Exploring the recognition of quadruplex DNA by an engineered Cys2-His2 zinc finger protein

We have recently described an engineered zinc finger protein (Gq1) that binds with high specificity to the intramolecular G-quadruplex formed by the human telomeric sequence 5'-(GGTTAG)(5)-3', and that inhibits the activity of the enzyme telomerase in vitro. Here we report site-directed mutagenesis, biophysical, and molecular modeling studies that provide new insights into quadruplex recognition by the zinc finger scaffold. We show that any one finger of Gq1 can be replaced with the corresponding finger of Zif268, without significant loss of quadruplex affinity or quadruplex versus duplex discrimination. Replacement of two fingers, with one being finger 2, of Gq1 by Zif268 results in significant impairment of quadruplex recognition and loss of discrimination. Molecular modeling suggests that the zinc fingers of Gq1 can bind to the human parallel-stranded quadruplex structure in a stable arrangement, whereas Zif268-quadruplex models show significantly weaker binding energy. Modeling also suggests that an important role of the key protein finger residues in the Gq1-quadruplex complex is to maintain Gq1 in an optimum conformation for quadruplex recognition.     

Novel zinc finger nuclease created by combining the Cys2His2- and His4-type zinc finger domains

To improve the DNA hydrolytic activity of the zinc finger nuclease, we have created a new artificial zinc finger nuclease (ZWH4) by connecting two distinct zinc finger domains possessing different types of Zn(II) binding sites (Cys₂His₂- and His₄-types). The overall fold of ZWH4 is similar to that of the wild-type Sp1 zinc finger (Sp1(zf123)) as revealed by circular dichroism spectroscopy. The gel mobility shift assay demonstrated that ZWH4 binds to the GC box DNA, although the DNA-binding affinity is lower than that of Sp1(zf123). Evidently, ZWH4 hydrolyzes the covalently closed circular plasmid DNA (form I) containing the GC box (pBSGC) to the linear duplex DNA (form III) in the presence of a higher concentration (50 times) of the protein than DNA for a 24-h reaction. Of special interest is the fact that the novel mixed zinc finger protein containing the Cys₂His₂- and His₄-type domains was first created. The present results provide the useful information for the redesign strategy of an artificial nuclease based on the zinc finger motif.     

Beyond the "recognition code": structures of two Cys2His2 zinc finger/TATA box complexes

BACKGROUND: Several methods have been developed for creating Cys2His2 zinc finger proteins that recognize novel DNA sequences, and these proteins may have important applications in biological research and gene therapy. In spite of this progress with design/selection methodology, fundamental questions remain about the principles that govern DNA recognition. One hypothesis suggests that recognition can be described by a simple set of rules--essentially a "recognition code"--but careful assessment of this proposal has been difficult because there have been few structural studies of selected zinc finger proteins. RESULTS: We report the high-resolution cocrystal structures of two zinc finger proteins that had been selected (as variants of Zif268) to recognize a eukaryotic TATA box sequence. The overall docking arrangement of the fingers within the major groove of the DNA is similar to that observed in the Zif268 complex. Nevertheless, comparison of Zif268 and the selected variants reveal significant differences in the pattern of side chain-base interactions. The new structures also reveal side chain-side chain interactions (both within and between fingers) that are important in stabilizing the protein-DNA interface and appear to play substantial roles in recognition. CONCLUSIONS: These new structures highlight the surprising complexity of zinc finger-DNA interactions. The diversity of interactions observed at the protein-DNA interface, which is especially striking for proteins that were all derived from Zif268, challenges fundamental concepts about zinc finger-DNA recognition and underscores the difficulty in developing any meaningful recognition code.     

Independence of metal binding between tandem Cys2His2 zinc finger domains.

Most Cys2His2 zinc finger proteins contain tandem arrays of metal binding domains. The tandem nature of these arrays suggests that metal binding by these domains may not be independent but rather that metal binding may occur in a cooperative manner. This is especially true in light of the crystal structure of a three zinc finger array bound to DNA that revealed several types of interactions between domains. To address this question, peptides containing two tandem domains have been prepared. While metal binding studies do show that the two finger peptide has a metal ion affinity about threefold higher than that for a single domain peptide with the same sequence, additional studies reveal that this behavior is due to increased single site affinities in the context of the two domain peptide rather than to cooperativity. These studies indicate that domains of this type are independent of one another with regard to metal binding, at least in the absence of DNA. This observation has implications with regard to the question of whether the activities of proteins of this class might be modulated by available zinc concentrations.     

NAR Breakthrough Article: A systematic survey of the Cys2His2 zinc finger DNA-binding landscape

Cys₂His₂ zinc fingers (C2H2-ZFs) comprise the largest class of metazoan DNA-binding domains. Despite this domain''s well-defined DNA-recognition interface, and its successful use in the design of chimeric proteins capable of targeting genomic regions of interest, much remains unknown about its DNA-binding landscape. To help bridge this gap in fundamental knowledge and to provide a resource for design-oriented applications, we screened large synthetic protein libraries to select binding C2H2-ZF domains for each possible three base pair target. The resulting data consist of >160 000 unique domain–DNA interactions and comprise the most comprehensive investigation of C2H2-ZF DNA-binding interactions to date. An integrated analysis of these independent screens yielded DNA-binding profiles for tens of thousands of domains and led to the successful design and prediction of C2H2-ZF DNA-binding specificities. Computational analyses uncovered important aspects of C2H2-ZF domain–DNA interactions, including the roles of within-finger context and domain position on base recognition. We observed the existence of numerous distinct binding strategies for each possible three base pair target and an apparent balance between affinity and specificity of binding. In sum, our comprehensive data help elucidate the complex binding landscape of C2H2-ZF domains and provide a foundation for efforts to determine, predict and engineer their DNA-binding specificities.     

Profiling the DNA-binding specificities of engineered Cys2His2 zinc finger domains using a rapid cell-based method

The C₂H₂ zinc finger is the most commonly utilized framework for engineering DNA-binding domains with novel specificities. Many different selection strategies have been developed to identify individual fingers that possess a particular DNA-binding specificity from a randomized library. In these experiments, each finger is selected in the context of a constant finger framework that ensures the identification of clones with a desired specificity by properly positioning the randomized finger on the DNA template. Following a successful selection, multiple zinc-finger clones are typically recovered that share similarities in the sequences of their DNA-recognition helices. In principle, each of the clones isolated from a selection is a candidate for assembly into a larger multi-finger protein, but to date a high-throughput method for identifying the most specific candidates for incorporation into a final multi-finger protein has not been available. Here we describe the development of a specificity profiling system that facilitates rapid and inexpensive characterization of engineered zinc-finger modules. Moreover, we demonstrate that specificity data collected using this system can be employed to rationally design zinc fingers with improved DNA-binding specificities.     

DNA-induced alpha-helix capping in conserved linker sequences is a determinant of binding affinity in Cys(2)-His(2) zinc fingers

High-affinity, sequence-specific DNA binding by Cys(2)-His(2) zinc finger proteins is mediated by both specific protein-base interactions and non-specific contacts between charged side-chains and the phosphate backbone. In addition, in DNA complexes of multiple zinc fingers, protein-protein interactions between the finger units contribute to the binding affinity. We present NMR evidence for another contribution to high- affinity binding, a highly specific DNA-induced helix capping involving residues in the linker sequence between fingers. Capping at the C terminus of the alpha-helix in each zinc finger, incorporating a consensus TGEKP linker sequence that follows each finger, provides substantial binding energy to the DNA complexes of zinc fingers 1-3 of TFIIIA (zf1-3) and the four zinc fingers of the Wilms' tumor suppressor protein (wt1-4). The same alpha-helix C-capping motif is observed in the X-ray structures of four other protein-DNA complexes. The structures of each of the TGEKP linkers in these complexes can be superimposed on the linker sequences in the zf1-3 complex, revealing a remarkable similarity in both backbone and side-chain conformations. The canonical linker structures from the zinc-finger-DNA complexes have been compared to the NMR structure of the TGEKP linker connecting fingers 1 and 2 in zf1-3 in the absence of DNA. This comparison reveals that additional stabilization likely arises in the DNA complexes from hydrogen bonding between the backbone amide of E3 and the side-chain O(gamma) of T1 in the linker. We suggest that these DNA-induced C-capping interactions provide a means whereby the multiple-finger complex, which must necessarily be domain-flexible in the unbound state as it searches for the correct DNA sequence, can be "snap-locked" in place once the correct DNA sequence is encountered. These observations provide a rationale for the high conservation of the TGEKP linker sequences in Cys(2)-His(2) zinc finger proteins.     

Towards therapeutic applications of engineered zinc finger proteins

It has long been the goal of molecular biologists to design DNA-binding proteins for the specific control of gene expression. The zinc finger design is ideally suited for such purposes, discriminating between closely related sequences both in vitro and in vivo. Whereas other DNA-binding proteins generally make use of the 2-fold symmetry of the double helix, zinc fingers do not and so can be linked linearly in tandem to recognize DNA sequences of different lengths, with high fidelity. This modular design offers a large number of combinatorial possibilities for the specific recognition of DNA. By fusing zinc finger peptides to repression or activation domains, genes can be selectively targeted and switched off and on. Several recent applications of such engineered zinc finger proteins (ZFPs) are described, including the activation of vascular endothelial growth factor (VEGF) in a human cell line and an animal model. Clinical trials have recently begun on using VEGF-activating ZFPs to treat human peripheral arterial disease, by stimulating vascular growth. Also in progress are pre-clinical studies using ZFPs to target the defective genes in two monogenic disorders, SCID and SCA. The aim is to replace them in each case by a correct copy from an extrachromosomal DNA donor by means of homologous recombination. Promising results are reported.     

Prediction of DNA-binding specificity in zinc finger proteins

Zinc finger proteins interact via their individual fingers to three base pair subsites on the target DNA. The four key residue positions -1, 2, 3 and 6 on the alpha-helix of the zinc fingers have hydrogen bond interactions with the DNA. Mutating these key residues enables generation of a plethora of combinatorial possibilities that can bind to any DNA stretch of interest. Exploiting the binding specificity and affinity of the interaction between the zinc fingers and the respective DNA can help to generate engineered zinc fingers for therapeutic purposes involving genome targeting. Exploring the structure-function relationships of the existing zinc finger-DNA complexes can aid in predicting the probable zinc fingers that could bind to any target DNA. Computational tools ease the prediction of such engineered zinc fingers by effectively utilizing information from the available experimental data. A study of literature reveals many approaches for predicting DNA-binding specificity in zinc finger proteins. However, an alternative approach that looks into the physico-chemical properties of these complexes would do away with the difficulties of designing unbiased zinc fingers with the desired affinity and specificity. We present a physico-chemical approach that exploits the relative strengths of hydrogen bonding between the target DNA and all combinatorially possible zinc fingers to select the most optimum zinc finger protein candidate.     

Selectivity of arsenite interaction with zinc finger proteins

Arsenic is a carcinogenic element also used for the treatment of acute promyelocytic leukemia. The reactivity of proteins to arsenic must be associated with the various biological functions of As. Here, we investigated the selectivity of arsenite to zinc finger proteins (ZFPs) with different zinc binding motifs (C2H2, C3H, and C4). Single ZFP domain proteins were used for the direct comparison of the reactivity of different ZFPs. The binding constants and the reaction rates have been studied quantitatively. Results show that both the binding affinity and reaction rates of single-domain ZFPs follow the trend of C4 > C3H ? C2H2. Compared with the C2H2 motif ZFPs, the binding affinities of C3H and C4 motif ZFPs are nearly two orders of magnitude higher and the reaction rates are approximately two-fold higher. The formation of multi-domain ZFPs significantly enhances the reactivity of C2H2 type ZFPs, but has negligible effects on C3H and C4 ZFPs. Consequently, the reactivities of the three types of multi-domain ZFPs are rather similar. The 2D NMR spectra indicate that the As(III)-bound ZFPs are also unfolded, suggesting that arsenic binding interferes with the function of ZFPs.