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

Abstract

Adenovirus early region 1A (AdE1A) binds to the C-terminal 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 β-turns. In this present study the effect of CtBP1 binding on the conformation of C-terminal region of Ad12E1A has been investigated. Using one- and two-dimensional 1H NMR spectroscopy, the conformation of 20-residue peptides equivalent to amino acids I 241–V 260 and E 247–N 266 of Ad12E1A were examined in the absence of CtBP1. Whilst the latter peptide forms a series of β-turns in its C-terminal half as reported previously, the former peptide is α-helical over the region D 243–Q 253. Upon interaction with CtBP1 the conformation of the backbone in the region 255PVDLCVK 261 of the Ad12E1A E 247–N 266 peptide reorganises from a predominately β-turn to an α-helical conformation. This structural isomerisation is characterised by a shift upfield of 0.318 ppm for the δ-CH 3 proton resonance of V 256. 2-D NOESY experiments showed new signals in the amide-α 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 254VPVDLS 259 was assessed for their importance in determining structure and consequently affinity of the peptide for CtBP. It has been concluded that Ad12E1A residues 255P–V 260 serve initially as a recognition site for CtBP and then as an anchor through a β-turns → α-helix conformational rearrangement. In addition it has been predicted that regions N-terminal to the PXDLS motif in AdE1As from different virus serotypes and from mammalian proteins form α-helices.