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Structure of a Designed Dimeric Zinc Finger Protein Bound to DNA
Abstract
Proteins that employ dimerization domains to bind cooperatively to DNA have a number of potential advantages over monomers with regards to gene regulation. Using a combination of structure-based design and phage display, a dimeric Cys(2)His(2) zinc finger protein has been created that binds cooperatively to DNA via an attached leucine zipper dimerization domain. This chimera, derived from components of Zif268 and GCN4, displayed excellent DNA-binding specificity, and we now report the 1.5 A resolution cocrystal structure of the Zif268-GCN4 homodimer bound to DNA. This structure shows how phage display has annealed the DNA binding and dimerization domains into a single functional unit. Moreover, this chimera provides a potential platform for the creation heterodimeric zinc finger proteins that can regulate a desired target gene through cooperative DNA recognition.
... Here, the spacing as well as overlapping of the different subsites vary in every cases along with the size of the subsites (Corbi et al., 2004). First of all, the binding site of a (2002) Residue grafting GCN4-aPP Solvent exposed residues of the alpha helix of avian pancreatic polypeptide (aPP) were replaced by the residues of GCN4 important for GCN4-DNA interaction Formation of six finger motif for the recognition of a longer DNA sequence Wolfe, Grant, and Pabo (2003) specific ZnF protein is fused with the desired target sequence. Then it is connected to two fixed fingers of a three finger motif, and the randomized third finger positions itself accordingly. ...
... In these constructs, the dimerization domain of Gal4 remains attached to ZnF1 and 2 of Zif268 and ZnF2 and 3 of Zif268 respectively. These STFs can bind to a predicted site that is composed of two 6 base-paired ZnF subsites with a 13 base-pair spacer in between with high specificity and affinity (Wolfe et al., 2003). In another six finger fusion protein 268//NRE which act as a genome specific TF (Fig. 1), Finger 2, 6 and 8 of the Zif268 remains tethered to the three finger domains of NRE via an appropriate linker (Kang & Kim, 2000). ...
In this chapter, we discuss different design strategies of synthetic proteins, especially synthetic transcription factors. Design and engineering of synthetic transcription factors is particularly relevant for the need-based manipulation of gene expression. With recent advances in structural biology techniques and with the emergence of other precision biochemical/physical tools, accurate knowledge on structure-function relations is increasingly becoming available. Besides discussing the underlying principles of design, we go through individual cases, especially those involving four major groups of transcription factors—basic leucine zippers, zinc fingers, helix-turn-helix and homeodomains. We further discuss how synthetic biology can come together with structural biology to alter the genetic blueprint of life.
... Another method utilised to increase the length of the DNA target site is by attaching a dimerisation domain. In an alternative approach to tuning DNA-binding specificity, two ZnFs were linked to the GCN4 leucine zipper domain; the resultant dimeric protein bound a 10-bp DNA site with high specificity (Wolfe et al., 2003;Figure 1.10) The design of novel ZnF-based DNA-binding proteins has recently matured to the point where researchers are using these engineered proteins to regulate the expression of genes in model organisms to manipulate phenotype in a defined way. ...
... 10. Structure of the chimeric Zif23-GCN4 protein. The designed protein contains two classical ZnFs of Zif268 fused to the leucine zipper of GCN4 (PDB code 1LLM;Wolfe et al., 2003). ...
... The same e↵ect can also be achieved by engineering dimerizing ZFs that bind cooperatively. An early example used structure-based design to engineer a two-finger ZF which dimerized via a LZ motif to form a four-finger complex (Wolfe et al. 2003(Wolfe et al. , 2000. ...
Gene-regulatory networks are ubiquitous in nature and critical for bottom-up engineering of synthetic networks. Transcriptional repression is a fundamental function that can be tuned at the level of DNA, protein, and cooperative protein–protein interactions, necessitating high-throughput experimental approaches for in-depth characterization. Here, we used a cell-free system in combination with a high-throughput microfluidic device to comprehensively study the different tuning mechanisms of a synthetic zinc-finger repressor library, whose affinity and cooperativity can be rationally engineered. The device is integrated into a comprehensive workflow that includes determination of transcription-factor binding-energy landscapes and mechanistic modeling, enabling us to generate a library of well-characterized synthetic transcription factors and corresponding promoters, which we then used to build gene-regulatory networks de novo. The well-characterized synthetic parts and insights gained should be useful for rationally engineering gene-regulatory networks and for studying the biophysics of transcriptional regulation.
... The same e↵ect can also be achieved by engineering dimerizing ZFs that bind cooperatively. An early example used structure-based design to engineer a two-finger ZF which dimerized via a LZ motif to form a four-finger complex (Wolfe et al. 2003(Wolfe et al. , 2000. ...
Gene regulatory networks are ubiquitous in nature and critical for bottom-up engineering of synthetic networks. Transcriptional repression is a fundamental function in gene regulatory networks and can be tuned at the level of DNA, protein, and cooperative protein - protein interactions, necessitating high-throughput experimental approaches for in-depth characterization. Here we used a cell-free system in combination with a high-throughput microfluidic device to comprehensively study the different tuning mechanisms of a synthetic zinc-finger repressor library, whose affinity, specificity, and cooperativity can be rationally engineered. The device is integrated into a comprehensive workflow that includes determination of transcription factor binding energy landscapes and mechanistic modeling. By integrating these methods we generated a library of well-characterized synthetic transcription factors and corresponding promoters, and used these standardized parts to build gene regulatory networks de novo in a cell-free environment. The well-characterized synthetic parts and insights gained should be useful for rationally engineering gene regulatory networks and for studying the biophysics of transcriptional regulation.
... The team TU Darmstadt 2016 [233] combined auxotrophic incorporation of a non-canonical amino acid and a reporter for low levels of the ncAA (BBa_K1416000, BBa_K1976025) [234] designed by the team Austin Texas 2014 [235]. Amber codons were placed at the beginning of a Colicin E2 immunity protein [236] and the mutated Zif23-GCN4 repressor (F4OMT), a dimeric Cis2His2 zinc finger protein [237]. In case of the absence of the ncAA, both proteins cannot be translated, resulting in expression of the reporter system mVenus [238] under control of a Zif23-GCN4-controlled promoter and subsequent initiation of the suicide reaction. ...
Background
Biosafety is a key aspect in the international Genetically Engineered Machine (iGEM) competition, which offers student teams an amazing opportunity to pursue their own research projects in the field of Synthetic Biology. iGEM projects often involve the creation of genetically engineered bacterial strains. To minimize the risks associated with bacterial release, a variety of biosafety systems were constructed, either to prevent survival of bacteria outside the lab or to hinder horizontal or vertical gene transfer.
Main body
Physical containment methods such as bioreactors or microencapsulation are considered the first safety level. Additionally, various systems involving auxotrophies for both natural and synthetic compounds have been utilized by iGEM teams in recent years. Combinatorial systems comprising multiple auxotrophies have been shown to reduced escape frequencies below the detection limit. Furthermore, a number of natural toxin-antitoxin systems can be deployed to kill cells under certain conditions. Additionally, parts of naturally occurring toxin-antitoxin systems can be used for the construction of ‘kill switches’ controlled by synthetic regulatory modules, allowing control of cell survival. Kill switches prevent cell survival but do not completely degrade nucleic acids. To avoid horizontal gene transfer, multiple mechanisms to cleave nucleic acids can be employed, resulting in ‘self-destruction’ of cells. Changes in light or temperature conditions are powerful regulators of gene expression and could serve as triggers for kill switches or self-destruction systems. Xenobiology-based containment uses applications of Xeno-DNA, recoded codons and non-canonical amino acids to nullify the genetic information of constructed cells for wild type organisms. A ‘minimal genome’ approach brings the opportunity to reduce the genome of a cell to only genes necessary for survival under lab conditions. Such cells are unlikely to survive in the natural environment and are thus considered safe hosts. If suitable for the desired application, a shift to cell-free systems based on Xeno-DNA may represent the ultimate biosafety system.
Conclusion
Here we describe different containment approaches in synthetic biology, ranging from auxotrophies to minimal genomes, which can be combined to significantly improve reliability. Since the iGEM competition greatly increases the number of people involved in synthetic biology, we will focus especially on biosafety systems developed and applied in the context of the iGEM competition.
... Conversely, five other down-stream peptides that contained only seven heptads of the myosin rod showed limited α-helical content. 23 This problem of stabilizing coiled coils that lack their native trigger sequence has been circumvented for some coiled-coils by incorporation of a short stable coiled-coil, such as that seen in the well-studied transcription factor GCN4. 18,24,25 This strategy could be 6 applied to sections of LMM but is not predicted to eliminate self-assembly into paracrystals at low ionic strength. ...
Sarcomeric myosins have the remarkable ability to form regular bipolar thick filaments that, together with actin thin filaments, constitute the fundamental contractile unit of skeletal and cardiac muscle. This has been established for over fifty years and yet a molecular model for the thick filament has not been attained. In part this is due to the lack of a detailed molecular model for the coiled-coil that constitutes the myosin rod. The ability to self-assemble resides in the C-terminal of the section of myosin known as light meromyosin (LMM) which exhibits strong salt dependent aggregation that has inhibited structural studies. Here we evaluate the feasibility of generating a complete model for the myosin rod by combining overlapping structures of five sections of coiled-coil covering 164 amino acid residues which constitute 20% of LMM. Each section contains ∼7-9 heptads of myosin. The problem of aggregation was overcome by incorporating the globular folding domains, Gp7 and Xrcc4 which enhance crystallization. The effect of these domains on the stability and conformation of the myosin rod was examined through biophysical studies and overlapping structures. In addition, a computational approach was developed to combine the sections into a contiguous model. The structures were aligned, trimmed to form a contiguous model, and simulated for >700 ns to remove the discontinuities and achieve an equilibrated conformation that represents the native state. This experimental and computational strategy lays the foundation for building a model for the entire myosin rod. This article is protected by copyright. All rights reserved.
... HDs can be fused to other DBDs to increase the complex sequences they can specify. For example, ZFs have been fused to the leucine zipper to create a heterodimeric functional unit (Wolfe et al. 2003). Thus, the combinations of chimeric DBDs utilizing HDs are endless where HDs can be seen as parts that can be added to the toolbox to build chimeric transcription factors or chimeric nucleases. ...
Homeodomains (HDs) are a large family of DNA-binding domains contained in transcription factors that are most notable for regulating body development and patterning in metazoans. HDs consist of three alpha helices preceded by an N- terminal arm, where the third helix (the recognition helix) and the N-terminal arm are responsible for defining DNA-binding specificity. Here we attempted to engineer the HDs by fully randomizing positions in the recognition helix to specify each of the 64 possible 3’ triplet sites (i.e. TAANNN). We recovered HD variants that preferentially recognize or are compatible with 44 of the possible sites, a dramatic increase from the previously observed range of specificities. Many of these HD variants contain combinations of novel specificity determinants that are uncommon or absent in extant HDs, where these determinants can be grafted into alternate HD backbones with an accompanying alteration in their specificity. The identified determinates expand our understanding of HD recognition, allowing for the creation of more explicit recognition models for this family. Additionally, we demonstrate that HDs can recognize a broader range of DNA sequences than anticipated, thus raising questions about the fitness barrier that restricts the evolution HD-DNA recognition in nature. Finally, these new HD variants have utility as DNA-binding domains to direct targeting of customizable sequence-specific nuclease as demonstrated by site-specific lesions created in zebrafish. Thus HDs can guide sequence-specific enzymatic function precisely and predictably within a complex genome when used in engineered artificial enzymes.
... Another potential way to increase the targeting range of the available 2F-modules is by fusing them with a dimerization domain such as a coiled coil domain that would allow cooperative binding of 2F-modules as well as skipping bases between the two half sites 236,237 . ...
The utility of a model organism for studying biological processes is closely tied to its amenability to genome manipulation. Although tools for targeted genome engineering in mice have been available since 1987, most organisms including zebrafish have lacked efficient reverse genetic tools, which has stymied their broad implementation as a model system to study biological processes. The development of zinc finger nucleases (ZFNs) that can create double-strand breaks at desired sites in a genome has provided a universal platform for targeted genome modification. ZFNs are artificial restriction endonucleases that comprise of an array of 3- to 6-C2H2-zinc finger DNA-binding domains fused with the dimeric cleavage domain of the type IIs endonuclease FokI. C2H2-zinc fingers are the most common, naturally occurring DNA-binding domain, and their specificity can be engineered to recognize a variety of DNA sequences providing a strategy for targeting the appended nuclease domain to desired sites in a genome. The utility of ZFNs for gene editing relies on their activity and precision in vivo both of which depend on the generation of ZFPs that bind desired target sites high specificity and affinity.
Although various methods are available that allow construction of ZFPs with novel specificities, ZFNs assembled using existing approaches often display negligible in vivo activity, presumably resulting from ZFPs with either low affinity or suboptimal specificity. A root cause of this deficiency is the presence of interfering interactions at the finger-finger interface upon assembly of multiple fingers. In this study we have employed bacterial-one-hybrid (B1H)-based selections to identify two-finger zinc finger units (2F-modules) containing optimized interface residues that can be combined with published finger archives to rapidly yield ZFNs that can target more than 95% of the zebrafish and human protein-coding genes while maintaining a success rate higher than that of ZFNs constructed using available methods. In addition to genome engineering in model organisms, this advancement in ZFN design will aid in the development of ZFN-based therapeutics.
In the process of creating this archive, we have undertaken a broader study of zinc finger specificity to better understand fundamental aspects of DNA recognition. In the process we have created the largest protein-DNA interaction dataset for zinc fingers to be described that will facilitate the development of better predictive models of recognition. Ultimately, these predictive models would enable the rational design of synthetic zinc finger proteins for targeted gene regulation or genomic modification, and the prediction of genomic binding sites for naturally occurring zinc finger proteins for the construction of more accurate gene regulatory networks.
... Binding of the Cl-orders these residues to their bound-like conformations by mimicking the DNA phosphates prior to binding. The next step is to perform similar MD simulations of the remaining ZF-DNA complex structures human YY1 (Houbaviy et al. 1996), tramtrack protein (Fairall et al. 1993), ZF-TATA box complex (Wolfe et al. 2001), designed ZF (Kim and Berg 1996) and designed dimeric ZF chimera (Wolfe et al. 2003) to further analyze the effects of counter ions in the formation of the protein-DNA encounter complex by mimicking DNA phosphate groups and directing the protein towards the DNA. ...
The C2H2 zinc finger (ZF) transcription factors (TF) form the largest family of DNA binding proteins in eukaryotes. TFs are key proteins involved in gene regulation that bind to specific DNA sites. A major obstacle towards understanding the molecular basis of transcriptional regulation is the lack of a recognition code for protein-DNA interactions. We aim to understand molecular mechanisms of DNA recognition and to quantitatively estimate recognition rules for TF-DNA interactions. We identified key residues playing an important role in ZF-DNA interactions and found that they are prealigned to conformations observed in the bound state prior to binding. A binding site for Cl- ions corresponding to the pocket where DNA phosphates are found most buried in the complex of ZFs is identified. Bound ions constrain conformations of important residues consistent with observations of increased binding affinity with increased ionic strength in protein-DNA interactions. These results suggest a general mechanism where ZFs, through their key residues, rapidly form encounter complexes amenable for a fast readout of the DNA. We developed a novel experimentally-based approach using crystal structures and binding data on the C2H2 ZFs and decoded ten fundamental specific interactions for protein-DNA recognition. These are: Five hydrogen bonds, three desolvation penalties, a non-polar energy, and a novel water accessibility factor. The code is applied to three data sets with a total of 89 ZF mutants on three ZFs of EGR. Guided by simulations of individual ZFs, we mapped the interactions into homology models with all feasible intra- and inter- molecular bonds and selected the structure with the lowest free energy for each ZF. The interactions reproduce changes in affinity of 35 mutants of finger I (FI) (R2 = 0.99), 23 mutants of FII (R2 = 0.97) and 31 human ZFs on FIIII (R2 = 0.95). The method predicts bound ZF-DNA complexes for all mutants, decoding molecular basis of ZF-DNA specificity. These findings reveal recognition rules that depend on DNA sequence/structure, molecular water at the interface and induced fit of the C2H2 TFs. In summary, our method provides the first robust framework to decode the molecular basis of TFs binding to DNA.
... leucine zipper) dimerization region of Gcn4 (residues 251-281) to a hairpin polyamide to generate the first synthetic ATF [113,130]. Furthermore, fusions of leucine zipper IDs to ZF DBDs produced homodimers (GCN4 pair) and heterodimers (Fos-Jun) [131,132]. Peptides selected by ...
Transcription factors control the fate of a cell by regulating the expression of genes and regulatory networks. Recent successes in inducing pluripotency in terminally differentiated cells as well as directing differentiation with natural transcription factors has lent credence to the efforts that aim to direct cell fate with rationally designed transcription factors. Because DNA-binding factors are modular in design, they can be engineered to target specific genomic sequences and perform pre-programmed regulatory functions upon binding. Such precision-tailored factors can serve as molecular tools to reprogramme or differentiate cells in a targeted manner. Using different types of engineered DNA binders, both regulatory transcriptional controls of gene networks, as well as permanent alteration of genomic content, can be implemented to study cell fate decisions. In the present review, we describe the current state of the art in artificial transcription factor design and the exciting prospect of employing artificial DNA-binding factors to manipulate the transcriptional networks as well as epigenetic landscapes that govern cell fate.
... A common cellular strategy to modulate TF activity is through cooperative binding between TFs, a process often mediated by leucine zipper (LZ) protein–protein interaction domains (10). Whereas the effect of LZs on the structure and function of ZF DBDs has been characterized biochemically, the use of LZs to modulate ZF-TF activity in cells has not been explored (11–13). We chose two well-characterized homodimerizing LZ domains with different binding affinities, the protein interaction domains of human c-Jun (Kd=448µM) and Saccharomyces cerevisiae GCN4 (Kd = 8 nM), to tune our engineered ZF-TFs (14,15). ...
The ability to perform molecular-level computation in mammalian cells has the potential to enable a new wave of sophisticated
cell-based therapies and diagnostics. To this end, we developed a Boolean logic framework utilizing artificial Cys2–His2 zinc finger transcription factors (ZF-TFs) as computing elements. Artificial ZFs can be designed to specifically bind different
DNA sequences and thus comprise a diverse set of components ideal for the construction of scalable networks. We generate ZF-TF
activators and repressors and demonstrate a novel, general method to tune ZF-TF response by fusing ZF-TFs to leucine zipper
homodimerization domains. We describe 15 transcriptional activators that display 2- to 463-fold induction and 15 transcriptional
repressors that show 1.3- to 16-fold repression. Using these ZF-TFs, we compute OR, NOR, AND and NAND logic, employing hybrid
promoters and split intein-mediated protein splicing to integrate signals. The split intein strategy is able to fully reconstitute
the ZF-TFs, maintaining them as a uniform set of computing elements. Together, these components comprise a robust platform
for building mammalian synthetic gene circuits capable of precisely modulating cellular behavior.
... The second zinc finger serves as a transition from the DNA binding domain to the protein interaction domain by interacting with both DNA and the other subunit of Zif268-GCN4. A high-affinity probe for Ikaros (BS-4) [34] has wider spacing between the cognate DNA sequences (three base pairs) than Zif268-GCN4. However , binding ZF2 and ZF3 of Ikaros to the BS-4 DNA is likely to place ZF4 in close proximity with the corresponding ZF4 from the interacting Ikaros partner with probably the same orientation as Zif268-GCN4. ...
The zinc finger motif was used as a vehicle for the initial discovery of Ikaros in the context of T-cell differentiation and has been central to all subsequent analyses of Ikaros function. The Ikaros gene is alternately spliced to produce several isoforms that confer diversity of function and consequently have complicated analysis of the function of Ikaros in vivo. Key features of Ikaros in vivo function are associated with six C2H2 zinc fingers; four of which are alternately incorporated in the production of the various Ikaros isoforms. Although no complete structures are available for the Ikaros protein or any of its family members, considerable evidence has accumulated about the structure of zinc fingers and the role that this structure plays in the functions of the Ikaros family of proteins. This review summarizes the structural aspects of Ikaros zinc fingers, individually, and in tandem to provide a structural context for Ikaros function and to provide a structural basis to inform the design of future experiments with Ikaros and its family members.
... (A–E) Typical content of 3D-footprint entries, illustrated with dimeric complex 1llm_CD, a Zif23-GCN4 chimera (32), and with non-redundant monomeric complex 1a0a_B, positive regulatory protein PHO4 (33). (A) An interface graph dissecting atomic contacts and nucleotides at the interface responsible for specific DNA discrimination, where solid bases indicate indirect readout mechanisms. ...
3D-footprint is a living database, updated and curated on a weekly basis, which provides estimates of binding specificity
for all protein–DNA complexes available at the Protein Data Bank. The web interface allows the user to: (i) browse DNA-binding
proteins by keyword; (ii) find proteins that recognize a similar DNA motif and (iii) BLAST similar DNA-binding proteins, highlighting
interface residues in the resulting alignments. Each complex in the database is dissected to draw interface graphs and footprint
logos, and two complementary algorithms are employed to characterize binding specificity. Moreover, oligonucleotide sequences
extracted from literature abstracts are reported in order to show the range of variant sites bound by each protein and other
related proteins. Benchmark experiments, including comparisons with expert-curated databases RegulonDB and TRANSFAC, support
the quality of structure-based estimates of specificity. The relevant content of the database is available for download as
flat files and it is also possible to use the 3D-footprint pipeline to analyze protein coordinates input by the user. 3D-footprint
is available at http://floresta.eead.csic.es/3dfootprint with demo buttons and a comprehensive tutorial that illustrates the main uses of this resource.
... For some applications shorter 2 × 2 units based on the same design could be used (Urnov et al., 2005). Another approach to linking zinc fingers into longer arrays involves using dimerising domains such as GAL4 dimerisation domain (Pomerantz et al., 1998), leucine zipper dimerisation domains (Wolfe et al., 2000Wolfe et al., , 2003 ), hormone receptor dimerising domain (Beerli et al., 2000b; Xu et al., 2001) or phage display selected dimerisation peptides (Wang and Pabo, 1999). It has to be noted that increased binding affinity can also be achieved by using two non-covalently linked 3-finger proteins, which cooperatively bind to adjacent sites (Liu et al., 2001; Reynolds et al., 2003). ...
The Cys(2)His(2) zinc finger is one of the most common DNA-binding motifs in Eukaryota. A simple mode of DNA recognition by the Cys(2)His(2) zinc finger domain provides an ideal scaffold for designing proteins with novel sequence specificities. The ability to bind specifically to virtually any DNA sequence combined with the potential of fusing them with effector domains has led to the technology of engineering of chimeric DNA-modifying enzymes and transcription factors. This in turn has opened the possibility of using the engineered zinc finger-based factors as novel human therapeutics. One such synthetic factor-designer zinc finger transcription activator of the vascular endothelial growth factor A gene-has recently entered clinical trials to evaluate the ability of stimulating the growth of blood vessels in treating the peripheral arterial obstructive disease. This review concentrates on the aspects of natural Cys(2)His(2) zinc fingers evolution and fundamental steps in design of engineered zinc finger proteins. The applications of engineered zinc finger proteins are discussed in a context of the mechanism mediating their effect on the targeted DNA. Furthermore, the regulation of the expression of zinc finger proteins and their targeting to various cellular compartments and to chromatin and non-chromatin target templates are described. Also possible future applications of designer zinc finger proteins are discussed.
... Perhaps surprisingly, dimerization affects not only the ZF domains that participate in the dimer interface but the ZF domains which are adjacent to the interacting domains are also bound in a non-wild-type manner. Wolfe et al. (65) noted that the DNA molecules in the 1llm and 1f2i complexes were not as significantly under-wound as in the wild-type Zif268 complex, which would explain why all ZF domains were affected. The three ZF domains from chain B of the 1p47 tandem dimer complex (Table 1) similarly exhibit non-wild-type docking. ...
Predicting the binding specificity of transcription factors is a critical step in the characterization and computational identification
and of cis-regulatory elements in genomic sequences. Here we use protein–DNA structures to predict binding specificity and consider
the possibility of predicting position weight matrices (PWM) for an entire protein family based on the structures of just
a few family members. A particular focus is the sensitivity of prediction accuracy to the docking geometry of the structure
used. We investigate this issue with the goal of determining how similar two docking geometries must be for binding specificity
predictions to be accurate. Docking similarity is quantified using our recently described interface alignment score (IAS).
Using a molecular-mechanics force field, we predict high-affinity nucleotide sequences that bind to the second zinc-finger
(ZF) domain from the Zif268 protein, using different C2H2 ZF domains as structural templates. We identify a strong relationship between IAS values and prediction accuracy, and define
a range of IAS values for which accurate structure-based predictions of binding specificity is to be expected. The implication
of our results for large-scale, structure-based prediction of PWMs is discussed.
... In essence, beta-branched residues in a (Ile, Val) and gamma-branched residues in d (Leu) specify dimers, a reversal of side-chains at these positions specifies tetramers, and a core formed entirely of betabranched residues forms trimers. Native and mutant GCN4 zippers have been used successfully for the production of chimeric proteins, allowing, among many others, studies of dimeric DNA-binding proteins (Wolfe et al., 2003) and histidine kinases (Wang et al., 2002), dimeric (Perera et al., 2003) and trimeric (Yang et al., 2000) virus envelope proteins, and tetrameric high-affinity artificial antibodies (Pack et al., 1995). In all these cases, the zippers were used to trigger oligomerization of the proteins of interest, and as a side effect, protein stability and solubility was increased. ...
We repeatedly experienced difficulties in obtaining pure protein of a defined oligomeric state when expressing domains that
consist partially or entirely of coiled coils. We therefore modified an established expression vector, pASK-IBA, to generate
N- and C-terminal fusions of the cloned domain in heptad register with the GCN4 leucine zipper. GCN4 is a well-characterized
coiled coil, for which stable dimeric, trimeric and tetrameric forms exist. To test this expression system, we produced a
series of constructs derived from the trimeric autotransporter adhesin STM3691 of Salmonella (SadA), which has a highly repetitive structure punctuated by coiled-coil regions. The constructs begin and end with predicted
coiled-coil segments of SadA, each fused in the correct heptad register to the trimeric form of GCN4, GCN4pII. All constructs
were expressed at high levels, trimerized either natively or after refolding from inclusion bodies, and yielded crystals that
diffracted to high resolution. Thus, fusion to GCN4pII allows for the efficient expression and crystallization of proteins
containing trimeric coiled coils. The structure of short constructs can be solved conveniently by molecular replacement using
the known GCN4 structure as a search model. The system can be adapted for constructs with dimeric or tetrameric coiled coils,
using the corresponding GCN4 variants.
Sequence-specific DNA-binding transcription factors are central to gene regulation. They are often associated with consensus binding sites that predict far more genomic sites than are bound in vivo. One explanation is that most sites are blocked by nucleosomes, such that only sites in nucleosome-depleted regulatory regions are bound. We compared the binding of the yeast transcription factor Gcn4 in vivo using published ChIP-seq data (546 sites) and in vitro, using a modified SELEX method ("G-SELEX"), which utilizes short genomic DNA fragments to quantify binding at all sites. We confirm that Gcn4 binds strongly to an AP-1-like sequence (TGACTCA) and weakly to half-sites. However, Gcn4 binds only some of the 1078 exact matches to this sequence, even in vitro. We show that there are only 166 copies of the high-affinity RTGACTCAY site (exact match) in the yeast genome, all occupied in vivo, largely independently of whether they are located in nucleosome-depleted or nucleosomal regions. Generally, RTGACTCAR/YTGACTCAY sites are bound much more weakly and YTGACTCAR sites are unbound, with biological implications for determining induction levels. We conclude that, to a first approximation, Gcn4 binding can be predicted using the high-affinity site, without reference to chromatin structure. We propose that transcription factor binding sites should be defined more precisely using quantitative data, allowing more accurate genome-wide prediction of binding sites and greater insight into gene regulation.
Protein-protein interactions control a wide variety of natural biological processes. α-Helical coiled coils frequently mediate such protein-protein interactions. Due to the relative simplicity of their sequences and structures and the ease with which properties such as strength and specificity of interaction can be controlled, coiled coils can be designed de novo to deliver a variety of non-natural protein-protein-interaction domains. Herein, several de novo designed coiled coils are tested for their ability to mediate protein-protein interactions in Escherichia coli cells. The set includes a parallel homodimer, a parallel homotetramer, an antiparallel homotetramer and a newly designed heterotetramer, all of which have been characterized in vitro by biophysical and structural methods. Using a transcription repression assay based on reconstituting the Lac repressor, we find that the modules behave as designed in the cellular environment. Each design imparts a different property to the resulting Lac repressor-coiled coil complexes, resulting in the benefit of being able to reconfigure the system in multiple ways. Modification of the system also allows the interactions to be controlled: assembly can be tuned by controlling the expression of the constituent components; and complexes can be disrupted through helix sequestration. The small and straightforward de novo designed components that we deliver are highly versatile and have considerable potential as protein-protein-interaction domains in synthetic biology where proteins must be assembled in highly specific ways. The relative simplicity of the designs makes them amenable to future modifications to introduce finer control over their assembly and to adapt them for different contexts.
Programmable transcription factors have been instrumental in recent elucidations of developmental and regulatory pathways pertaining to biology and medicine. Programmable DNA-binding domains (DBDs) are cheap and relatively easy to use. Moreover, fusing transcriptional and epigenetic modulators to programmable DBDs can be done with ease. Recent improvements of these modulators have increased the efficacy and appeal of their biomedical applications. Screening and spatiotemporal control of genomic regulation using programmable DBDs have drastically improved the breadth and resolution of (epi)genomic knowledge. The widespread adoption of programmable DBDs for (epi)genomic investigations will lead to a deeper and more comprehensive understanding of diseases and other biological phenomena.
Molecular engineering of protein assemblies, including the fabrication of nanostructures and synthetic signaling pathways, relies on the availability of modular parts that can be combined to give different structures and functions. Protein interactions are an important modular part, yet a limited number of well-characterized interaction components are available. Coiled-coil protein interaction modules have been demonstrated to be useful for biomolecular design, and many parallel homodimers and heterodimers are available in the coiled-coil toolkit. In this work, we sought to design a set of orthogonal antiparallel homodimeric coiled coils using a computational approach. There are very few antiparallel homodimers described in the literature, and none have been measured for cross-reactivity. We tested the ability of the distance-dependent statistical potential DFIRE to predict orientation preferences for coiled-coil dimers of known structure. The DFIRE model was then combined with the CLASSY multi-state protein design framework to engineer sets of three orthogonal antiparallel homodimeric coiled coils. Experimental measurements confirmed the successful design of three peptides that preferentially formed antiparallel homodimers that, furthermore, did not interact with one additional previously reported antiparallel homodimer. Two designed peptides that formed higher-order structures suggest how future design protocols could be improved. The successful designs represent a significant expansion of the existing protein-interaction toolbox for molecular engineers.
Protein–DNA interactions play a major role in all aspects of genetic activity within an organism, such as transcription, packaging, rearrangement, replication and repair. The molecular detail of protein–DNA interactions can be best visualized through crystallography, and structures emphasizing insight into the principles of binding and base-sequence recognition are essential to understanding the subtleties of the underlying mechanisms. An increasing number of high-quality DNA-binding protein structure determinations have been witnessed despite the fact that the crystallographic particularities of nucleic acids tend to pose specific challenges to methods primarily developed for proteins. Crystallographic structure solution of protein–DNA complexes therefore remains a challenging area that is in need of optimized experimental and computational methods. The potential of the structure-solution program
ARCIMBOLDO
for the solution of protein–DNA complexes has therefore been assessed. The method is based on the combination of locating small, very accurate fragments using the program
Phaser
and density modification with the program
SHELXE
. Whereas for typical proteins main-chain α-helices provide the ideal, almost ubiquitous, small fragments to start searches, in the case of DNA complexes the binding motifs and DNA double helix constitute suitable search fragments. The aim of this work is to provide an effective library of search fragments as well as to determine the optimal
ARCIMBOLDO
strategy for the solution of this class of structures.
Protein design is a valuable tool for understanding the fundamental factors that dictate protein structure and function. The field of protein design has seen significant progress with a growing range of applications in areas of research from fundamental biochemistry to biotechnology and medicine. Rapid advancement of computer applications in biotechnology now enables one to model the evolution of a particular protein on a human time scale, instead of an evolutionary time scale. The effort of designing proteins into redesign is categorized, which involves rationally engineering desired functionality into an existing protein scaffold and de novo design, which involves building a peptidic or protein system that is not directly related to any sequence found in nature yet folds into a predicted structure and carries out desired reactions.
This work presents a protein structure that has been designed purely for aesthetic reasons, symbolizing decades of coiled-coil research and praising its most fundamental model system, the GCN4 leucine zipper. The GCN4 leucine zipper is a highly stable coiled coil which can be tuned to adopt different oligomeric states via mutation of its core residues. For these reasons it is used in structural studies as a stabilizing fusion adaptor. On the occasion of the 50(th) birthday of Andrei N. Lupas, we used it to create the first personalized protein structure: we fused the sequence ANDREI-N-LVPAS in heptad register to trimeric GCN4 adaptors and determined its structure by X-ray crystallography. The structure demonstrates the robustness and versatility of GCN4 as a fusion adaptor. We learn how proline can be accommodated in trimeric coiled coils, and put the structure into the context of the other GCN4-fusion structures known to date.
The synthetic biology toolkit contains a growing number of parts for regulating transcription and translation, but very few that can be used to control protein association. Here we report characterization of 22 previously published heterospecific synthetic coiled-coil peptides called SYNZIPs. We present biophysical analysis of the oligomerization states, helix orientations, and affinities of 27 SYNZIP pairs. SYNZIP pairs were also tested for interaction in two cell-based assays. In a yeast two-hybrid screen, >85% of 253 comparable interactions were consistent with prior in vitro measurements made using coiled-coil microarrays. In a yeast-signaling assay controlled by coiled-coil mediated scaffolding, 12 SYNZIP pairs were successfully used to down-regulate the expression of a reporter gene following treatment with α-factor. Characterization of these interaction modules dramatically increases the number of available protein interaction parts for synthetic biology and should facilitate a wide range of molecular engineering applications. Summary characteristics of 27 SYNZIP peptide pairs are reported in specification sheets available in the Supporting Information and at the SYNZIP Web site [http://keatingweb.mit.edu/SYNZIP/].
The novel method of detecting weak coal gas concentration signal is proposed based on Duffing chaotic oscillator theory in the paper. The chaotic feature of Duffing oscillator is introduced, the basic principle of periodic signal detection with Duffing chaotic oscillator is described, and the chaotic threshold of Duffing oscillator is discussed and determined by the Melnikov function. The autocorrelation theory and chaotic signal amplitude detection method are combined to decrease the detecting errors for weak coal gas concentration signals. Theory analysis and simulation results show that the higher sensitivity and good anti-noise performance are obtained by using of the Duffing chaotic oscillator due to its sensitivity to the initial measurement condition while the tested signal with periodic perturbation is used as the driving force of Duffing equation.
Integrating vectors such as viruses and transposons insert transgenes semi-randomly and can potentially disrupt or deregulate genes. For these techniques to be of therapeutic value, a method for controlling the precise location of insertion is required. The piggyBac (PB) transposase is an efficient gene transfer vector active in a variety of cell types and proven to be amenable to modification. Here we present the design and validation of chimeric PB proteins fused to the Gal4 DNA binding domain with the ability to target transgenes to pre-determined sites. Upstream activating sequence (UAS) Gal4 recognition sites harbored on recipient plasmids were preferentially targeted by the chimeric Gal4-PB transposase in human cells. To analyze the ability of these PB fusion proteins to target chromosomal locations, UAS sites were randomly integrated throughout the genome using the Sleeping Beauty transposon. Both N- and C-terminal Gal4-PB fusion proteins but not native PB were capable of targeting transposition nearby these introduced sites. A genome-wide integration analysis revealed the ability of our fusion constructs to bias 24% of integrations near endogenous Gal4 recognition sequences. This work provides a powerful approach to enhance the properties of the PB system for applications such as genetic engineering and gene therapy.
The Cys(2)His(2)-type zinc finger is a common DNA binding motif that is widely used in the design of artificial zinc finger proteins. In almost all Cys(2)His(2)-type zinc fingers, position 4 of the α-helical DNA-recognition site is occupied by a Leu residue involved in formation of the minimal hydrophobic core. However, the third zinc finger domain of native Zif268 contains an Arg residue instead of the conserved Leu. Our aim in the present study was to clarify the role of this Arg in the formation of a stable domain structure and in DNA binding by substituting it with a Lys, Leu, or Hgn, which have different terminal side-chain structures. Assessed were the metal binding properties, peptide conformations, and DNA-binding abilities of the mutants. All three mutant finger 3 peptides exhibited conformations and thermal stabilities similar to the wild-type peptide. In DNA-binding assays, the Lys mutant bound to target DNA, though its affinity was lower than that of the wild-type peptide. On the other hand, the Leu and Hgn mutants had no ability to bind DNA, despite the similarity in their secondary structures to the wild-type. Our results demonstrate that, as with the Leu residue, the aliphatic carbon side chain of this Arg residue plays a key role in the formation of a stable zinc finger domain, and its terminal guanidinium group appears to be essential for DNA binding mediated through both electrostatic interaction and hydrogen bonding with DNA phosphate backbone.
A method is presented for determining conditions for the cocrystallization of zinc finger proteins with DNA. The method describes steps beginning with protein expression, through purification, design of DNA for cocrystallization, and the conditions to screen for cocrystallization.
Molecular dynamics (MD) simulations for Zif268 (a zinc-finger-protein binding specifically to the GC-rich DNA)-d(A(1) G(2) C(3) G(4) T(5) G(6) G(7) G(8) C(9) A(10) C(11) )(2) and TATA(ZF) (a zinc-finger-protein recognizing the AT-rich DNA)-d(A(1) C(2) G(3) C(4) T(5) A(6) T(7) A(8) A(9) A(10) A(11) G(12) G(13) )(2) complexes have been performed for investigating the DNA binding affinities and specific recognitions of zinc fingers to GC-rich and AT-rich DNA sequences. The binding free energies for the two systems have been further analyzed by using the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method. The calculations of the binding free energies reveal that the affinity energy of Zif268-DNA complex is larger than that of TATA(ZF) -DNA one. The affinity between the zinc-finger-protein and DNA is mainly driven by more favorable van-der-Waals and nonpolar/solvation interactions in both complexes. However, the affinity energy difference of the two binding systems is mainly caused by the difference of van-der-Waals interactions and entropy components. The decomposition analysis of MM-PBSA free energies on each residue of the proteins predicts that the interactions between the residues with the positive charges and DNA favor the binding process; while the interactions between the residues with the negative charges and DNA behave in the opposite way. The interhydrogen-bonds at the protein-DNA interface and the induced intrafinger hydrogen bonds between the residues of protein for the Zif268-DNA complex have been identified at some key contact sites. However, only the interhydrogen-bonds between the residues of protein and DNA for TATA(ZF) -DNA complex have been found. The interactions of hydrogen-bonds, electrostatistics and van-der-Waals type at some new contact sites have been identified. Moreover, the recognition characteristics of the two studied zinc-finger-proteins have also been discussed.
The solution structures of nucleocapsid (NC)-like CCHC zinc-binding domains bound to nucleic acid targets have revealed that these domains bind guanosine residues within single-stranded nucleic acids. Here, we have performed initial studies examining the potential use of NC-like CCHC zinc-binding domains as modules to construct single-stranded nucleic acid binding peptides. The affinity for guanosine-containing single-stranded deoxyribooligonucleotides increases with the number of CCHC domains in the peptide. The length of the linker between domains affects the spacing of guanosine residues in oligonucleotides that are preferentially bound. These studies provide a proof of principle that NC-like CCHC zinc-binding domains can be utilized as a basis for designing peptides that bind specific single-stranded nucleic acid sequences.
Zinc (Zn) homeostasis in adults is achieved principally through a balance between intestinal absorption and excretion involving adaptive mechanisms programmed by levels of dietary Zn. Zn absorption in infants is not as tightly regulated as that in adults, which may induce potential toxicity in infants due to the relatively high capacity of Zn absorption. We hypothesized that intestinal Zn homeostasis is developmentally regulated and depends on intestinal maturation, which in turn affects Zn transporter regulation.
Cultured human fetal (FHs 74 Int, F) and adult (Caco-2: undifferentiated, U; differentiated, D) intestinal cells were used to determine developmental differences in Zn uptake and effects of Zn exposure on Zn transporters.
Zn uptake rates in F and U cells were higher compared with D cells (F, 9-fold; U, 3-fold). F cells were more intolerant to Zn exposure than were U or D cells (LD50 = 67.9 +/- 5.3; 117.0 +/- 5.2; 224.4 +/- 3.7 micromol/L, respectively). Two mechanisms were involved in developmental regulation of Zn homeostasis: differential Zn transporter expression and differential response to Zn exposure. In F cells, zinc-regulated transporter (ZRT)/iron-regulated transporter (IRT)-like protein (Zip)4 expression was undetectable; Zn (50 micromol/L) increased levels of Zn transporter (ZnT)1, ZnT2, and metallothionein-1 mRNA and ZnT1 protein. U and D cells had higher mRNA expression of ZnT1 (U: 5-fold; D: 7-fold, respectively) and ZnT2 (U: 2-fold; D: 9-fold, respectively) than F cells, and D cells also had higher Zip4 expression (3-fold) than U cells. In U cells, Zn exposure increased Zip4 protein level, but not membrane-associated abundance. However, in D cells, Zn exposure decreased both the Zip4 protein level and membrane-associated abundance.
Zn absorption is developmentally regulated through intestinal Zn efflux and sequestration and import mechanisms, which may be responsible for differences in Zn absorption observed between infants and adults.
The versatile coiled-coil protein motif is widely used to induce and control macromolecular interactions in biology and materials science. Yet the types of interaction patterns that can be constructed using known coiled coils are limited. Here we greatly expand the coiled-coil toolkit by measuring the complete pairwise interactions of 48 synthetic coiled coils and 7 human bZIP coiled coils using peptide microarrays. The resulting 55-member protein "interactome" includes 27 pairs of interacting peptides that preferentially heteroassociate. The 27 pairs can be used in combinations to assemble sets of 3 to 6 proteins that compose networks of varying topologies. Of special interest are heterospecific peptide pairs that participate in mutually orthogonal interactions. Such pairs provide the opportunity to dimerize two separate molecular systems without undesired crosstalk. Solution and structural characterization of two such sets of orthogonal heterodimers provide details of their interaction geometries. The orthogonal pair, along with the many other network motifs discovered in our screen, provide new capabilities for synthetic biology and other applications.
Engineered transcription factors and endonucleases based on designed Cys(2)His(2) zinc finger domains have proven to be effective tools for the directed regulation and modification of genes. The introduction of this technology into both research and clinical settings necessitates the development of rapid and accurate means of evaluating both the binding affinity and binding specificity of designed zinc finger domains. Using a fluorescence anisotropy-based DNA-binding assay, we examined the DNA-binding properties of two engineered zinc finger proteins that differ by a single amino acid. We demonstrate that the protein with the highest affinity for a particular DNA site need not be the protein that binds that site with the highest degree of specificity. Moreover, by comparing the binding characteristics of the two proteins at varying salt concentrations, we show that the ionic strength makes significant and variable contributions to both affinity and specificity. These results have significant implications for zinc finger design as they highlight the importance of considering affinity, specificity, and environmental requirements in designing a DNA-binding domain for a particular application.
The aim of this thesis is to consider how fundamental engineering principles might best be applied to the design and construction of engineered biological systems. I begin by applying these principles to a key application area of synthetic biology: metabolic engineering. Abstraction is used to compile a desired system function, reprogramming bacterial odor, to devices with human-defined function, then to biological parts, and finally to genetic sequences. Standardization is used to make the process of engineering a multi-component system easier. I then focus on devices that implement digital information processing through transcriptional regulation in Escherichia coli. For simplicity, I limit the discussion to a particular type of device, a transcriptional inverter, although much of the work applies to other devices as well. First, I discuss basic issues in transcriptional inverter design. Identification of key metrics for evaluating the quality of a static device behavior allows informed device design that optimizes digital performance. Second, I address the issue of ensuring that transcriptional devices work in combination by presenting a framework for developing standards for functional composition. The framework relies on additional measures of device performance, such as error rate and the operational demand the device places on the cellular chassis, in order to proscribe standard device signal thresholds. Third, I develop an experimental, proof-of-principle implementation of a transcriptional inverter based on a synthetic transcription factor derived from a zinc finger DNA binding domain and a leucine zipper dimerization domain. Zinc fingers and leucine zippers offer a potential scalable solution to the challenge of building libraries of transcription-based logic devices for arbitrary information processing in cells.
The human retinoid X receptor alpha (hRXRalpha) is a member of the nuclear receptor super-family of ligand-activated transcription factors. The Doyle laboratory has previously engineered a variety of functional hRXRalpha variants that activate gene expression in response to synthetic ligands (LG335 and γ-oxo-1-pyrenebutyric acid), compounds that are poor activators of wild-type hRXRalpha. The variants generally no longer respond to the wild-type ligand 9-cis retinoic acid. To enable targeting of these engineered receptors to arbitrary DNA sequences, we developed a program, ESPSearch, for identifying short or specific sequences in DNA or protein. ESPSearch enables identification of combinations of known zinc finger motifs to target arbitrary genes, as well having several other applications. The ability to target any DNA sequence means that the engineered receptors can be directed to control any gene. The ligand binding, self-association, coactivator interactions, and unfolding properties of the ligand binding domain of wild-type hRXRalpha were characterized. Our expression and purification protocol improves upon existing methods, providing high purity protein in a single step with more than twice prior yields. A general fluorescence-based method for measuring ligand affinity with hRXRalpha was developed, and used to determine binding constants for the small molecules. The presence of a peptide containing the binding motif from coactivator proteins (LxxLL) differentially increased the affinity of the receptor for the ligands. Assays to determine the self-association give a Kd for the dimer-tetramer equilibrium of 35 µM. hRXRalpha was found to denature irreversibly when heated, but shifts in apparent Tm due to ligands correlates strongly with the ligand binding affinities. Our results clarify disparities in existing reports and provide a benchmark for comparison. Reliable analysis of our data led to the development of a computer program for rigorous, automated data fitting. Nine functional variants of hRXRalpha were characterized to probe correlations between biophysical properties and the observed functional activity of the receptors, which differ significantly from wild-type. Although the correlation between ligand binding affinity and melting temperature was strong for all variants, there was essentially no correlation between ligand binding and activation of the variants. The mutations, which are all contained within the binding pocket, have significant long-range effects on the protein, causing changes in ligand-LxxLL interactions and oligomerization of the variants. Experimental and computational analysis of selected mutations suggests that they are highly coupled, complicating protein design. However, the large variation in properties amongst the variants also suggests that hRXRalpha can be mutated extensively while still retaining function. The long-range impact of binding pocket mutations will need to be taken into account in future engineering projects, as hRXRalpha is a flexible, dynamic protein. Ph.D. Committee Chair: Doyle, Donald; Committee Member: Bommarius, Andreas; Committee Member: Harvey, Stephen; Committee Member: Hud, Nicholas; Committee Member: Kubanek, Julia
In the last decade, the efforts in clarifying the interaction between zinc finger proteins and DNA targets strongly stimulated the creativity of scientists in the field of protein engineering. In particular, the versatility and the modularity of zinc finger (ZF) motives make these domains optimal building blocks for generating artificial zinc finger peptides (ZFPs). ZFPs can act as transcription modulators potentially able to control the expression of any desired gene, when fused to an appropriate effector domain. Artificial ZFPs open the possibility to re-program the expression of specific genes at will and can represent a powerful tool in basic science, biotechnology and gene therapy. In this review we will focus on old, novel and possible future applications of artificial ZFPs.
ESPSearch is a computer program for rapidly identifying nucleic acid or amino acid sequences of any length within any source sequence from promoters to entire genomes to protein libraries. ESPSearch utilizes a user-constructed database to identify many sequences simultaneously, including target sequences with wildcards and mismatches and user-specified patterns of those recognized sequences. Here we use ESPSearch to identify a variety of possible binding sites for dimeric artificial transcription factors within several p53 recognition sites and the promoter of the BAX gene. Heterodimeric and homodimeric proteins are designed using human zinc fingers by identifying groups of zinc finger binding sites meeting particular pattern constraints. ESPSearch is also used to estimate the specificity of each artificial transcription factor by searching the entire genome. Next, the specificity of several possible small interfering RNA (siRNA) sequences is determined by searching both the whole genome and the library of known human mRNAs. Finally, ESPSearch identifies proteins containing different forms of the LXXLL motif used in nuclear receptor-coactivator interactions from the human proteome, making use of user-defined groups of amino acids. ESPSearch could also be applied to other tasks involving sequence and pattern recognition on small and large scales. ESPSearch is freely available at http://web.chemistry.gatech.edu/-doyle/espsearch/.
We have identified four mutations in Xenopus TFIIIA that increase the stability of TFIIIA-5 S rRNA gene complexes. In each case, the mutation has a relatively modest effect on equilibrium binding affinity. In three cases, these equilibrium binding effects can be ascribed primarily to decreases in the rate constant for protein-DNA complex dissociation. In the fourth case, however, a substitution of phenylalanine for the wild-type leucine at position 148 in TFIIIA results in much larger compensating changes in the kinetics of complex assembly and dissociation. The data support a model in which a relatively unstable population of complexes with multi-component dissociation kinetics forms rapidly; complexes then undergo a slow conformational change that results in very stable, kinetically homogeneous TFIIIA-DNA complexes. The L148F mutant protein acts as a particularly potent transcriptional activator when it is fused to the VP16 activation domain and expressed in yeast cells. Substitution of L148 to tyrosine or tryptophan produces an equally strong transcriptional activator. Substitution to histidine results in genetic and biochemical effects that are more modest than, but similar to, those observed with the L148F mutation. We propose that an amino acid with a planar side chain at position 148 can intercalate between adjacent base pairs in the intermediate element of the 5 S rRNA gene. Intercalation occurs slowly but results in a very stable DNA-protein complex. These results suggest that transcriptional activation by a cis-acting sequence element is largely dependent on the kinetic, rather than the thermodynamic, stability of the complex formed with an activator protein. Thus, transcriptional activation is dependent in large part on the lifetime of the activator-DNA complex rather than on binding site occupancy at steady state. Introduction of intercalating amino acids into zinc finger proteins may be a useful tool for producing artificial transcription factors with particularly high in vivo activity.
Artificial DNA binding peptides recognizing separated sequences would expand varieties of the target genes for desirable transcriptional control. Here we demonstrated that polyarginine linker between two 3-zinc finger domains gives DNA binding selectivity to the separated target sequences. We created a six-zinc finger peptide, Sp1ZF6(Arg)8, by connecting two DNA binding domains of transcription factor Sp1 with a bulky and cationic polyarginine linker. The DNA binding properties to continuous and discontinuous target sequences were examined and compared to those of Sp1ZF6(Gly)10 containing a flexible and neutral polyglycine linker. The dissociation constants indicate that Sp1ZF6(Arg)8 has an obvious DNA binding preference to discontinuous target sequences but not Sp1ZF6(Gly)10. Footprinting analyses also showed that Sp1ZF6(Arg)8 binds properly only to the discontinuous target sites, while Sp1ZF6(Gly)10 does not distinguish them. The results provide helpful information for linker design of future zinc finger peptides to various states of DNA as gene expression regulators.
The divergently transcribed DIT1 and DIT2 genes of Saccharomyces cerevisiae, which belong to the mid-late class of sporulation-specific genes, are subject to Ssn6-Tup1-mediated repression in mitotic
cells. The Ssn6-Tup1 complex, which is required for repression of diverse sets of coordinately regulated genes, is known to
be recruited to target genes by promoter-specific DNA-binding proteins. In this study, we show that a 42-bp negative regulatory
element (NRE) present in the DIT1-DIT2 intergenic region consists of two distinct subsites and that a multimer of each subsite supports efficient Ssn6-Tup1-dependent
repression of a CYC1-lacZ reporter gene. By genetic screening procedures, we identified DFG16, YGR122w, VPS36, and the DNA-binding proteins Rim101 and Nrg1 as potential mediators of NRE-directed repression. We show that Nrg1 and Rim101
bind simultaneously to adjacent target sites within the NRE in vitro and act as corepressors in vivo. We have found that the
ability of Rim101 to be proteolytically processed to its active form and mediate NRE-directed repression not only depends
on the previously characterized RIM signaling pathway but also requires Dfg16, Ygr122w, and components of the ESCRT trafficking
pathway. Interestingly, Rim101 was processed in bro1 and doa4 strains but was unable to mediate efficient repression.
Zinc finger fusion proteins, having a Ca-binding site from troponin C, were created to develop Ca-responsive regulation of DNA binding. The typical zinc finger folding of a novel fusion protein with a single finger, F2-Tn, was investigated using UV-vis spectroscopy of the Co-substituted form and CD experiments. Detailed structural analyses of F2-Tn/Zn2+ using NMR experiments and structural calculations clarify that our fusion protein gives a native zinc finger folding with the artificial Ca-binding domain intervening two helices. The Ca-responsive DNA-binding affinity of troponin-fused protein with two fingers (using F1F2-Tn) was investigated by electrophoretic mobility shift assay (EMSA). EMSA analyses of F1F2-Tn were performed under the conditions of various concentrations of the Ca ion. F1F2-Tn has a Kd value of 5.8 nM in the absence of Ca ion and shows a higher Kd value of 13 nM in the presence of 100 equiv of Ca ion. The artificially designed fusion zinc finger protein with a Ca-binding domain has Ca-responsive DNA-binding affinity. It is leading to a better understanding of the construction of zinc finger-based artificial transcriptional factors with a Ca switch.
Engineered biological systems have been used to manipulate information, construct materials, process chemicals, produce energy, provide food, and help maintain or enhance human health and our environment. Unfortunately, our ability to quickly and reliably engineer biological systems that behave as expected remains quite limited. Foundational technologies that make routine the engineering of biology are needed. Vibrant, open research communities and strategic leadership are necessary to ensure that the development and application of biological technologies remains overwhelmingly constructive.
Although many zinc finger motifs have been developed to recognize specific DNA triplets, a rational way to selectively skip a particular non-recognized gap in the DNA sequence has never been established. We have now created a 6-zinc finger peptide with an alpha-helix linker, Sp1ZF6(EAAAR)4, which selectively binds to the discontinuous recognition sites in the same phase (10 bp gap) against the opposite phase (5 bp gap) of the DNA helix. The linker peptide (EAAAR)4 forms an alpha-helix structure stabilized by salt bridges, and the helical length is estimated to be about 30 A, corresponding to that of the 10 bp DNA. The gel shift assays demonstrate that Sp1ZF6(EAAAR)4 preferably binds to the 10 bp-gapped target rather than the 5 bp-gapped target. The CD spectra show that the alpha-helical content of the (EAAAR)4 linker is higher in the complex with the 10 bp-gapped target than in the complex with the 5 bp-gapped target. The present results indicate that the alpha-helical linker is suitable for binding to the recognition sites in the same phase and that the linker induces the loss of binding affinity to the recognition sites with the opposite phase. The engineering of a helix-structured linker in the 6-zinc finger peptides should be one of the most promising approaches for selectively targeting discontinuous recognition sites depending on their phase situations.
The design of DNA-binding proteins for the specific control of the gene expression is one of the big challenges for several research laboratories in the post-genomic era. An artificial transcription factor with the desired DNA binding specificity could work as a powerful tool and drug to regulate the target gene. The zinc-finger proteins, which typically contain many fingers linked in a tandem fashion, are some of the most intensively studied DNA-binding proteins. In particular, the Cys(2)His(2)-type zinc finger is one of the most common DNA-binding motifs in eukaryotes. A simple mode of DNA recognition by the Cys(2)His(2)-type zinc-finger domain provides an ideal framework for designing proteins with new functions. Our laboratory has utilized several design strategies to create new zinc-finger peptides/proteins by redesigning the Cys(2)His(2)-type zinc-finger motif. This review focuses on the aspects of design strategies, mainly from our recent results, for the creation of artificial zinc-finger proteins, and discusses the possible application of zinc-finger technology for gene regulation and gene therapy.
Publisher Summary X-ray data can be collected with zero-, one-, and two-dimensional detectors, zero-dimensional (single counter) being the simplest and two-dimensional the most efficient in terms of measuring diffracted X-rays in all directions. To analyze the single-crystal diffraction data collected with these detectors, several computer programs have been developed. Two-dimensional detectors and related software are now predominantly used to measure and integrate diffraction from single crystals of biological macromolecules. Macromolecular crystallography is an iterative process. To monitor the progress, the HKL package provides two tools: (1) statistics, both weighted (χ 2 ) and unweighted (R-merge), where the Bayesian reasoning and multicomponent error model helps obtain proper error estimates and (2) visualization of the process, which helps an operator to confirm that the process of data reduction, including the resulting statistics, is correct and allows the evaluation of the problems for which there are no good statistical criteria. Visualization also provides confidence that the point of diminishing returns in data collection and reduction has been reached. At that point, the effort should be directed to solving the structure. The methods presented in the chapter have been applied to solve a large variety of problems, from inorganic molecules with 5 A unit cell to rotavirus of 700 A diameters crystallized in 700 × 1000 × 1400 A cell.
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.
The Zif268 zinc finger-DNA complex has served as a model system for understanding how Cys2His2 type zinc fingers recognize DNA. Structural studies of the Zif268-DNA complex revealed that residues at four positions in
the α helix of each zinc finger play key roles in recognition, but there has been no information about the precise contributions
of individual residues. Here we report the results of binding studies involving five mutants of Zif268 that have changes in
the base-contacting residues of finger one. These studies let us evaluate the contributions that Arg18(position −1 of the α helix), Asp20 (position 2), Glu21 (position 3), and Arg24 (position 6) make to the overall energy of DNA binding. Our results confirm the important role played by these arginines.
By comparing the affinities of the wild type and mutant peptides for various sites, we also prove that Asp20 and Glu21 play important roles in determining binding site specificity.
We have targeted the activation of an endogenous chromosomal locus including the human erythropoietin gene using synthetic transcription factors. These transcription factors are targeted to particular DNA sequences in the 5'-flanking region of the erythropoietin gene through engineering of a zinc finger DNA binding domain. The DNA binding domain is linked to a VP16 transcriptional activation domain. We find that these synthetic transcription factors invariably activate transiently transfected templates in which sequences within the 5' flank of the erythropoietin gene are fused to a luciferase reporter. The efficiency of activation under these circumstances at a defined site is dependent on DNA binding affinity. In contrast, only a subset of these same zinc finger proteins is able to activate the endogenous chromosomal locus. The activity of these proteins is influenced by their capacity to gain access to their recognition elements within the chromatin infrastructure. Zinc finger transcription factors will provide a powerful tool to probe the determinants of chromatin accessibility and remodeling within endogenous chromosomal loci.
Cys2His2 zinc finger proteins offer a stable and versatile framework for the design of proteins that recognize desired target sites on double-stranded DNA. Individual fingers from these proteins have a simple beta beta alpha structure that folds around a central zinc ion, and tandem sets of fingers can contact neighboring subsites of 3-4 base pairs along the major groove of the DNA. Although there is no simple, general code for zinc finger-DNA recognition, selection strategies have been developed that allow these proteins to be targeted to almost any desired site on double-stranded DNA. The affinity and specificity of these new proteins can also be improved by linking more fingers together or by designing proteins that bind as dimers and thus recognize an extended site. These new proteins can then be modified by adding other domains--for activation or repression of transcription, for DNA cleavage, or for other activities. Such designer transcription factors and other new proteins will have important applications in biomedical research and in gene therapy.
The relationship between the structure of zinc-finger protein (ZFP) transcription factors and DNA sequence binding specificity has been extensively studied. Advances in this field have made it possible to design ZFPs de novo that will bind to specific targeted DNA sequences. It has been proposed that such designed ZFPs may eventually be useful in gene therapy. A principal advantage of this approach is that activation of an endogenous gene ensures expression of the natural array of splice variants. Preliminary studies in tissue culture have validated the feasibility of this approach. The studies reported here were intended to test whether engineered transcription factors are effective in a whole-organism model. ZFPs were designed to regulate the endogenous gene encoding vascular endothelial growth factor-A (Vegfa). Expression of these new ZFPs in vivo led to induced expression of the protein VEGF-A, stimulation of angiogenesis and acceleration of experimental wound healing. In addition, the neovasculature resulting from ZFP-induced expression of Vegfa was not hyperpermeable as was that produced by expression of murine Vegfa(164) cDNA. These data establish, for the first time, that specifically designed transcription factors can regulate an endogenous gene in vivo and evoke a potentially therapeutic biophysiologic effect.
The CCP4 (Collaborative Computational Project, number 4) program suite is a collection of programs and associated data and subroutine libraries which can be used for macromolecular structure determination by X-ray crystallography. The suite is designed to be flexible, allowing users a number of methods of achieving their aims and so there may be more than one program to cover each function. The programs are written mainly in standard Fortran77. They are from a wide variety of sources but are connected by standard data file formats. The package has been ported to all the major platforms under both Unix and VMS. The suite is distributed by anonymous ftp from Daresbury Laboratory and is widely used throughout the world.
We present a comprehensive software package, 3DNA, for the analysis, reconstruction and visualization of three‐dimensional
nucleic acid structures. Starting from a coordinate file in Protein Data Bank (PDB) format, 3DNA can handle antiparallel and
parallel double helices, single‐stranded structures, triplexes, quadruplexes and other complex tertiary folding motifs found
in both DNA and RNA structures. The analysis routines identify and categorize all base interactions and classify the double
helical character of appropriate base pair steps. The program makes use of a recently recommended reference frame for the
description of nucleic acid base pair geometry and a rigorous matrix‐based scheme to calculate local conformational parameters
and rebuild the structure from these parameters. The rebuilding routines produce rectangular block representations of nucleic
acids as well as full atomic models with the sugar–phosphate backbone and publication quality ‘standardized’ base stacking
diagrams. Utilities are provided to locate the base pairs and helical regions in a structure and to reorient structures for
effective visualization. Regular helical models based on X‐ray diffraction measurements of various repeating sequences can
also be generated within the program.
The nuclear factor of activated T cells (NFAT) and the AP-1 heterodimer, Fos-Jun, cooperatively bind a composite DNA site and synergistically activate the expression of many immune-response genes. A 2.7-A-resolution crystal structure of the DNA-binding domains of NFAT, Fos and Jun, in a quaternary complex with a DNA fragment containing the distal antigen-receptor response element from the interleukin-2 gene promoter, shows an extended interface between NFAT and AP-1, facilitated by the bending of Fos and DNA. The tight association of the three proteins on DNA creates a continuous groove for the recognition of 15 base pairs.
Hopes were high within the French research community when Claude Allègre was appointed minister of research last year. But a good start has given way to frustration with the slow speed of change.
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.
The yeast transcriptional activator GCN4 is 1 of over 30 identified eukaryotic proteins containing the basic region leucine zipper (bZIP) DNA-binding motif. We have determined the crystal structure of the GCN4 bZIP element complexed with DNA at 2.9 A resolution. The bZIP dimer is a pair of continuous alpha helices that form a parallel coiled coil over their carboxy-terminal 30 residues and gradually diverge toward their amino termini to pass through the major groove of the DNA-binding site. The coiled-coil dimerization interface is oriented almost perpendicular to the DNA axis, giving the complex the appearance of the letter T. There are no kinks or sharp bends in either bZIP monomer. Numerous contacts to DNA bases and phosphate oxygens are made by basic region residues that are conserved in the bZIP protein family. The details of the bZIP dimer interaction with DNA can explain recognition of the AP-1 site by the GCN4 protein.
Map interpretation remains a critical step in solving the structure of a macromolecule. Errors introduced at this early stage may persist throughout crystallographic refinement and result in an incorrect structure. The normally quoted crystallographic residual is often a poor description for the quality of the model. Strategies and tools are described that help to alleviate this problem. These simplify the model-building process, quantify the goodness of fit of the model on a per-residue basis and locate possible errors in peptide and side-chain conformations.
The Fos and Jun families of eukaryotic transcription factors heterodimerize to form complexes capable of binding 5'-TGAGTCA-3' DNA elements. We have determined the X-ray crystal structure of a heterodimer of the bZIP regions of c-Fos and c-Jun bound to DNA. Both subunits form continuous alpha-helices. The carboxy-terminal regions form an asymmetric coiled-coil, and the amino-terminal regions make base-specific contacts with DNA in the major groove. Comparison of the two crystallographically distinct protein-DNA complexes show that the coiled-coil is flexibly joined to the basic regions and that the Fos-Jun heterodimer does not recognize the asymmetric 5'-TGAGTCA-3' recognition element in a unique orientation. There is an extensive network of electrostatic interactions between subunits within the coiled-coil, consistent with proposals that these interactions determine preferential formation of the heterodimer over either of the homodimers.
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.
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.
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.
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.
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.
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.
Zinc-finger proteins of the Cys2-His2 type represent a class of malleable DNA-binding proteins that may be selected to bind diverse sequences. Typically, zinc-finger proteins containing three zinc-finger domains, like the murine transcription factor Zif268 and the human transcription factor Sp1, bind nine contiguous base pairs. To create a class of proteins that would be generally applicable to target unique sites within complex genomes, we have utilized structure-based modeling to design a polypeptide linker that fuses two three-finger proteins. Two six-fingered proteins were created and demonstrated to bind 18 contiguous bp of DNA in a sequence-specific fashion. Expression of these proteins as fusions to activation or repression domains allows transcription to be specifically up- or down-modulated within human cells. Polydactyl zinc-finger proteins should be broadly applicable as genome-specific transcriptional switches in gene therapy strategies and the development of novel transgenic plants and animals.
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.
Zinc fingers of the Cys2-His2 class comprise one of the largest families of eukaryotic DNA-binding motifs and recognize a diverse set of DNA sequences. These proteins have a relatively simple modular structure and key base contacts are typically made by a few residues from each finger. These features make the zinc finger motif an attractive system for designing novel DNA-binding proteins and for exploring fundamental principles of protein-DNA recognition.
Here we report the X-ray crystal structures of zinc finger-DNA complexes involving three variants of Zif268, with multiple changes in the recognition helix of finger one. We describe the structure of each of these three-finger peptides bound to its corresponding target site. To help elucidate the differential basis for site-specific recognition, the structures of four other complexes containing various combinations of these peptides with alternative binding sites have also been determined.
The protein-DNA contacts observed in these complexes reveal the basis for the specificity demonstrated by these Zif268 variants. Many, but not all, of the contacts can be rationalized in terms of a recognition code, but the predictive value of such a code is limited. The structures illustrate how modest changes in the docking arrangement accommodate the new sidechain-base and sidechain-phosphate interactions. Such adaptations help explain the versatility of naturally occurring zinc finger proteins and their utility in design.
It is well-known that DNA binding of native nine zinc-finger protein TEIIIA is dominated by interaction of select few fingers. Newly designed zinc-finger peptide Sp1ZF9 containing nine Cys2-His2 type motifs has been manipulated. The DNA-binding property of Sp1ZF9 was compared with those of native three zinc-finger Sp1(530-623) and artificial six zinc-finger Sp1ZF6 peptides. Although the equilibrium time was less than 0.5 h for Sp1(530-623)-DNA complex, Sp1ZF6 and Sp1ZF9 required approximately 48 and 72 h, respectively, for full complex formation. Evidently, the footprinting analysis demonstrated that Sp1ZF9 and Sp1ZF6 bind at least 27 and 18 contiguous base pairs of DNA sequence, respectively. Sp1ZF9 showed two step bindings to DNA, namely first the recognition of GC (5'-GGG-GCG-GGGCC-3') sequence by the N-terminal Sp1 domain and next the recognition of the corresponding target sequences by the middle and C-terminal Sp1 domains. In contrast with unimolecular binding of Sp1ZF9 and Sp1ZF6, two Sp1(530-623) molecules bind to one GCIII (5'-GGG-GCG-GGG-GGG-GCG-GGG-GGG++ +-GCG-GGGCC-3') site region. Semispecific complex formed at the beginning of Sp1ZF9-DNA interaction has also been characterized by kinetic analysis using surface plasmon resonance. Interestingly, the association rate constants for GC and GCIII complexes of Sp1ZF9 are smaller than those of the corresponding Sp1(530-623) complexes. Of special interest is the fact that new nine zinc-finger peptide Sp1ZF9 can bind to DNA sequence of approximately 30 base pairs. Such multi zinc-finger peptides may be useful as genome-specific transcriptional switches in future.
To create a universal system for the control of gene expression, we have studied methods for the construction of novel polydactyl zinc finger proteins that recognize extended DNA sequences. Elsewhere we have described the generation of zinc finger domains recognizing sequences of the 5'-GNN-3' subset of a 64-member zinc finger alphabet. Here we report on the use of these domains as modular building blocks for the construction of polydactyl proteins specifically recognizing 9- or 18-bp sequences. A rapid PCR assembly method was developed that, together with this predefined set of zinc finger domains, provides ready access to 17 million novel proteins that bind the 5'-(GNN)6-3' family of 18-bp DNA sites. To examine the efficacy of this strategy in gene control, the human erbB-2 gene was chosen as a model. A polydactyl protein specifically recognizing an 18-bp sequence in the 5'-untranslated region of this gene was converted into a transcriptional repressor by fusion with Kr uppel-associated box (KRAB), ERD, or SID repressor domains. Transcriptional activators were generated by fusion with the herpes simplex VP16 activation domain or with a tetrameric repeat of VP16's minimal activation domain, termed VP64. We demonstrate that both gene repression and activation can be achieved by targeting designed proteins to a single site within the transcribed region of a gene. We anticipate that gene-specific transcriptional regulators of the type described here will find diverse applications in gene therapy, functional genomics, and the generation of transgenic organisms.
We have taken a comprehensive approach to the generation of novel DNA binding zinc finger domains of defined specificity. Herein we describe the generation and characterization of a family of zinc finger domains developed for the recognition of each of the 16 possible 3-bp DNA binding sites having the sequence 5'-GNN-3'. Phage display libraries of zinc finger proteins were created and selected under conditions that favor enrichment of sequence-specific proteins. Zinc finger domains recognizing a number of sequences required refinement by site-directed mutagenesis that was guided by both phage selection data and structural information. In many cases, residues not expected to make base-specific contacts had effects on specificity. A number of these domains demonstrate exquisite specificity and discriminate between sequences that differ by a single base with >100-fold loss in affinity. We conclude that the three helical positions -1, 3, and 6 of a zinc finger domain are insufficient to allow for the fine specificity of the DNA binding domain to be predicted. These domains are functionally modular and may be recombined with one another to create polydactyl proteins capable of binding 18-bp sequences with subnanomolar affinity. The family of zinc finger domains described here is sufficient for the construction of 17 million novel proteins that bind the 5'-(GNN)6-3' family of DNA sequences. These materials and methods should allow for the rapid construction of novel gene switches and provide the basis for a universal system for gene control.
Peptides that mediate dimerization of attached zinc finger DNA-binding domains have been evolved in vitro starting from random sequences. We first used phage display to select dimerization elements from libraries of random 15-residue polypeptides that were fused to the N terminus of the zinc finger domains. We then reoptimized these peptides by sequentially randomizing five-residue blocks (proceeding across the peptide in three steps) and selecting variant peptides that further stabilized the protein-DNA complex. Biochemical experiments confirmed that the selected peptides promote dimerization of the zinc fingers on an appropriate DNA target site. These results demonstrate that dimerization units can be obtained readily from random polypeptide libraries of moderate complexity. Our success reemphasizes the utility of searching random peptide libraries in protein design projects, and the sequences presented here may be useful when designing novel transcription factors.
Transcriptional activation often requires the rapid assembly of complexes between dimeric transcription factors and specific DNA sites. Here we show that members of the basic region leucine zipper and basic region helix-loop-helix zipper transcription factor families follow an assembly pathway in which two protein monomers bind DNA sequentially and form their dimerization interface while bound to DNA. Nonspecific protein or DNA competitors have little effect on the rate of assembly along this pathway, but slow a competing pathway in which preformed dimers bind DNA. The sequential monomer-binding pathway allows the protein to search for and locate a specific DNA site more quickly, resulting in greater specificity prior to equilibrium.
We have developed a bacterial "two-hybrid" system that readily allows selection from libraries larger than 10(8) in size. Our bacterial system may be used to study either protein-DNA or protein-protein interactions, and it offers a number of potentially significant advantages over existing yeast-based one-hybrid and two-hybrid methods. We tested our system by selecting zinc finger variants (from a large randomized library) that bind tightly and specifically to desired DNA target sites. Our method allows sequence-specific zinc fingers to be isolated in a single selection step, and thus it should be more rapid than phage display strategies that typically require multiple enrichment/amplification cycles. Given the large library sizes our bacterial-based selection system can handle, this method should provide a powerful tool for identifying and optimizing protein-DNA and protein-protein interactions.
Several strategies have been reported for the design and selection of novel DNA-binding proteins. Most of these studies have used Cys(2)His(2) zinc finger proteins as a framework, and have focused on constructs that bind DNA in a manner similar to Zif268, with neighboring fingers connected by a canonical (Krüppel-type) linker. This linker does not seem ideal for larger constructs because only modest improvements in affinity are observed when more than three fingers are connected in this manner. Two strategies have been described that allow the productive assembly of more than three canonically linked fingers on a DNA site: connecting sets of fingers using linkers (covalent), or assembling sets of fingers using dimerization domains (non-covalent).
Using a combination of structure-based design and phage display, we have developed a new dimerization system for Cys(2)His(2) zinc fingers that allows the assembly of more than three fingers on a desired target site. Zinc finger constructs employing this new dimerization system have high affinity and good specificity for their target sites both in vitro and in vivo. Constructs that recognize an asymmetric binding site as heterodimers can be obtained through substitutions in the zinc finger and dimerization regions.
Our modular zinc finger dimerization system allows more than three Cys(2)His(2) zinc fingers to be productively assembled on a DNA-binding site. Dimerization may offer certain advantages over covalent linkage for the recognition of large DNA sequences. Our results also illustrate the power of combining structure-based design with phage display in a strategy that assimilates the best features of each method.
Cys2His2 zinc fingers are one of the most common DNA-binding motifs found in eukaryotic transcription factors. These proteins typically contain several fingers that make tandem contacts along the DNA. Each finger has a conserved beta beta alpha structure, and amino acids on the surface of the alpha-helix contact bases in the major groove. This simple, modular structure of zinc finger proteins, and the wide variety of DNA sequences they can recognize, make them an attractive framework for attempts to design novel DNA-binding proteins. Several studies have selected fingers with new specificities, and there clearly are recurring patterns in the observed side chain-base interactions. However, the structural details of recognition are intricate enough that there are no general rules (a "recognition code") that would allow the design of an optimal protein for any desired target site. Construction of multifinger proteins is also complicated by interactions between neighboring fingers and the effect of the intervening linker. This review analyzes DNA recognition by Cys2His2 zinc fingers and summarizes progress in generating proteins with novel specificities from fingers selected by phage display.
Protein-protein interactions often play a crucial role in stabilizing protein-DNA complexes and thus facilitate site-specific DNA recognition. We have worked to incorporate such protein-protein contacts into our design and selection strategies for short peptide extensions that promote cooperative binding of zinc finger proteins to DNA. We have determined the crystal structure of one of these fusion protein-DNA complexes. The selected peptide extension was found to mediate dimerization by reaching across the dyad axis and contacting a hydrophobic patch on the surface of the zinc finger bound to the adjacent DNA site. The peptide-zinc finger protein interactions observed in this structure are similar to those of some homeodomain heterodimers. We also find that the region of the zinc finger surface contacted by the selected peptide extension corresponds to surfaces that also make key interactions in the zinc finger proteins GLI and SWI5.
A large fraction of known transcription factors form 2:1 complexes with DNA. In our studies of the assembly of such ternary (protein-protein-DNA) complexes formed by bZIP and bHLHZip proteins, we found that the proteins recognize DNA as monomers. Here we show that protein monomer-DNA complexes are favored at high DNA concentrations. Further, we show that, due to fast rates of association with protein monomers, DNA and other polyanions accelerate the rate of protein dimer formation. Finally, we find that DNA-assisted formation of protein dimers provides a mechanism by which dimeric transcription factors can rapidly discriminate between specific and nonspecific sites.
The availability of rapid and robust methods for controlling gene function is of prime importance not only for assigning functions to newly discovered genes, but also for therapeutic intervention. Traditionally, gene function has been probed by often-laborious methods that either increase the level of a gene product or decrease it. Advances now make it possible to rapidly produce zinc-finger proteins capable of recognizing virtually any 18 bp stretch of DNA--a sequence long enough to specify a unique address in any genome. The attachment of functional domains also allows the design of tailor-made transcription factors for specific genes. Recent studies demonstrate that artificial transcription factors are capable of controlling the expression of endogenous genes in their native chromosomal context with a high degree of specificity in both animals and plants. Dominant regulatory control of expression of any endogenous gene can be achieved rapidly and can be also placed under chemical control. A wide range of potential applications is now within reach.