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C O Pabo

Publications and source records attributed to C O Pabo.

At least 37 records · Page 2Linked to original sources
PubMed1998

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.

Binding Sites↗
PubMed1998

Structure-based design of a dimeric zinc finger protein.

Designing DNA-binding proteins with novel sequence specificities may provide valuable tools for biological research and gene therapy. Computer modeling was used to design a dimeric zinc finger protein, ZFGD1, containing zinc fingers 1 and 2 from Zif268 and a portion of the dimerization domain of GAL4. ZFGD1 binds with high affinity and specificity to the predicted binding site, which contains two 6 base-pair symmetry-related zinc finger subsites separated by a 13 base-pair spacer. The DNA-binding specificity of this fusion protein is determined primarily by the zinc fingers and can be systematically altered through the substitution of the zinc fingers with variants selected by phage display. This zinc finger-GAL4 fusion may serve as a prototype for designed DNA-binding proteins that could exploit advantages of homo- and heterodimer formation, and the adaptability of the Cys2His2 zinc finger motif, to target virtually any site in the genome.

Binding Sites↗
PubMed1998

Comparison of X-ray and NMR structures for the Antennapedia homeodomain-DNA complex.

Homeodomains are one of the key families of eukaryotic DNA-binding motifs and provide an important model system for studying protein-DNA interactions. We have crystallized the Antennapedia homeodomain-DNA complex and solved this structure at 2.4 A resolution. NMR and molecular dynamics studies had implied that this homeodomain achieves specificity through an ensemble of rapidly fluctuating DNA contacts. The crystal structure is in agreement with the underlying NMR data, but our structure reveals a well-defined set of contacts and also reveals the locations and roles of water molecules at the protein-DNA interface. The synthesis of X-ray and NMR studies provides a unified, general model for homeodomain-DNA interactions.

Amino Acid Sequence↗
PubMed1997

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 II 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.

Amino Acid Sequence↗
PubMed1997

Engrailed (Gln50-->Lys) homeodomain-DNA complex at 1.9 A resolution: structural basis for enhanced affinity and altered specificity.

BACKGROUND: 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 A 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.

Animals↗
PubMed1997

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.

DNA-Binding Proteins↗
PubMed1997

A general strategy for selecting high-affinity zinc finger proteins for diverse DNA target sites.

A method is described for selecting DNA-binding proteins that recognize desired sequences. The protocol involves gradually extending a new zinc finger protein across the desired 9- or 10-base pair target site, adding and optimizing one finger at a time. This procedure was tested with a TATA box, a p53 binding site, and a nuclear receptor element, and proteins were obtained that bind with nanomolar dissociation constants and discriminate effectively (greater than 20,000-fold) against nonspecific DNA. This strategy may provide important information about protein-DNA recognition as well as powerful tools for biomedical research.

Amino Acid Sequence↗
PubMed1996

Zif268 protein-DNA complex refined at 1.6 A: a model system for understanding zinc finger-DNA interactions.

BACKGROUND: Zinc fingers of the Cys2 His2 class recognize a wide variety of different DNA sequences and are one of the most abundant DNA-binding motifs found in eukaryotes. The previously determined 2.1 A structure of a complex containing the three zinc fingers from Zif268 has served as a basis for many modeling and design studies, and Zif268 has proved to be a very useful model system for studying how TFIIIA-like zinc fingers recognize DNA. RESULTS: We have refined the structure of the Zif268 protein-DNA complex at 1.6 A resolution. Our structure confirms all the basic features of the previous model and allows us to focus on some critical details at the protein-DNA interface. In particular, our refined structure helps explain the roles of several acidic residues located in the recognition helices and shows that the zinc fingers make a number of water-mediated contacts with bases and phosphates. Modeling studies suggest that the distinctive DNA conformation observed in the Zif268-DNA complex is correlated with finger-finger interactions and the length of the linkers between adjacent fingers. Circular dichroism studies indicate that at least some of the features of this distinctive DNA conformation are induced upon complex formation. CONCLUSIONS: Our 1.6 A structure should provide an excellent framework for analyzing the effects of Zif268 mutations, for modeling related zinc finger-DNA complexes, and for designing and selecting Zif268 variants that will recognize other DNA sites.

Amino Acid Sequence↗
PubMed1996

Oct-1 POU domain-DNA interactions: cooperative binding of isolated subdomains and effects of covalent linkage.

Structural and biochemical studies of Oct-1 POU domain-DNA interactions have raised important questions about cooperativity and the role of the linker connecting the POU-specific domain and the POU homeo domain. To analyze these interactions, we have studied binding of the isolated domains. Surprisingly, we find that two unlinked polypeptides corresponding to the POU-specific domain and the POU homeo domain bind cooperatively to the octamer site and have a coupling energy of 1.6 kcal/mole. We suggest that overlapping DNA contacts near the center of the octamer site may be the source of this cooperativity, as there are no protein-protein contacts between the domains in the crystal structure of the Oct-1 POU domain-DNA complex. These studies also have allowed us to describe the thermodynamic contribution of the linker (present in the intact POU domain) in terms of an effective concentration (3.6 mM). The broader implications for understanding cooperativity in protein-DNA recognition and gene regulation are discussed.

Base Sequence↗
PubMed1995

Analysis of homeodomain function by structure-based design of a transcription factor.

The homeodomain is a 60-amino acid module which mediates critical protein-DNA and protein-protein interactions for a large family of regulatory proteins. We have used structure-based design to analyze the ability of the Oct-1 homeodomain to nucleate an enhancer complex. The Oct-1 protein regulates herpes simplex virus (HSV) gene expression by participating in the formation of a multiprotein complex (C1 complex) which regulates alpha (immediate early) genes. We recently described the design of ZFHD1, a chimeric transcription factor containing zinc fingers 1 and 2 of Zif268, a four-residue linker, and the Oct-1 homeodomain. In the presence of alpha-transinduction factor and C1 factor, ZFHD1 efficiently nucleates formation of the C1 complex in vitro and specifically activates gene expression in vivo. The sequence specificity of ZFHD1 recruits C1 complex formation to an enhancer element which is not efficiently recognized by Oct-1. ZFHD1 function depends on the recognition of the Oct-1 homeodomain surface. These results prove that the Oct-1 homeodomain mediates all the protein-protein interactions that are required to efficiently recruit alpha-transinduction factor and C1 factor into a C1 complex. The structure-based design of transcription factors should provide valuable tools for dissecting the interactions of DNA-bound domains in other regulatory circuits.

Amino Acid Sequence↗
PubMed1995

Crystal structure of a paired domain-DNA complex at 2.5 A resolution reveals structural basis for Pax developmental mutations.

The 2.5 A resolution structure of a cocrystal containing the paired domain from the Drosophila paired (prd) protein and a 15 bp site shows structurally independent N-terminal and C-terminal subdomains. Each of these domains contains a helical region resembling the homeodomain and the Hin recombinase. The N-terminal domain makes extensive DNA contacts, using a novel beta turn motif that binds in the minor groove and a helix-turn-helix unit with a docking arrangement surprisingly similar to that of the lambda repressor. The C-terminal domain is not essential for prd binding and does not contact the optimized site. All known developmental missense mutations in the paired box of mammalian Pax genes map to the N-terminal subdomain, and most of them are found at the protein-DNA interface.

Amino Acid Sequence↗
PubMed1995

Structure-based design of transcription factors.

Computer modeling suggested that transcription factors with novel sequence specificities could be designed by combining known DNA binding domains. This structure-based strategy was tested by construction of a fusion protein, ZFHD1, that contained zinc fingers 1 and 2 from Zif268, a short polypeptide linker, and the homeodomain from Oct-1. The fusion protein bound optimally to a sequence containing adjacent homeodomain (TAATTA) and zinc finger (NGGGNG) subsites. When fused to an activation domain, ZFHD1 regulated promoter activity in vivo in a sequence-specific manner. Analysis of known protein-DNA complexes suggests that many other DNA binding proteins could be designed in a similar fashion.

Amino Acid Sequence↗
PubMed1994

Distinctive DNA conformation with enlarged major groove is found in Zn-finger-DNA and other protein-DNA complexes.

We have analyzed DNA conformations in a series of protein-DNA complexes, and we find that a distinctive conformation--with an enlarged major groove--occurs in a number of different complexes. During this analysis, we also developed a simplified model of DNA structure that illustrates the relative position of (i) the base pairs, (ii) the phosphate backbone, and (iii) the double-helical axis. This model highlights the key structural features of each duplex, facilitating the analysis and comparison of structures that are distinct from canonical A-DNA or B-DNA. Comparing DNA conformations in this way revealed that an otherwise unrelated set of protein-DNA complexes have interesting structural similarities, including an enlarged major groove. We refer to this class of structures as Beg-DNA (where eg means enlarged groove). Since related features occur in such a diverse set of protein-DNA complexes, we suggest that this conformation may have a significant role in protein-DNA recognition.

Base Sequence↗
PubMed1994

Crystal structure of MyoD bHLH domain-DNA complex: perspectives on DNA recognition and implications for transcriptional activation.

The crystal structure of a MyoD basic-helix-loop-helix (bHLH) domain-DNA complex has been solved and refined at 2.8 A resolution. This structure proves that bHLH and bHLH-leucine zipper (bHLH-ZIP) proteins are remarkably similar; it helps us understand subtle differences in binding preferences for these proteins; and it has surprising implications for our understanding of transcription. Specifically, Ala-114 and Thr-115, which are required for positive control in the myogenic proteins, are buried at the protein-DNA interface. These residues are not available for direct protein-protein contacts, but they may determine the conformation of Arg-111. Comparisons with Max suggest that the conformation of this arginine, which is different in the two structures, may play an important role in myogenic transcription.

Amino Acid Sequence↗
PubMed1994

Crystal structure of the Oct-1 POU domain bound to an octamer site: DNA recognition with tethered DNA-binding modules.

The structure of an Oct-1 POU domain-octamer DNA complex has been solved at 3.0 A resolution. The POU-specific domain contacts the 5' half of this site (ATGCAAAT), and as predicted from nuclear magnetic resonance studies, the structure, docking, and contacts are remarkably similar to those of the lambda and 434 repressors. The POU homeodomain contacts the 3' half of this site (ATGCAAAT), and the docking is similar to that of the engrailed, MAT alpha 2, and Antennapedia homeodomains. The linker region is not visible and there are no protein-protein contacts between the domains, but overlapping phosphate contacts near the center of the octamer site may favor cooperative binding. This novel arrangement raises important questions about cooperativity in protein-DNA recognition.

Amino Acid Sequence↗
PubMed1994

DNA recognition by beta-sheets in the Arc repressor-operator crystal structure.

Transcription of the ant gene during lytic growth of bacteriophage P22 (ref. 1) is regulated by the cooperative binding of two Arc repressor dimers to a 21-base-pair operator site. Here we report the co-crystal structure of this Arc tetramer-operator complex at 2.6 A resolution. As expected from genetic and structural studies and from the co-crystal structure of the homologous Escherichia coli MetJ repressor, each Arc dimer uses an antiparallel beta-sheet to recognize bases in the major groove. However, the Arc and MetJ complexes differ in several important ways: the beta-sheet-DNA interactions of Arc are far less symmetrical; DNA binding by Arc is accompanied by important conformational changes in the beta-sheet; and Arc uses a different part of its protein surface for dimer-dimer interactions.

Base Sequence↗
PubMed1994

Zinc finger phage: affinity selection of fingers with new DNA-binding specificities.

A phage display system was developed and used to select zinc finger proteins with altered DNA-binding specificities. The three zinc fingers of the Zif268 protein were expressed on the surface of filamentous phage, and a library of variants was prepared by randomizing critical amino acids in the first zinc finger. Affinity selections, using DNA sites with base changes in the region recognized by the first finger, yielded Zif268 variants that bound tightly and specifically to the new sites. This phage system provides a tool for the study of protein-DNA interactions and may offer a general method for selecting zinc finger proteins that recognize desired target sites on double-stranded DNA.

Amino Acid Sequence↗