Structural determinants outside the PXDLS sequence affect the interaction of adenovirus E1A: C-terminal interacting protein and Drosophila repressorswith C-terminal binding protein

C-Terminal binding protein (CtBP) interacts with a highly conserved amino a cid motif (PXDLS) at the C terminus of adenovirus early region 1A (AdE1A) p rotein. This amino acid sequence has recently been demonstrated in the mamm alian protein C-terminal interacting protein (CtIP) and a number of Drosoph ila repressors including Snail, Knirps and Hairy. In the study described he re we have examined the structures of synthetic peptides identical to the C tBP binding sites on these proteins using NMR spectroscopy. It has been sho wn that peptides identical to the CtBP binding site in CtIP and at the N te rminus of Snail form a series of p-turns similar to those seen in AdE1A. Th e PXDLS motif towards the C terminus of Snail forms an a-helix. However, th e motifs in Knirps and Hairy did not adopt well-defined structures in TFE/w ater mixtures as shown by the absence of medium range NOEs and a high propo rtion of signal overlap. The affinities of peptides for Drosophila and mamm alian CtBP were compared using enzyme-linked immunosorbent assay. CtIP, Sna il (N-terminal peptide) and Knirps peptides all bind to mammalian CtBP with high affinity (K-i of 1.04, 1.34 and 0.52 muM, respectively). However, dif ferent effects were observed with dCtBP, most notably the affinity for the Snail (N-terminal peptide) and Knirps peptides were markedly reduced (K-i o f 332 and 56 muM, respectively) whilst the Hairy peptide bound much more st rongly (Ki for dCtBP of 6.22 compared to 133 muM for hCtBP). In addition we have shown that peptides containing identical PXDLS motifs but with differ ent N and C terminal sequences have appreciably different affinities for ma mmalian CtBP and different structures in solution. We conclude that the fac tors governing the interactions of CtBPs with partner proteins are more com plex than simple possession of the PXDLS motif. In particular the overall s econdary structures and amino acid side chains in the binding sites of part ner proteins are of importance as well as possible global structural effect s in both members of the complex. These data are considered evidence for a multiplicity of CtBPs and partner proteins in the cell. (C) 2001 Published by Elsevier Science B.V.