Engrailed (Gln50→Lys) homeodomain–DNA complex at 1.9 Å resolution: structural basis for enhanced affinity and altered specificity

AbstractBackground: The homeodomain is one of the key DNA-binding motifs used in eukaryotic gene regulation, and homeodomain proteins play critical roles in development. The residue at position 50 of many homeodomains appears to determine the differential DNA-binding specificity, helping to distinguish among binding sites of the form TAATNN. However, the precise role(s) of residue 50 in the differential recognition of alternative sites has not been clear. None of the previously determined structures of homeodomain–DNA complexes has shown evidence for a stable hydrogen bond between residue 50 and a base, and there has been much discussion, based in part on NMR studies, about the potential importance of water-mediated contacts. This study was initiated to help clarify some of these issues.Results: The crystal structure of a complex containing the engrailed Gln50→Lys variant (QK50) with its optimal binding site TAATCC (versus TAATTA for the wild-type protein) has been determined at 1.9 Å resolution. The overall structure of the QK50 variant is very similar to that of the wild-type complex, but the sidechain of Lys50 projects directly into the major groove and makes several hydrogen bonds to the O6 and N7 atoms of the guanines at base pairs 5 and 6. Lys50 also makes an additional water-mediated contact with the guanine at base pair 5 and has an alternative conformation that allows a hydrogen bond with the O4 of the thymine at base pair 4.Conclusions: The structural context provided by the folding and docking of the engrailed homeodomain allows Lys50 to make remarkably favorable contacts with the guanines at base pairs 5 and 6 of the binding site. Although many different residues occur at position 50 in different homeodomains, and although numerous position 50 variants have been constructed, the most striking examples of altered specificity usually involve introducing or removing a lysine sidechain from position 50. This high-resolution structure also confirms the critical role of Asn51 in homeodomain–DNA recognition and further clarifies the roles of water molecules near residues 50 and 51

Transcriptional repression by zinc finger peptides - Exploring the potential for applications in gene therapy

A series of studies were performed to determine whether zinc finger peptides could efficiently repress transcription from RNA polymerase Il promoters in vivo and to determine how such repression might depend on the position of the zinc finger binding site with respect to those of the TATA box or the initiator element. Promoter constructs were prepared with Zif268 binding sites inserted at various positions, and the activity of a reporter gene was measured in transfection studies. We found that the peptide containing the three zinc fingers of Zif268 could efficiently repress activated transcription when bound to a site near the TATA box (19-fold repression) or when bound to a site near the initiator element (18-fold repression). Repression was even more effective when the zinc finger peptide was bound to both of these sites (63-fold repression). Novel zinc finger peptides that had been selected via phage display also served as repressors of activated transcription, but repression with these proteins was somewhat less efficient than with the Zif268 peptide.

Getting a handhold on DNA: Design of poly-zinc finger proteins with femtomolar dissociation constants

Structure-based design was used to link zinc finger peptides and make poly-finger proteins that have dramatically enhanced affinity and specificity. Our studies focused on a fusion in which the three-finger Zif268 peptide was linked to a designed three-finger peptide (designated “NRE”) that specifically recognizes a nuclear hormone response element. Gel shift assays indicate that this six-finger peptide, 268//NRE, binds to a composite 18-bp DNA site with a dissociation constant in the femtomolar range. We find that the slightly longer linkers used in this fusion protein provide a dramatic improvement in DNA-binding affinity, working much better than the canonical “TGEKP” linkers that have been used in previous studies. Tissue culture transfection experiments also show that the 268//NRE peptide is an extremely effective repressor, giving 72-fold repression when targeted to a binding site close to the transcription start site. Using this strategy, and linking peptides selected via phage display, should allow the design of novel DNA-binding proteins—with extraordinary affinity and specificity—for use in biological research and gene therapy

Design of TATA box-binding protein/zinc finger fusions for targeted regulation of gene expression

Fusing the TATA box-binding protein (TBP) to other DNA-binding domains may provide a powerful way of targeting TBP to particular promoters. To explore this possibility, a structure-based design strategy was used to construct a fusion protein, TBP/ZF, in which the three zinc fingers of Zif268 were linked to the COOH terminus of yeast TBP. Gel shift experiments revealed that this fusion protein formed an extraordinarily stable complex when bound to the appropriate composite DNA site (half-life up to 630 h). In vitro transcription experiments and transient cotransfection assays revealed that TBP/ZF could act as a site-specific repressor. Because the DNA-binding specificities of zinc finger domains can be systematically altered by phage display, it may be possible to target such TBP/zinc finger fusions to desired promoters and thus specifically regulate expression of endogenous genes