Structural determinants present in the C-terminal binding protein binding site of adenovirus early region 1A proteins.
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.
Cancer Research Campaign Institute for Cancer Studies, University of Birmingham, Edgbaston, Birmingham B15 2TA, United Kingdom. D.P.Molloy@cancer.bham.ac.uk
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Molloy
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Milner
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Yakub
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Chinnadurai
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Gallimore
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Grand
D017500:Adenovirus E1A Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D004797:Enzyme-Linked Immunosorbent Assay; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation
D017500:Adenovirus E1A Proteins