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 bin d to a highly conserved six-amino acid motif very close to the C termi nus of adenovirus early region 1A (Ad EIA) proteins. We have developed an enzyme-linked immunosorbent assay that has facilitated the screeni ng of synthetic peptides identical or similar to the binding site on A d E1A for their ability to bind CtBP and thus inhibit its interaction with Ad12 Elk It has been shown that amino acids both C-terminal and N -terminal to the original proposed binding site contribute to the inte raction of peptides with CtBP, Single amino acid substitutions across the binding site appreciably alter the K-d of the peptide for CtBP, in dicative of a marked reduction in the affinity of the peptide for CtBP , The solution structures of synthetic peptides equivalent to the C te rmini of both Ad5 and Ad12 E1A and two substituted forms of these have been determined by proton NMR spectroscopy. Both the Ad12 and Ad5 pep tides dissolved in trifluoroethanol/water mixtures were found to adopt regular secondary structural conformations seen as a series of beta-t urns. An Ad12 peptide bearing a substitution that resulted in only ver y weak binding to CtBP (Ad12 L258G) was found to be random coil in sol ution, However, a second mutant (Ad12 V256K), which bound to CtBP rath er more strongly (although not as well as the wild type), adopted a co nformation similar to that of the wild type, We conclude that secondar y structure (beta-turns) and an appropriate series of amino acid side chains are necessary for recognition by CtBP.