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

Abstract

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 K d 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.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 (17,1...

Two Nonconsensus Sites in the Epstein-Barr Virus Oncoprotein EBNA3A Cooperate to Bind the Co-repressor Carboxyl-terminal-binding Protein (CtBP)

“…A recent study of the CtBPbinding site in E1A indicated that the initial proline residue in PXDLS was extremely important for the efficient binding and mutation to alanine dramatically reduced the affinity between CtBP and a peptide (19). Consistent with this observation, various cross-species comparisons of well characterized CtBPbinding sites showed that the initial proline is almost invariably conserved (15,18).…”

“…All these various factors that regulate transcription and bind to CtBP contain a conserved Pro-X-Asp-Leu-Ser ("PXDLS") CtBPinteraction domain that is necessary and probably sufficient for the interaction. The conserved proline and leucine residues are generally considered essential for efficient binding (15,18,19). A second mammalian CtBP was recently described and the two family members, which are referred to as CtBP1 and CtBP2, are largely homologous although they may have distinct tissue distributions (20).…”

Acetylation of adenovirus E1A regulates binding of the transcriptional corepressor CtBP

“…Most interactions involving CtBP occur through the binding motif, PXDLS. Recent studies, however, have suggested that the binding site in E1A for CtBP extends over a slightly larger sequence than originally suggested (48). In many instances, this motif is followed at the carboxyl-terminal end by a Lys residue, but the function of this Lys has never before been addressed.…”

“…It is likely, therefore, that the fraction of E1A that is acetylated at Lys-239 is highly deficient in its ability to bind CtBP. To determine the effects of the E1A mutations and acetylation more quantitatively, we used an ELISA (48). Briefly, this method involved mixing a GST-fusion protein containing the carboxyl-terminal 67 amino acids of E1A (GST-E1ACter) with immobilized full-length CtBP in the presence of varying concentrations of wild-type and mutated E1A peptides.…”

Adenovirus E1A: remodelling the host cell, a life or death experience

“…There are a number of reasons for this, over expression of E1A in eukaryote cells leads to apoptosis (White, 1998), the majority of E1A expressed in E. coli is insoluble or it may have only limited structure until it is complexed with partner proteins (Bayley and Mymryk, 1994). Using peptide mimicry and NMR spectroscopy we have resolved structures for the N-terminal portion of CR3, the TBP binding site (Molloy et al, 1999), this was found to form an a helix whilst the CtBP binding site (including the PXDLS motif, Shaeper et al, 1995) formed a series of b turns (Molloy et al, 1998). Bioinformatic predictions suggest that E1A also has secondary structure at the Nterminus over the region where a number of cellular proteins bind (Molloy, personal communication).…”