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J Biol Chem, Vol. 274, Issue 6, 3503-3512, February 5, 1999

The Structure of the Site on Adenovirus Early Region 1A Responsible for Binding to TATA-binding Protein Determined by NMR Spectroscopy

David P. Molloy, K. John Smith^§, Anne E. Milner, Phillip H. Gallimore, and Roger J. A. Grand

From the  CRC Institute for Cancer Studies, University of Birmingham, Edgbaston, Birmingham B15 2TA, United Kingdom and ^§ School of Biochemistry, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom

Previous detailed mutational analysis has shown that the binding site on adenovirus (Ad) early region 1A (E1A) for TATA-binding protein (TBP) is located toward the N terminus of conserved region 3 (CR3). Here we demonstrate that 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 the zinc finger motif, can disrupt binding of E1A to TBP. These findings suggest 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 on E1A. The interaction between Ad12 E1A and TBP was confirmed by direct co-precipitation experiments. In ¹H NMR studies of CR3 peptides, regular patterns of NOEs were observed from which their conformational preferences in aqueous solution were determined. Both Ad5 and Ad12 peptides were shown to contain regions of helical backbone structure in 50% trifluoroethanol. In each case, the type and intensities of NOE cross-peaks observed correlated best to -helical turns. These helices are more extensive in larger peptides and extend from Glu¹⁴¹ to Val¹⁴⁷ and from Arg¹⁴⁴ to Pro¹⁵² in the full-length Ad5 and Ad12 13S E1A proteins, respectively. The structure of a 19-residue Ad5 CR3 peptide carrying the V147L mutation in the full-length protein that abolishes TBP binding was examined. No significant differences between the substituted and wild type peptides were observed, suggesting that this substitution in the intact protein may cause disruption of global rather than local structures.

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