Binding studies with mutants of Zif268. Contribution of individual side chains to binding affinity and specificity in the Zif268 zinc finger-DNA complex.

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 alpha 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 alpha 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.     

Rearrangement of side-chains in a Zif268 mutant highlights the complexities of zinc finger-DNA recognition.

Structural and biochemical studies of Cys(2)His(2) zinc finger proteins initially led several groups to propose a "recognition code" involving a simple set of rules relating key amino acid residues in the zinc finger protein to bases in its DNA site. One recent study from our group, involving geometric analysis of protein-DNA interactions, has discussed limitations of this idea and has shown how the spatial relationship between the polypeptide backbone and the DNA helps to determine what contacts are possible at any given position in a protein-DNA complex. Here we report a study of a zinc finger variant that highlights yet another source of complexity inherent in protein-DNA recognition. In particular, we find that mutations can cause key side-chains to rearrange at the protein-DNA interface without fundamental changes in the spatial relationship between the polypeptide backbone and the DNA. This is clear from a simple analysis of the binding site preferences and co-crystal structures for the Asp20-->Ala point mutant of Zif268. This point mutation in finger one changes the specificity of the protein from GCG TGG GCG to GCG TGG GC(G/T), and we have solved crystal structures of the D20A mutant bound to both types of sites. The structure of the D20A mutant bound to the GCG site reveals that contacts from key residues in the recognition helix are coupled in complex ways. The structure of the complex with the GCT site also shows an important new water molecule at the protein-DNA interface. These side-chain/side-chain interactions, and resultant changes in hydration at the interface, affect binding specificity in ways that cannot be predicted either from a simple recognition code or from analysis of spatial relationships at the protein-DNA interface. Accurate computer modeling of protein-DNA interfaces remains a challenging problem and will require systematic strategies for modeling side-chain rearrangements and change in hydration.     

Signatures of Protein-DNA Recognition in Free DNA Binding Sites

One obstacle to achieving complete understanding of the principles underlying sequence-dependent recognition of DNA is the paucity of structural data for DNA recognition sequences in their free (unbound) state. Here, we carried out crystallization screening of 50 DNA duplexes containing cognate protein binding sites and obtained new crystal structures of free DNA binding sites for three distinct modes of DNA recognition: anti-parallel β strands (MetR), helix-turn-helix motif + hinge helices (PurR), and zinc fingers (Zif268). Structural changes between free and protein-bound DNA are manifested differently in each case. The new DNA structures reveal that distinctive sequence-dependent DNA geometry dominates recognition by MetR, protein-induced bending of DNA dictates recognition by PurR, and deformability of DNA along the A-B continuum is important in recognition by Zif268. Together, our findings show that crystal structures of free DNA binding sites provide new information about the nature of protein-DNA interactions and thus lend insights towards a structural code for DNA recognition.     

Determination of the base recognition positions of zinc fingers from sequence analysis.

The CC/HH zinc finger is a small independently folded DNA recognition motif found in many eukaryotic proteins, which ligates zinc through two cysteine and two histidine ligands. A database of 1340 zinc fingers from 221 proteins has been constructed and a program for analysis of aligned sequences written. This paper describes sequence analysis aimed at determining the amino acid positions that recognize the DNA bases, by comparing two types of sequence variation. Using the idea that long runs of adjacent zinc fingers have arisen from internal gene duplication, the conservation of each position of the finger within the runs was calculated. The conservation of each position of the finger between homologous proteins from different species was also noted. A correlation of the two types of conservation showed clusters of related amino acids. One cluster of three positions was found to be especially variable within long runs, but highly conserved between corresponding fingers of homologous proteins; these positions are predicted to be the base contact positions. They match the amino acid positions that contact the bases in the co-crystal structure determined by Pavletich and Pabo [Science, 240, 809-817 (1991)]. An adjacent cluster of four positions on the plot may also be associated with DNA binding. This analysis shows that the base recognition positions can be identified even in the absence of a known structure for a zinc finger. These results are applicable to zinc fingers where the structure of the complex is unknown, in particular suggesting that the individual finger--DNA interaction seen in the Zif268--DNA structure has been conserved in many zinc finger--DNA interactions.     

Combining structure-based design with phage display to create new Cys(2)His(2) zinc finger dimers

BACKGROUND: 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). RESULTS: 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. CONCLUSIONS: 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.     

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.     

Solution structures of two CCHC zinc fingers from the FOG family protein U-shaped that mediate protein-protein interactions

BACKGROUND: Zinc finger domains have traditionally been regarded as sequence-specific DNA binding motifs. However, recent evidence indicates that many zinc fingers mediate specific protein-protein interactions. For instance, several zinc fingers from FOG family proteins have been shown to interact with the N-terminal zinc finger of GATA-1. RESULTS: We have used NMR spectroscopy to determine the first structures of two FOG family zinc fingers that are involved in protein-protein interactions: fingers 1 and 9 from U-shaped. These fingers resemble classical TFIIIA-like zinc fingers, with the exception of an unusual extended portion of the polypeptide backbone prior to the fourth zinc ligand. [15N,(1)H]-HSQC titrations have been used to define the GATA binding surface of USH-F1, and comparison with other FOG family proteins indicates that the recognition mechanism is conserved across species. The surface of FOG-type fingers that interacts with GATA-1 overlaps substantially with the surface through which classical fingers typically recognize DNA. This suggests that these fingers could not contact both GATA and DNA simultaneously. In addition, results from NMR, gel filtration, and sedimentation equilibrium experiments suggest that the interactions are of moderate affinity. CONCLUSIONS: Our results demonstrate unequivocally that zinc fingers comprising the classical betabetaalpha fold are capable of mediating specific contacts between proteins. The existence of this alternative function has implications for the prediction of protein function from sequence data and for the evolution of protein function.     

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.     

Structure of a designed dimeric zinc finger protein bound to DNA

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.     

Selected peptide extension contacts hydrophobic patch on neighboring zinc finger and mediates dimerization on DNA.

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.     

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.     

Keep Your Fingers Off My DNA: Protein–Protein Interactions Mediated by C2H2 Zinc Finger Domains

Cys2-His2 (C2H2) zinc finger domains (ZFs) were originally identified as DNA-binding domains, and uncharacterized domains are typically assumed to function in DNA binding. However, a growing body of evidence suggests an important and widespread role for these domains in protein binding. There are even examples of zinc fingers that support both DNA and protein interactions, which can be found in well-known DNA-binding proteins such as Sp1, Zif268, and Ying Yang 1 (YY1). C2H2 protein–protein interactions (PPIs) are proving to be more abundant than previously appreciated, more plastic than their DNA-binding counterparts, and more variable and complex in their interactions surfaces. Here we review the current knowledge of over 100 C2H2 zinc finger-mediated PPIs, focusing on what is known about the binding surface, contributions of individual fingers to the interaction, and function. An accurate understanding of zinc finger biology will likely require greater insights into the potential protein interaction capabilities of C2H2 ZFs.     

Engineered zinc finger proteins that respond to DNA modification by HaeIII and HhaI methyltransferase enzymes

Zinc finger modules are capable of specifically interacting with DNA that contains 5-methylcytosine (5-mC) in place of cytosine, suggesting that zinc finger-DNA binding could be regulated by extrinsic methylation of DNA. Here, we have used phage display to engineer zinc finger proteins that detect and discriminate DNA methylation by the prokaryotic enzymes HaeIII and HhaI. In these systems, zinc finger-DNA complexes are induced by DNA modification using the appropriate enzyme, which can therefore act as a switch. To further develop the specificity of the switch, zinc finger discrimination between 5-mC and thymine in DNA sequences is demonstrated despite the presence of the characteristic major groove methyl group that is common to both bases. Specificity was achieved using a DNA-binding strategy involving synergy between adjacent zinc fingers. We propose that engineered zinc fingers that recognise particular DNA modifications, such as sequence-specific DNA methylation, could be integrated into artificial regulatory circuits for the control of gene expression and other biological processes.     

Looking into DNA recognition: zinc finger binding specificity

We present a quantitative, theoretical analysis of the recognition mechanisms used by two zinc finger proteins: Zif268, which selectively binds to GC-rich sequences, and a Zif268 mutant, which binds to a TATA box site. This analysis is based on a recently developed method (ADAPT), which allows binding specificity to be analyzed via the calculation of complexation energies for all possible DNA target sequences. The results obtained with the zinc finger proteins show that, although both mainly select their targets using direct, pairwise protein–DNA interactions, they also use sequence-dependent DNA deformation to enhance their selectivity. A new extension of our methodology enables us to determine the quantitative contribution of these two components and also to measure the contributions of individual residues to overall specificity. The results show that indirect recognition is particularly important in the case of the TATA box binding mutant, accounting for 30% of the total selectivity. The residue-by-residue analysis of the protein–DNA interaction energy indicates that the existence of amino acid–base contacts does not necessarily imply sequence selectivity, and that side chains without contacts can nevertheless contribute to defining the protein's target sequence.     

序列特异的三锌指多肽的构建及其在大肠杆菌中的表达

在获得单一锌指突变体的基础上,以小鼠转录因子Zif268的三锌指DNA结合区为模板,利用重叠(Over-lap)PCR技术,获得了关键氨基酸位点同时突变的三锌指突变体ZF123、2ZF123。ZF123、2ZF123分别克隆进pUC-18质粒,序列测定正确后,以pGEX-2T为表达质粒,在大肠杆菌JM109中实现了功能性的表达。经SDS-PAGE分析,表达出了分子量34.0kD的融合蛋白,扫描分析其含量在20%左右。菌体经超声波破碎后,对可溶性融合蛋白进行了纯化得到了游离的目的蛋白,为进一步的DNA结合特性分析、杂交转录因子的构建等奠定了基础。     

Quantitative parameters for amino acid-base interaction: implications for prediction of protein-DNA binding sites.

Inspection of the amino acid-base interactions in protein-DNA complexes is essential to the understanding of specific recognition of DNA target sites by regulatory proteins. The accumulation of information on protein-DNA co-crystals challenges the derivation of quantitative parameters for amino acid-base interaction based on these data. Here we use the coordinates of 53 solved protein-DNA complexes to extract all non-homologous pairs of amino acid-base that are in close contact, including hydrogen bonds and hydrophobic interactions. By comparing the frequency distribution of the different pairs to a theoretical distribution and calculating the log odds, a quantitative measure that expresses the likelihood of interaction for each pair of amino acid-base could be extracted. A score that reflects the compatibility between a protein and its DNA target can be calculated by summing up the individual measures of the pairs of amino acid-base involved in the complex, assuming additivity in their contributions to binding. This score enables ranking of different DNA binding sites given a protein binding site and vice versa and can be used in molecular design protocols. We demonstrate its validity by comparing the predictions using this score with experimental binding results of sequence variants of zif268 zinc fingers and their DNA binding sites.