.0 × 10-6, and 332.0 × 10-6 M for peptides A4, D*1, A*6, C5, and A7, respectively.

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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Table I
Sequences of Ad5 and Ad12 synthetic peptides used in this study
The sequences of the 29 synthetic peptides identical to the C termini of Ad12 E1A and Ad5 E1A used in this study are shown. Amino acids Pro279-Cys284, which form the CtBP binding site on Ad5 E1A (21), are underlined in peptide E*8 (set 4). The corresponding sequence of Ad12 E1A (Pro255-Val260) is illustrated in boldface italics in the sequence of peptide A4 (set 1). Set 1, peptides equivalent to various lengths of the wild type Ad12 E1A sequence. Set 2, peptides carrying substitution of individual amino acids with alanine across the CtBP binding site on Ad12 E1A. Set 3, peptides equivalent to Ad12 carrying more radical substitutions across the binding site. Set 4, sequences of peptides equivalent to the C terminus of Ad5 E1A. The affinity of CtBP for each peptide was assessed by ELISA (see under "Experimental Procedures"), and the binding constants were evaluated from best fit of the data for each peptide (n = 4) using Equation 1 and are presented as Kd ± S.E.

In the second set of peptides (Table I) individual residues across the binding site were substituted with alanine. Substitution at positions 253, 257, 260, and 261 (peptides C*8, D*4, D*7, and D*8, respectively) had essentially little or no effect on the determined Kd. However, when Ala replaced residues Val254, Pro255, Val256, Lys258, or Ser259, the ability of the peptide to bind CtBP was appreciably impaired. This confirms that Val254 contributes to binding to CtBP, although not to the same extent as the succeeding five amino acids. Particularly disruptive of binding is the substitution of P255A (D*2), which presumably alters a secondary structural element within the peptide. The observation that the replacement D257A does not affect the interaction implies that the nature of the Asp side chain is not directly involved in binding to CtBP and that the conformational integrity of the peptide is maintained by Ala at that position in the sequence.

In set 3 peptides, rather more radical substitutions have been introduced into the proposed binding site (Table I). The replacement of Val256 by Glu increased the Kd of peptide C1 only modestly, whereas all of the other changes give peptides with much reduced affinity for CtBP. Some changes obviously have a greater effect than others. For example, the substitution L258A (D*5, set 2) or L258G (C4, set 3) gave peptides with affinities of 20 and 29 × 10-6 M, respectively, but the substitution L258E (C5, set 3) reduced the affinity considerably more (Kd = 120 × 10-6 M). These findings imply that in contrast to Asp257, the large hydrophobic moiety of Lys258 is required to participate directly in the binding interaction with CtBP.

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-alpha 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 dbeta 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 dalpha N(i, i+2) Val256-Leu258, Asp257-Ser259, Leu258-Val260, Ser259-Lys261, Val260-Arg262, dNN(i, i+2) Val256-Leu258, Leu258-Val260, and weak dalpha 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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Fig. 2.   1H NMR assignments for the 20-a.a. wild type Ad12 peptide. A, the amide-alpha region of a NOESY spectrum (mixing time, 200 ms duration) collected on a sample of the 20-a.a. Ad12 E1A wild type peptide (at a concentration of 7 mg/ml) of sequence 247EEEREQTVPVDLSVKRPRCN266 in 50% (v/v) TFE/40% H2O/10% 2H2O, pH 5.5, at 285 K is presented. NOEs corresponding to cross-peaks within a single residue are labeled with a single number corresponding to those positions in the sequence of full-length Ad12 266-a.a. E1A. NOEs that define medium range connectivities of the type dalpha N(i, i+2), dbeta N(i, i+2), and two dalpha N(i, i +3) are labeled with both residue numbers. B, summary of NOEs, 3JNHalpha coupling constants and backbone amide proton temperature shift coefficients observed for the 20-a.a. wild type Ad12 peptide based on the numbering system of Ad12 266-a.a. E1A. The sequential (i+1) and medium range (i+2 and i+3) NOEs are represented by the thickness of the bars. For Pro residues, the delta -CH2 signals substitute for the NH protons. More than 95% of proline adopts a trans backbone conformation. 3JNHalpha coupling constants were calculated from one-dimensional pulse-collect spectra in 50% d3-TFE, pH 5.5, at 285 K.


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Fig. 3.   Structural properties of the 20-a.a. wild type Ad12 peptide. The calculated structure for the 20-a.a. Ad12 peptide. Eight superimposed structures are shown out of the 47 that converged from the 100 calculated. In the left panel, both the backbone and side chain atoms are presented. In the right hand panel only the alpha -carbon atoms are shown and residues are labeled using the single-letter abbreviation and position in the sequence of Ad12 266-a.a. E1A. Initial structures for the peptide were calculated in the absence of hydrogen bonds. However, three hydrogen bonds were identified that corresponded to amide protons of low temperature shift coefficients (Ser259, 1.9 ppb/K; Val260, 2.5 ppb/K, Lys261, 2.0 ppb/K, which were included as COi---NHi+3 hydrogen bonds in the calculation of the structures shown. In the final 47 structures calculated, the average root mean square differences from the mean structure were 0.87 ± 0.12 Å for all atoms for the residues between Val254 and Arg262 and 1.52 ± 0.17 Å for all the non-hydrogen atoms. No distance restraint was violated by more than 0.5 Å.

For some peptides in TFE/water mixtures, most notably whale myoglobin G-H hairpin (25) and PDE RD1 splice region (28), NOEs of the type dalpha N(i, i+3) and dalpha N(i, i+4) define tight overlapping alpha -helical turns (29), whereas the relative quantity and magnitude of dalpha N(i, i+3) over dalpha N(i, i+2) would define a 310-helix (29) or type III turn (30, 31). For the Ad12 C terminus peptide, the relative magnitude of dalpha N(i, i+2) over dalpha N(i, i+3) medium range NOEs denote beta -turns extending over the consecutive 4 residues, where the distance from C-alpha (i) to C-alpha (i+3) in each turn is less than 7 Å (32). Turns of this type can also be identified by small 3JNH-alpha coupling constants for residues at positions 2 and/or 3 in the turn and low temperature shift coefficients for the backbone amide proton resonance for the residues at position (i+3) in each turn (33). In addition, the absence of aromatic residues in the C terminus of Ad12 E1A rules out type IV turns of the general form XArPArHp, where X, Ar, and Hp represent any amino acid, an aromatic residue and a small hydrophobic residue, respectively (34).

Although there is some variation in the backbone conformation Glu251-Val254, as a consequence of the weak NOEs dbeta N(i, i+2) Glu251-Thr253 and Gln252-Val254, it appears that the NOE dalpha N(i, i+3) Val254-Asp257 defines a beta -turn over the sequence Val254-Pro255-Val256-Asp257, where the average distance C-alpha i(Val254) to C-alpha i+3(Asp257) is 5.2 Å (Fig. 3). Although Pro has previously been shown commonly to occupy position (i+1) in types I and II beta -turns (35), the absence of Gly at position (i+2), which would introduce a 180° twist in the middle of the turn (35), and the presence of the dalpha N(i, i+3) NOE Val254-Asp257, which would be unobserved in a type II turn (29), indicate that the region between Val254 and Asp257 adopts a type I turn conformation. Over the C terminus of the well-ordered portion of the Ad12 peptide (Asp257-Arg262), the relative magnitude of medium range dalpha N(i, i+2) NOEs (see above) defines a series of overlapping beta -turns formed by residues between Val256 and Ser259, Asp257 and Val260, Leu258 and Lys261, and Ser259 and Arg262, with 3JNH-alpha coupling constants within the range 6-8 Hz (Fig. 2B). The side chain methyl groups of Val256, Leu258, and Val260 are allowed to form a hydrophobic cluster. The type I turn formed over residues (i)Asp257 to (i+3)Val260 is particularly well defined, because dalpha N(i, i+2) Leu258-Val260 and dalpha N(i, i+3) Asp257-Val260 connectivities were observed, and the average distance between Cys-alpha (i) (Asp257) and Cys-alpha (i+3) (Val260) is 4.8 Å.(Fig. 3). Analysis of the initial calculated structures indicated that some degree of intramolecular hydrogen bonding occurs within the peptide and three hydrogen bonds, each occurring over a CO(i)---HN(i+3) donor/acceptor pattern, were observed. These correlated with low temperature dependence of the backbone amide proton chemical shifts for Asp257, Val260, and Lys261 (Fig. 2B). These were included in the final calculations for the Ad12 peptide, and eight superimposed structures are illustrated (Fig. 3).

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 dalpha N(i, i+1) connectivities and a single weak NOE of the type dalpha 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-alpha coupling constants (~7-8 Hz, Fig. 4) and indicates that the peptide looses beta -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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Fig. 4.   Summary of the observed NOEs and 3JNHalpha coupling constants for the C4 peptide showing the sequential and a single medium range cross-peak, which are represented by the thickness of the bars (nomenclature as for Fig. 3). More than 95% of proline adopts a trans backbone conformation, and the delta -CH2 groups substitute for the NH protons in the dalpha N(i, i+1) series of connectivities. 3JNHalpha coupling constants were calculated from one-dimensional pulse-collect spectra in 50% d3-TFE, pH 5.5, at 285 K.

The structure of a second mutant peptide [A*7 V256K] was also studied. This peptide has a higher affinity for CtBP than C4 (a Kd of 12 µM compared with 29 µM; Table I) and might be expected to adopt on average a solution structure closer to that of wild type. A summary of the observed NOEs and the calculated structure of the peptide A*7 are illustrated in Fig. 5. In the absence of medium range connectivities, residues Glu251-Val254 show pronounced fraying. In addition, as a consequence of the V256K mutation, certain NOEs, for example, dalpha N(i, i+2) Val254-Val256, seen in the wild type structure are absent, whereas others, dbeta N(i, i+2) Pro255-Asp257 and Lys256-Leu258 are weak. Consequently, the precise backbone conformation varies over residues Val254-Asp257, disrupting the beta -turn adopted by the corresponding region of the wild type peptide. However, the NOEs dalpha N(i, i+2) Lys256-Leu258, Asp257-Ser259, Leu258-Val260, Val260-Arg262; dbeta N(i, i+2) Asp257-Ser259, Val260-Arg262, and dNN(i, i+2) Val260-Arg262 (Fig. 5A) again define a series of overlapping beta -turns formed by residues between Lys256-Ser259, Asp257-Val260, and Ser259-Arg262. Therefore, the averaged conformational preference of peptide A*7 over the region Lys256-Arg262 is similar to that adopted over the region Val256-Arg262 in the wild type peptide (see above). However, the increase in Kd for CtBP (Table I) is likely to result from a combination of a disruption of the beta -turn structure over residues Val254-Asp257 and acquisition of the positively charged, freely flexible side chain of lysine.


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Fig. 5.   . Structural properties of the 14-a.a. A*7 Ad12 peptide. A, summary of the observed NOEs, 3JNHalpha coupling constants and temperature shift coefficients for the A*7 peptide showing the sequential and medium range cross-peaks, which are represented by the thickness of the bars (nomenclature as for Fig. 3). More than 95% of proline adopts a trans backbone conformation, and the delta -CH2 groups substitute for the NH protons in the dalpha N(i, i+1) series of connectivities. 3JNHalpha coupling constants were calculated from one-dimensional pulse-collect spectra in 50% d3-TFE, pH 5.5, at 285 K. B, the calculated structure for the A*7 peptide. Nine superimposed structures are shown out of the 32 that converged from 75 calculated. In the left panel, both the backbone and side chain atoms are shown. In the right panel, only the alpha -carbon atoms are shown. Residues from the corresponding region of full-length Ad12 266-a.a. E1A are labeled using the single-letter abbreviation and position in the sequence. In the final 32 structures calculated, the average root mean square differences from the mean structure were 0.75 ± 0.21 Å for all atoms of residues between Lys256 and Arg262 and 1.42 ± 0.15 Å for all non-hydrogen atoms. No distance restraint was violated by more than 0.5 Å.

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 (dalpha N(i, i+2) Val278-Leu280) are unassignable, and as a consequence of shorter sequence length (compared with wild type Ad12), some NOEs are weak (dalpha N(i, i+2) Leu280-Leu282 and dbeta 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 dalpha N(i, i+2) Leu280-Leu282, Asp281-Ser283, Leu282-Cys284, Ser283-Lys285; dbeta N (i, i+2) Pro279-Asp281, Asp281-Ser283, and Cys284-Arg286 and 3JNH-alpha coupling constants in the range 5.8-8.0 Hz (Fig. 6A) define a series of overlapping beta -turns, again illustrated by good alignment between the superimposed C-alpha 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. beta -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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Fig. 6.   Structural properties of the 14-a.a Ad5 E*8 peptide. A, the NOEs observed, 3JNHalpha coupling constants, and temperature shift coefficients for the E*8 peptide using the nomenclature for Ad5 266-a.a E1A are summarized. The sequential (i+1) and medium range (i+2) connectivitites are represented by the thickness of the bars. Prolines at positions 279 and 287 adopt a trans backbone conformation, and the delta -CH2 signals substitute for the NH protons in the summary of the sequential (i+1) connectivities. 3JNHalpha coupling constants were calculated from one-dimensional pulse-collect spectra in 50% d3-TFE, pH 5.5, at 285 K. B, the calculated structure for the E8* peptide. Seven superimposed structures are shown out of 45 that converged from 100 calculated. Residues are labeled using the nomenclature for full-length Ad5 289-a.a. E1A using the single-letter abbreviation and position in the sequence. In the left panel, both the backbone and the side chain atoms are shown. In the right panel, only the alpha -carbon atoms are presented. From the final 45 structures calculated, the root mean square deviations from the mean structure for all atoms between Pro279 and Cys284 were 0.83 ± 0.17 Å and 1.75 ± 0.25 Å for all non-hydrogen atoms. No distance restraint was violated by more than 0.5 Å.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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 beta -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 beta -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 beta -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 dalpha N(i, i+2) NOE Val254-Val256 defines a beta -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, alpha -helices and beta -sheets are commonly connected by loops (see, for example, Ref. 37). Interestingly, beta -turns have been observed in Aspergillus oryzae alpha -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 alpha -helical conformations in small peptides that posses a high intrinsic helical capability (25), whereas beta -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 alpha -lactalbumin and adopts a helical conformation in the crystal structure of the native form of the protein (45).

The above discussion implies that beta -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 beta -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 alpha -MF adopts a beta -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 alpha -amylase forms two beta -turns (38), whereas the beta -turn formed by residues PPQEE at the C terminus of integrin alpha 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 beta -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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* 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.

parallel 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.
    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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