Synthetic peptides that specifically bind nuclear hormone receptors offer an alternative approach to small molecules for the modulation of receptor signaling and subsequent gene expression. Here we describe the design of a series of novel stapled peptides that bind the coactivator peptide site of estrogen receptors. Using a number of biophysical techniques, including crystal structure analysis of receptor-stapled peptide complexes, we describe in detail the molecular interactions and demonstrate that all-hydrocarbon staples modulate molecular recognition events. The findings have implications for the design of stapled peptides in general.

Z-DNA, the left-handed conformer of DNA, is stabilized by the negative supercoiling generated during the movement of an RNA polymerase through a gene. Recently, we have shown that the editing enzyme ADAR1 (double-stranded RNA adenosine deaminase, type 1) has two Z-DNA binding motifs, Zalpha and Zbeta, the function of which is currently unknown. Here we show that a peptide containing the Zalpha motif binds with high affinity to Z-DNA as a dimer, that the binding site is no larger than 6 bp and that the Zalpha domain can flip a range of sequences, including d(TA)3, into the Z-DNAconformation. Evidence is also presented to show that Zalpha and Zbeta interact to form a functional DNA binding site. Studies with atomic force microscopy reveal that binding of Zalpha to supercoiled plasmids is associated with relaxation of the plasmid. Pronounced kinking of DNA is observed, and appears to be induced by binding of Zalpha. The results reported here support a model where the Z-DNA binding motifs target ADAR1 to regions of negative supercoiling in actively transcribing genes. In this situation, binding by Zalpha would be dependent upon the local level of negative superhelicity rather than the presence of any particular sequence.

Double-stranded RNA deaminase I (ADAR1) contains the Z-DNA binding domain Z␣. Here we report the solution structure of free Z␣ and map the interaction surface with Z-DNA, confirming roles previously assigned to residues by mutagenesis. Comparison with the crystal structure of the (Z␣) 2͞Z-DNA complex shows that most Z-DNA contacting residues in free Z␣ are prepositioned to bind Z-DNA, thus minimizing the entropic cost of binding. Comparison with homologous (␣؉␤)helix-turn-helix͞B-DNA complexes suggests that binding of Z␣ to B-DNA is disfavored by steric hindrance, but does not eliminate the possibility that related domains may bind to both B-and Z-DNA. RNA editing in mammals alters codons in mRNA through site-specific deamination of adenosines and cytosines, leading to proteins with modified function. Adenosine to inosine (A 3 I) editing modulates the calcium permeability of neural glutamate receptors (1) and reduces the G-protein coupling efficacy of serotonin 2C receptors (2). Double-stranded RNA deaminases I and II (ADAR1͞2) catalyze these A 3 I conversions, but unknown auxiliary factors are thought to be involved in the control of editing efficiency in vivo (3). ADAR1, but not ADAR2, has two left-handed Z-DNA binding domains, Z␣ and Z␤, at its N terminus. These domains may contribute to the control of ADAR1-mediated editing in vivo (4). Z-DNA formation in vivo has been shown to be transcription dependent in prokaryotes and eukaryotes (5). Z-DNA can be generated transiently 5Ј to a moving RNA polymerase in alternating purine͞pyrimidine sequences (5), thereby providing a transient binding site for Z␣ and Z␤. Thus, Z-DNA binding may ensure that the catalytic activity of ADAR1 is targeted to sites where nascent pre-mRNA substrates emerge (5).Here we have determined the solution structure of free Z␣ and mapped the interaction surface between Z␣ and a 6-bp d(CG) substrate DNA by two-dimensional (2D) 15 N-heteronuclear single quantum correlation (HSQC) NMR spectroscopy. Z␣ binds this substrate with high affinity (K d ϭ 30 nM) and a stoichiometry of 2:1 (protein͞DNA) (6-10). The map of the interaction surface in solution agrees well with the crystal structure of Z␣ complexed with Z-DNA (7). Further the structure of Z␣ free in solution demonstrates that there are only minor conformational changes upon binding Z-DNA. Not only is the overall structure the same, but unexpectedly, most Z-DNA contacting residues are prepositioned in free Z␣ to fit Z-DNA. This study also examines why Z␣ preferentially binds Z-DNA rather than B-DNA despite its high structural homology to (␣ϩ␤) helix-turn-helix (␣ϩ␤HTH) B-DNA binding proteins. Materials and MethodsProtein Preparation. The Z␣ domain, comprising residues 119-200 of human ADAR1 (GenBank accession no. U10439), has been described (8). Z␣ was expressed as a fusion protein with a N-terminal (His) 6 -tag from a pET-21a vector (Novagen) in Escherichia coli strain HM174(DE3). For isotope labeling, bacteria were grown in M9 medium containing 1 g͞liter 15 NH 4 Cl and 1.5 g͞liter 13 C-gl...

RNA editing alters pre-mRNA through site-selective adenosine deamination, which results in codon changes that lead to the production of novel proteins. An enzyme that catalyzes this reaction, double-stranded RNA adenosine deaminase (ADAR1), contains two N-terminal Z-DNA-binding motifs, Zalpha and Zbeta, the function of which is as yet unknown. In this study, multidimensional NMR spectroscopy was used to show that the topology of Zalpha is alpha1beta1alpha2alpha3beta2beta3. Long-range NOEs indicate that beta1 and beta3 interact with each other. Site-directed mutagenesis was used to identify residues in alpha3, beta3 and the loop connecting beta2 to beta3 that affect Z-DNA binding. Also identified were 11 hydrophobic residues that are essential for protein stability. Comparison with known structures reveals some similarity between Zalpha and (alpha + beta) helix-turn-helix proteins, such as histone 5 and the family of hepatocyte nuclear factor-3 winged-helix-turn-helix transcription factors. Taken together, the structural and functional data suggest that recognition of Z-DNA by Zalpha involves residues in both the alpha3 helix and the C-terminal beta-sheet.