The structure of the site on adenovirus early region 1A responsible for binding to TATA-binding protein determined by NMR spectroscopy

Previous detailed mutational analysis has shown that the binding site on ad enovirus (Ad) early region 1A (E1A) for TATA-binding protein (TBP) is locat ed toward the N terminus of conserved region 3 (CR3), Here we demonstrate t hat synthetic peptides of between 15 and 22 amino acids, identical to amino acid sequences of CR3 present in the larger Ad5 E1A (13 S product) and in both the Ad12 E1A (13 and 12 S products) proteins that lie N-terminal to th e zinc finger motif, can disrupt binding of E1A to TBP, These findings sugg est that the peptides are biologically active in terms of interacting with TBP and must therefore comprise some, if not all, of the TBP binding site o n E1A, The interaction between Ad12 E1A and TBP was confirmed by direct co precipitation experiments, In H-1 NMR studies of CR3 peptides, regular patt erns of NOEs were observed from which their conformational preferences in a queous solution were determined. Both Ad5 and Ad12 peptides were shown to c ontain regions of helical backbone structure in 50% trifluoroethanol. In ea ch case, the type and intensities of NOE cross-peaks observed correlated be st to alpha-helical turns. These helices are more extensive in larger pepti des and extend from Glu(141) to Val(147) and from Arg(144) to Pro(152) in t he full-length Ad5 and Ad12 13S E1A proteins, respectively. The structure o f a 19-residue Ad5 CR3 peptide carrying the V147L mutation in the full-leng th protein that abolishes TBP binding was examined. No significant differen ces between the substituted and wild type peptides were observed, suggestin g that this substitution in the intact protein may cause disruption of glob al rather than local structures.