The metallo-peptide [G(29)T(S)]Fe-2 contains two copies of the DNA recognition peptide of the yeast bZIP protein GCN4 assembled into a dimer with a 4'-substituted bis(terpyridyl)iron(II) complex. [G(29)T(S)]Fe-2 contains the same DNA recognition peptide as GCN4, yet it possesses a function that GCN4 does not: it discriminates between the CRE (ATGACGTCAT) and AP-1 (ATGACTCAT) target sites, two bZIP target sites that differ by the presence or the absence of a single G . C base pair. In terms of its CRE/AP-1 specificity, [G(29)T(S)]Fe-2 resembles the bZIP proteins CREB and CRE-BP1, whose biological functions require accurate discrimination of these two target sites. Here are described a series of experiments that explore the molecular basis for the high CRE/AP-1 specificity of [G(29)T(S)]Fe-2 and its homologue [G(28)T(S)]Fe-2. Quantitative analysis of equilibrium dissociation constants reveals that the stabilities of the [G(28)T(S)]Fe-2 . CRE and [G(29)T(S)]Fe-2 . CRE complexes are no higher than those of the corresponding disulfide-dimer . CRE complexes. In addition, the phosphate interference patterns of the [G(28)TS]Fe-2 . CRE and [G(29)T(S)]Fe-2 . CRE complexes superpose on those of the corresponding disulfide-dimer . CRE complexes. Finally, helical phasing analysis reveals that the metallo-peptides and the disulfide-dimer peptides all induce equivalent distortions in the DNA. However, CRE/AP-1 specificity is eliminated when the bis(terpyridyl)iron(II) complex is replaced by a sterically less-demanding bipyridyl moiety. This result, analyzed in the context of recently solved structures of GCN4 bound to the CRE and AP-1 target sites, leads us to propose that CRE/AP-1 specificity results from interactions between the bis(terpyridyl)iron(II) complex and the proximal region of the peptide that disrupts one or more critical protein AP-l interactions. Remarkably, the mechanism of CRE/AP-1 specificity proposed for the metallo-peptide bZIP models miners, at least in part, the mechanism employed by the naturally CRE-selective proteins CREB and CRE-BP1. Our observation that subtle and indirect effects on the conformation of a short peptide can lead to large changes in DNA target specificity provides evidence that it may be possible to design surprisingly small molecules that bind DNA with high sequence-specificity as well as high affinity.
