The beta-beta-alpha fold: explorations in sequence space

The computational redesign of the second zinc finger of Zif268 to produce a 28 residue peptide (FSD-1) that assumes a betabetaalpha fold without metal binding was recently reported. In order to explore the tolerance of this metal-free fold towards sequence variability, six additional peptides resulting from the ORBIT computational protein design process were synthesized and characterized. The experimental stabilities of five of these peptides are strongly correlated with the energies calculated by ORBIT. However, when a peptide with a mutation in the beta-turn is examined, the calculated stability does not accurately predict the experimentally determined stability. The NMR solution structure of a peptide incorporating this mutation (FSD-EY) reveals that the register between the beta-strands is different from the model structure used to select and score the sequences. FSD-EY has a type I' turn instead of the target EbaaagbE turn (rubredoxin knuckle). Two additional peptides that have improved side-chain to backbone hydrogen bonding and turn propensity for the target turn were characterized. Both are of stability comparable to that of FSD-1. These results demonstrate the robustness of the ORBIT protein design methods and underscore the need for continued improvements in negative design.     

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.     

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。     

Importance of alpha-helix N-capping motif in stabilization of betabetaalpha fold

FSD-1 (full sequence design 1) is a protein folded in a betabetaalpha motif, designed on the basis of the second zinc finger domain of Zif268 by a substitution of its metal coordination site with a hydrophobic core. In this work, we analyzed the possibility of introducing the DNA recognition motif of the template zinc finger (S(13)RSDH(17)) into FSD-1 sequence in order to obtain a small DNA-binding module devoid of cross-link(s) or metal cofactors. The hybrid protein was unfolded, as judged by CD and NMR criteria. To reveal the role of each of the five amino acids, which form the N-capping motif of the alpha-helix, we analyzed conformational and stability properties of eight FSD-1 mutants. We used a shielded methyl group of Leu 18 and a CD signal at 215 nm as a convenient measure of the folded state. Glu 17-->His substitution at the N(3) in S(13)NEKE(17) variant decreased the folded structure content from 90% to 25% (equivalent to 1.8 kcal * mole(-1) destabilization) by disruption of N-capping interactions, and had the most significant effect among single mutants studied here. The N(cap) Asn 14 substitution with Arg considerably decreased stability, reducing structure content from 90% to 40% (1.4 kcal * mole(-1) destabilization) by disruption of a helix-capping hydrogen bond and destabilization of a helix macrodipole. The N(1) Glu 15-->Ser mutation also produced a considerable effect (1.0 kcal * mole(-1) destabilization), again emphasizing the significance of electrostatic interactions in alpha-helix stabilization.     

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.     

An arginine residue instead of a conserved leucine residue in the recognition helix of the finger 3 of Zif268 stabilizes the domain structure and mediates DNA binding

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.     

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.     

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.     

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.     

TAT蛋白质转导结构域与SV40启动子特异的三锌指结构融合蛋白的表达与纯化

利用蛋白质转导结构域(PTDs)可以将与之融合表达的蛋白质直接送入细胞中。将通过筛选噬菌体展示锌指库得到的特异作用于SV40启动子上9bp序列的三锌指结构的序列插入含有TAT蛋白的蛋白质转导结构域的表达载体pET—TAT-NLS中,构建融合蛋白的表达载体pET-TAT-NLS—clone3。融合蛋白在E．coli BL21(DE3)中得到了可溶性表达,含量约占总蛋白的18％;并通过镍亲和凝胶层析柱得到了较好的纯化融合蛋白。     

Zif268的锌指DNA结合区在大肠杆菌中的功能性表达

锌指蛋白是最大的蛋白家族,是识别核酸最常见的、最有效的结构元件。通过选择合适的表达载体及诱导表达条件,实现了小鼠转录因子Zif268的锌指DNA结合区在大肠杆菌中的部分可溶性表达。凝胶迁移率移动试验证实纯化的可溶部分锌指DNA结合区可以特异性识别、结合其天然靶序列,提示锌指DNA结合区在大肠杆菌中得到了功能性表达。锌指DNA结合区在大肠杆菌中的功能性表达成功为锌指蛋白DNA相互作用的胞内遗传筛选模型的建立奠定了基础。     

一种特异识别SV40启动子的人工转录因子的构建

转录因子是真核表达调控中非常重要的一类反式作用因子,通常由DNA结合域与效应域两部分组成,而锌指结构是DNA结合域的常见组成单元。人工转录因子就是基于转录因子的这种结构特点,人为地选择针对特定序列的DNA结合域与具有特定作用的效应域组合而成。利用噬菌体展示技术,筛选到与SV40启动子上9bp序列特异结合的锌指结构,再连接KOX1的KRAB域构建了一种人工转录因子。转染实验表明它对SV40下游的报告基因的表达有很显著的抑制作用。     

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.     

Understanding the folding and stability of a zinc finger-based full sequence design protein with replica exchange molecular dynamics simulations

Full sequence design protein FSD-1 is a designed protein based on the motif of zinc finger protein. In this work, its folding mechanism and thermal stability are investigated using the replica exchange molecular dynamics model with the water molecules being treated explicitly. The results show that the folding of the FSD-1 is initiated by the hydrophobic collapse, which is accompanied with the formation of the C-terminal alpha-helix. Then the folding proceeds with the formation of the beta-hairpin and the further package of the hydrophobic core. Compared with the beta-hairpin, the alpha-helix has much higher stability. It is also found that the N-capping motif adopted by the FSD-1 contributes to the stability of the alpha-helix dramatically. The hydrophobic contacts made by the side chain of Tyr3 in the native state are essential for the stabilization of the beta-hairpin. It is also found that the folding of the N-terminal beta-hairpin and the C-terminal alpha-helix exhibits weak cooperativity, which is consistent with the experimental data. Meanwhile, the folding pathway is compared between the FSD-1 and the target zinc finger peptide, and the possible role of the zinc ion on the folding pathway of zinc finger is proposed. Proteins 2007. (c) 2007 Wiley-Liss, Inc.     

CCHX zinc finger derivatives retain the ability to bind Zn(II) and mediate protein-DNA interactions

Classical (CCHH) zinc fingers are among the most common protein domains found in eukaryotes. They function as molecular recognition elements that mediate specific contact with DNA, RNA, or other proteins and are composed of a betabetaalpha fold surrounding a single zinc ion that is ligated by two cysteine and two histidine residues. In a number of variant zinc fingers, the final histidine is not conserved, and in other unrelated zinc binding domains, residues such as aspartate can function as zinc ligands. To test whether the final histidine is required for normal folding and the DNA-binding function of classical zinc fingers, we focused on finger 3 of basic Krüppel-like factor. The structure of this domain was determined using NMR spectroscopy and found to constitute a typical classical zinc finger. We generated a panel of substitution mutants at the final histidine in this finger and found that several of the mutants retained some ability to fold in the presence of zinc. Consistent with this result, we showed that mutation of the final histidine had only a modest effect on DNA binding in the context of the full three-finger DNA-binding domain of basic Krüppel-like factor. Further, the zinc binding ability of one of the point mutants was tested and found to be indistinguishable from the wild-type domain. These results suggest that the final zinc chelating histidine is not an essential feature of classical zinc fingers and have implications for zinc finger evolution, regulation, and the design of experiments testing the functional roles of these domains.     

Analysis of PDZ domain-ligand interactions using carboxyl-terminal phage display

PDZ domains mediate protein-protein interactions at specialized subcellular sites, such as epithelial cell tight junctions and neuronal post-synaptic densities. Because most PDZ domains bind extreme carboxyl-terminal sequences, the phage display method has not been amenable to the study of PDZ domain binding specificities. For the first time, we demonstrate the functional display of a peptide library fused to the carboxyl terminus of the M13 major coat protein. We used this library to analyze carboxyl-terminal peptide recognition by two PDZ domains. For each PDZ domain, the library provided specific ligands with sub-micromolar binding affinities. Synthetic peptides and homology modeling were used to dissect and rationalize the binding interactions. Our results establish carboxyl-terminal phage display as a powerful new method for mapping PDZ domain binding specificity.