The effect of CtBP1 binding on the structure of the C-terminal region of adenovirus 12 early region 1A

Adenovirus early region 1A (AdEA) binds to the C-tenninal binding protein 1 (CtBP1) primarily through a highly conserved PXDLS motif located close to its C-terminus. Purified synthetic peptides equivalent to this region of AdE1A have been shown to form a series of beta-turns. In this present study the effect of CtBPI binding on the conformation of C-terminal region of Ad12E1A has been investigated. Using one- and two-dimensional H-1 NMR spectroscopy, the conformation of 20-residue peptides equivalent to amino acids I-241-V-260 and E-247 -N 266 of Ad] 2E1A were examined in the absence of CtBP I. Whilst the latter peptide forms a series of beta-turns in its C-tenninal half as reported previously, the former peptide is a-helical over the region D-243-Q(253). Upon interaction with CtBP1 the conformation of the backbone in the region 211 (PVDLCVK261)-P-255 of the Ad12E1A E-247 -N-266 peptide reorganises from a predominately beta-turn to an a-helical conformation. This structural isomerisation is characterised by a shift upfield of 0.318 pprn for the delta-CH3 proton resonance of V-256. 2-D NOESY experiments showed new signals in the amide-alpha region which correlate to transferred NOEs from the protein to the peptide residues E-251, V-256 and K-261. In further analyses the contribution of individual amino acids within the sequence (VPVDLS259)-V-254 was assessed for their importance in determining structure and consequently affinity of the peptide for CtBP. It has been concluded that Ad12E1A residues (255)p-V-260 serve initially as a recognition site for CtBP and then as an anchor through a beta-turns ->alpha-helix conformational rearrangement. In addition it has been predicted that regions N-tenninal to the PXDLS motif in AdE1As from different virus serotypes and from mammalian proteins form alpha-helices. (C) 2007 Elsevier Inc. All rights reserved.