The question of whether a protein whose natural sequence is inverted adopts a stable fold is still under debate. We have determined the 2.1-Å crystal structure of the retro-GCN4 leucine zipper. In contrast to the two-stranded helical coiled-coil GCN4 leucine zipper, the retro-leucine zipper formed a very stable, parallel four-helix bundle, which now lends itself to further structural and functional studies. Since the early folding experiments by Anfinsen (1), it has been accepted that structure and function of a protein are determined by its amino acid sequence as read from the N terminus to the C terminus. But how does the structure change if the amino acid sequence of the protein is inverted? Inverted sequences are occasionally found in genomic DNA, but thus far, a native retro-protein has not been detected. Physicochemical properties that are related to the amino acid composition or the hydrophobicity profile should not be affected by the inversion of the sequence, supporting the idea that retro-sequences might fold into a native-like conformation. Modeling experiments suggested that reversal of the backbone direction may result in a topological mirror image of the native structure of the protein (2) or may produce the same topology as that of the parent protein (3, 4). Thus far, no structure elucidation of a retro-Lpeptide at atomic resolution has been reported.Coiled coils, including leucine zippers, consist of two to five intertwined ␣-helices and are frequently found in oligomeric proteins such as transcription factors as well as motility and structural proteins (5). We used the two-stranded coiled-coil domain of the yeast transcription activator GCN4 (6) to dimerize an artificial HIV enhancer-binding peptide, an operation that resulted in increased inhibition of HIV enhancer-controlled transcription (7). Because modeling studies suggested that the retro-sequence of the GCN4 leucine zipper also seemed to form a suitable dimerization module, a 35-residue retro-GCN4 was synthesized and characterized. Oxidized retro-leucine zipper extended with Cys-Gly-Gly, previously termed (r-LZ38) 2 (r-GCN4-p1Ј; Fig. 1A), crystallized and is now shown by x-ray structure analysis to fold into a parallel tetrameric coiled coil. Materials and MethodsUltracentrifugation. Sedimentation velocity experiments were performed with a Beckman-Spinco XL-A analytical ultracentrifuge. The peptide was dissolved in 20 mM Tris⅐HCl͞80 mM NaCl adjusted to pH 5.0, and measurements were made over a 10-to 100-M peptide concentration range. The centrifuge was operated at a speed of 50,000 rpm at 20°C. A partial specific volume, v 20 ϭ 0.74 cm 3 ⅐g Ϫ1, was calculated from the amino acid composition as was an estimate of the degree of hydration, d 1 ϭ 0.47 g water͞g protein. Sedimentation velocity traces were analyzed according to the method described in ref. 8, and a value of the sedimentation coefficient, s w,20 ϭ 1.77 Ϯ 0.5 Svedberg, was obtained by extrapolation to infinite dilution. The axial ratio was calculated as described in ref. 9.Crysta...

An artificial HIV enhancer-binding polypeptide has recently been dimerized by covalently linking it to the leucine zipper motif of the yeast transcriptional activator GCN4 (Liu N et al., 1997, Eur Siophys J 25:399-403). Although it seemed that the dimerization of this peptide could be best achieved by the use of the retro sequence of the leucine zipper, this approach was not implemented in the original construct. As the first step toward the synthesis of a basic region-retro leucine zipper HIV enhancer-binding fusion protein, we have now prepared the retro version of the leucine zipper (r-LZ35) and performed initial physicochemical characterization. Circular dichroism and sedimentation equilibrium studies showed that, at concentrations

An artificial HIV-1 enhancer-binding 42-residue peptide (R42) that had been derived from bacteriophage 434 repressor inhibited the cell-free in vitro transcription of HIV-1 enhancer-containing plasmids [Hehlgans, T., Stolz, M., Klauser, S., Cui, T., Salgam, P., Brenz Verca, S., Widmann, M., Leiser, A., Sta Èdler, K. & Gutte, B. (1993) FEBS Lett. 315, 51±55;Caderas, G. (1997) PhD Thesis, University of Zu Èrich]. Here we show that, after N-terminal extension of R42 with a viral nuclear localization signal, the resulting nucR42 peptide was active in intact cells. NucR42 could be detected immunologically in nuclear extracts and produced a 60±70% reduction of the rate of transcription of an HIV-1 enhancer-carrying plasmid in COS-1 cells that had been cotransfected with the HIV enhancer plasmid, an expression plasmid for nucR42, and a control. NucR42 was also synthesized chemically and the synthetic product characterized by HPLC, mass spectrometry, and quantitative amino acid analysis. Band shift, footprint, and in vitro transcription assays in the presence of exogenous NF-kBp50 indicated that the binding sites of nucR42 and NF-kB on the HIV enhancers overlapped and that a relatively small excess of nucR42 sufficed to displace NF-kBp50. Band shift and in vitro transcription experiments showed also that exchange of the 434 repressor-derived nine-residue recognition helix of nucR42 for four glycines abolished the HIV enhancer binding specificity whereas leucine zipper-or retro-leucine zipper-mediated dimerization of R42 analogues increased it suggesting the potential application of such dimeric HIV enhancer-binding peptides as intracellular inhibitors of HIV replication.Keywords: artificial HIV-1 enhancer-binding peptide; footprint; cell-free in vitro transcription; nuclear localization signal; COS-1 cells.Although a number of functional peptides have been obtained by de novo design [1], this approach was not practical for the construction of sequence-specific HIV enhancer-binding peptides. Design based on natural models seemed more promising. Wharton and Ptashne [2] were able to change the binding specificity of bacteriophage 434 repressor to that of bacteriophage P22 repressor by replacing four amino acids of the recognition helix of 434 repressor with the corresponding amino acids of the recognition helix of P22 repressor. Later, Choo et al.[3] engineered an 86-residue DNA-binding peptide comprising three Cys 2 His 2 zinc fingers to interact specifically with a unique nine-base-pair segment of a BCR-ABL fusion oncogene. This peptide also bound to the target DNA sequence in transformed cells in culture and stopped transcription. The three zinc fingers were chosen by screening a zinc finger phage library with each of the three triplets forming the nine-base-pair DNA segment followed by mutating one of the selected zinc fingers to improve its specificity. In the context of our present work it is noteworthy that there is at least one cellular zinc finger protein that specifically binds to the HIV-1 enhancers (HIV-EP1; [...

A 42 residue artificial peptide that binds to the HIV-1 enhancers has been described previously. The specificity of interaction of the peptide with its target DNA sequence has been demonstrated by a variety of techniques. Naturally occurring regulatory proteins frequently bind to DNA as dimers, thereby increasing the strength and specificity of the interaction, the dimer interface often being provided by a leucine zipper type coiled coil. As a suitable binding site for this kind of system is located to the 5' end of the HIV enhancer region, it was decided to design and synthesize a fusion peptide that not only contained the DNA binding sequence of the original 42 residue peptide but also incorporated a leucine zipper based on that of the GCN4 transcriptional activator, that should, therefore, be capable of dimerizing. The resultant peptide, LZ66, has now been shown to be fully active in band shift and in vitro transcription assays and to exhibit about double the inhibitory activity of the parent 42 residue peptide. Preliminary CD measurements revealed that the peptide has a high alpha-helical content and that it adopts a stable conformation down to the low micromolar peptide concentration range. Sedimentation equilibrium studies confirmed that the principles involved in the design of the peptide are valid and that the peptide is indeed dimeric in solution.

We have designed and synthesized HIV‐1 enhancer‐binding polypeptides that were derived from bacteriophage 434 repressor. These peptides were 39–54 residues long and contained either the recognition helix or the entire helix‐turn‐helix motif of the DNA‐binding domain of 434 repressor. The dissociation constant of the complex formed between the standard peptide (R42) and a synthetic 70‐bp HIV enhancer DNA was ca. 10−8 M. The specificity of the interaction of R42 with the two HIV enhancers was demonstrated by competitive band shift assays, stepwise displacement of the p50 subunit of transcription factor NF‐κB from its two HIV enhancer binding sites, and DNase I footprinting; R42 seemed to protect best the two TTTCC sequences of the HIV enhancers against digestion by DNase I. R42 analogues with mutated recognition helix had lower DNA binding specificity. It remains to be investigated whether our artificial HIV enhancer‐binding polypeptides are active in vivo.