Acetylation at a lysine residue adjacent to the CtBP binding motif within adenovirus 12 E1A causes structural disruption and limited reduction of CtBP binding

Abstract

C-terminal binding protein (CtBP) has been shown to bind to a highly conserved five-amino-acid motif (PXDLS) located very close to the C-terminus of adenovirus early region 1A proteins. It has also been demonstrated that amino acids C-terminal and N-terminal to this original proposed binding site contribute to the interaction. However, conflicting evidence has been presented to show that acetylation of an adjacent lysine residue in Ad5E1A may or may not influence binding. It has now been demonstrated here that acetylation of a lysine, equivalent to position 261 in Ad12 E1A and position 285 in Ad5E1A, in a synthetic peptide disrupts the binding to CtBP1 and CtBP2 and alters the K(i) of the peptide, indicative of a reduction in the affinity of the peptide for CtBP1 and CtBP2, but only to a rather limited extent (less than 2-fold). The solution structures of synthetic peptides equivalent to wild-type and acetylated forms of the Ad12 E1A peptide have been determined by proton NMR spectroscopy. The wild-type form of the peptide adopts a series of beta-turns over the region Val(254)-Arg(262). Within the acetylated isoform, the beta-turn conformation is less extensive, Val(260)-Arg(262) adopting a random confirmation. We conclude that secondary structure (beta-turns) and an appropriate series of amino acid side chains over an extended binding site (PXDLSXK) are necessary for recognition by CtBP, acetylation of lysine interfering with both of these features, but not to such an extent as to totally inhibit interaction. Moreover, it is possible that the beta-turn conformation at the C-terminus of AdE1A contributes to binding to alpha importin and nuclear import. Acetylation of lysine (261) could disrupt interaction through structural destabilization as well as charge neutralization and subsequent nuclear localization.

Acetylation of Lysine 564 Adjacent to the C-terminal Binding Protein-binding Motif in EVI1 Is Crucial for Transcriptional Activation of GATA2

“…Therefore, the exact position of the acetylated lysine residue relevant to the transcriptional regulation is not important, but the important lysine residue is probably localized within a few amino acids just after the CtBP-binding motif. On the other hand, it has been reported that the acetylation at Lys 239 adjacent to the CtBPbinding motif in E1A causes a limited reduction in its CtBP binding ability as revealed by in vitro binding assays using synthetic peptides (29). In EVI1, CtBP2 was retained in the protein complex with EVI1 and P/CAF on the DNA at the GATA2 promoter region; however, HDAC1 was not included in this protein complex.…”

PLDLS-dependent interaction of E1A with CtBP: regulation of CtBP nuclear localization and transcriptional functions

“…However, the cytoplasmic localization of K239A-E1A may complicate the interpretation (Madison et al, 2002). A recent study using K239-acetylated E1A peptides suggested that Lys239 acetylation may modestly reduce the affinity for CtBP in vitro by inducing structural changes (Molloy et al, 2006).…”

C-terminal binding proteins: central players in development and disease

“…It is also noteworthy that CtBP activity can be regulated by acetylation of CtIPs such as E1A and RIP140. In both cases, acetylation of lysine residues flanking the PXDLS-binding motif located in these proteins results in decreased CtBP association and reduced transcriptional repression (58)(59)(60). Thus, CtBP activity can be regulated indirectly by enhancing or inhibiting its ability to bind CtBP-interacting partners.…”