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Selected peptide extension contacts hydrophobic patch on neighboring zinc finger and mediates dimerization on DNA
Abstract
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.
... Zinc fingers were first identified as conserved motifs present in the DNA binding protein TFIIIA. A C 2 H 2 zinc finger is also called the TFIIIA or Kruppel-like finger and consists of 20-30 amino acids with a secondary structure formed by tetrahedral binding of Zn 2+ to two cysteine and two histidine residues (Klug and Schwabe, 1995;Pabo et al., 2001;Laity et al., 2001). It consists of two β strands and an α helix and is generally described as X 2 CX 2-4 CX 12 HX 2-8 H where "X" is any amino acid and the numbers represent the spacing among the conserved residues (Fig 1.9). ...
... The ZXDC and ZXDA proteins are zinc finger transcription factors each containing ten C 2 H 2 type zinc fingers (ZnFs). The C 2 H 2 type zinc fingers are generally involved in DNA binding, RNA binding, selective protein-protein interactions or a combination of more than one macromolecular interaction Hata et al., 2000;Wang et al., 2001;Cassiday et al., 2002). ...
... We observed the presence of ZXDC at the MHC II promoter prior to and after IFN γ treatment . Given the important role of C 2 H 2 type zinc fingers in macromolecular interactions, they are generally involved in DNA binding, RNA binding or selective protein-protein interactions that play a significant role in the regulation of target genes Hata et al., 2000;Wang et al., 2001). Dr. Srikarthika Jambunathan in our lab identified that ZXDC preferentially binds to a purine rich sequence, AGGGT/A by SELEX assay. ...
... Parmi les protéines à doigts de zinc décrites dans la littérature, une protéine de 72 acides aminés ZIF12, basée sur la protéine Zif268, décrite par B. Wang et C. Pabo [45], [46] Figure 24). Les deux doigts de zinc correspondent à la protéine Zif268, un facteur de transcription connu pour cibler les séquences 5'-TGG-3' et 5'-GCG-3' [37] . ...
... Grâce à cette extension N-terminale, la protéine possède une affinité élevée en présence de la séquence d'ADN cible, avec une constante de dissociation de 15nM déterminée par électrophorèse (retard sur gel), contre 15µM pour la même protéine sans l'extension [45] . La structure cristallographique de la structure du complexe ADN-(ZIF12)2 obtenue par B. Wang et C. Pabo [46] montre deux caractéristiques intéressantes. Premièrement, l'interface de dimérisation entre l'extension N-terminale et le doigt de zinc de l'autre monomère implique l'hélice α du doigt de zinc, mais pas les acides aminés interagissant avec l'ADN ( Figure 26). ...
L’ADN est le support de l’information de tout être vivant. La possibilité de pouvoir cibler et visualiser in vivo une séquence spécifique d’ADN et plus particulièrement un gène est un enjeu de taille pour le suivi médical aussi bien que pour la compréhension du vivant. Pour y parvenir, la détection par luminescence est particulièrement attrayante de par sa facilité de visualisation avec des outils simples.L’objet de cette thèse était d’apporter la preuve de concept de sondes luminescentes pour la détection séquence spécifique d’ADN double brin, basées d’une part sur les propriétés de luminescence des lanthanides, particulièrement intéressantes pour la détection en milieu biologique, et d’autre part sur les propriétés de reconnaissance de l’ADN par les protéines à doigts de zinc. Nous nous sommes intéressé au ciblage d’un duplex d’ADN palindromique de 12 paires de bases par un couple de protéines à doigts de zinc intégrant un système FRET, où un complexe de lanthanide(III) sur une protéine joue le rôle de donneur et un fluorophore organique sur l’autre protéine joue celui d’accepteur.Pour cela, une nouvelle famille de complexes de lanthanide(III) bioconjugables a été élaborée et des protéines à doigts de zinc fonctionnalisée par différents chromophores ont été synthétisés chimiquement par synthèse peptidique supportée sur résine et assemblage par ligation chimique native de trois fragments. Les caractérisations spectroscopiques des systèmes développés ont permis de mettre en évidence l’interaction des sondes avec la séquence d’ADN palindromique et de valider la preuve de concept d’une détection de cette séquence par un FRET basé sur des lanthanides.
... Incorporation of an ID allows for ATF dimerization upon binding to 18 base pair sequences as homo-and heterodimers [55]. An ID can be a small molecule, a short peptide, a domain or even a full protein. ...
... This is consistent with previous findings on the binding specificity of cooperatively assembling DNA-binding molecules [115]. Based on affinity measurements of the cooperative binding of two ZF pairs, it would be reasonable to expect that an ATF bearing two ZFs and an ID would suffice to target 12-bp binding sites [55]. ...
Transcription factors (TFs) reprogram cell states by exerting control over gene regulatory networks and the epigenetic landscape of a cell. Artificial transcription factors (ATFs) are designer regulatory proteins comprised of modular units that can be customized to overcome challenges faced by natural TFs in establishing and maintaining desired cell states. Decades of research on DNA-binding proteins and synthetic molecules has provided a molecular toolkit for ATF design and the construction of genome-scale libraries of ATFs capable of phenotypic manipulation and reprogramming of cell states. Here, we compare the unique strengths and limitations of different ATF platforms, highlight the advantages of cooperative assembly, and present the potential of ATF libraries in revealing gene regulatory networks that govern cell fate choices. This article is protected by copyright. All rights reserved.
... This reduces the number of modules that need to be introduced and consequently eases the metabolic burden of the cells. Another novelty of this study was the use of three finger arrays instead of the previously used two-finger arrays (Wang et al., 2001;Wolfe et al., 2000). This permits the recognition of 18 bp long DNA elements. ...
... (Khalil et al., 2012) Require protein engineering and laborious selection and DNA hybrid assembly (Khalil et al., 2012;Reyon et al., 2012;Sanjana et al., 2012) Recognition of long sequences confers orthogonality. (Wang et al., 2001;Wolfe et al., 2000) TALE based sTFs ...
The modular nature of the transcriptional unit makes it possible to design robust modules with predictable input-output characteristics using a ‘parts- off a shelf’ approach. Customized regulatory circuits composed of multiple such transcriptional units have immense scope for application in diverse fields of basic and applied research. Synthetic transcriptional engineering seeks to construct such genetic cascades. Here, we discuss the three principle strands of transcriptional engineering: promoter and transcriptional factor engineering, and programming inducibilty into synthetic modules. In this context, we review the scope and limitations of some recent technologies that seek to achieve these ends. Our discussion emphasizes a requirement for rational combinatorial engineering principles and the promise this approach holds for the future development of this field.
... The importance of these tryptophan residues is further supported by their conservation among more than 40 Zic proteins identified in a wide range of eumetazoan species [15]. Previous studies have revealed that human GLI1 (PDBID: 2GLI) and yeast Zap1 (PDBID: 1ZW8) also possess ZFs with two tryptophans in the corresponding region and that these tryptophans are located in the hydrophobic core formed by two adjacent ZFs [16]. These data raise the possibility that domains containing the consensus sequence " Cys-X-Trp-Xn-Cys-Xn-His- Xn-His-Xn-Cys-X-Trp-Xn-Cys-Xn-His-Xn-His " , which we have named the tandem CWCH2 (tCWCH2) motif, are involved in the interaction between the two adjacent ZFs. ...
... ZFs utilizing in vitro-evolving protein-to-protein interac- tions [16], it was found that the hydrophobic residues in zif268 that interacted between the artificial proteins and the zif268 C2H2 ZF were similar to those involved in the GLI inter-ZF interaction. Superimposition revealed that the position corresponding to the tryptophan in tCWCH2 is occupied by a valine, suggesting that either intramolecular or intermolecular protein-toprotein interactions, mediated by hydrophobic residues, are generally utilized for C2H2 ZFs. ...
The C2H2 zinc finger (ZF) domain is widely conserved among eukaryotic proteins. In Zic/Gli/Zap1 C2H2 ZF proteins, the two N-terminal ZFs form a single structural unit by sharing a hydrophobic core. This structural unit defines a new motif comprised of two tryptophan side chains at the center of the hydrophobic core. Because each tryptophan residue is located between the two cysteine residues of the C2H2 motif, we have named this structure the tandem CWCH2 (tCWCH2) motif.
Here, we characterized 587 tCWCH2-containing genes using data derived from public databases. We categorized genes into 11 classes including Zic/Gli/Glis, Arid2/Rsc9, PacC, Mizf, Aebp2, Zap1/ZafA, Fungl, Zfp106, Twincl, Clr1, and Fungl-4ZF, based on sequence similarity, domain organization, and functional similarities. tCWCH2 motifs are mostly found in organisms belonging to the Opisthokonta (metazoa, fungi, and choanoflagellates) and Amoebozoa (amoeba, Dictyostelium discoideum). By comparison, the C2H2 ZF motif is distributed widely among the eukaryotes. The structure and organization of the tCWCH2 motif, its phylogenetic distribution, and molecular phylogenetic analysis suggest that prototypical tCWCH2 genes existed in the Opisthokonta ancestor. Within-group or between-group comparisons of the tCWCH2 amino acid sequence identified three additional sequence features (site-specific amino acid frequencies, longer linker sequence between two C2H2 ZFs, and frequent extra-sequences within C2H2 ZF motifs).
These features suggest that the tCWCH2 motif is a specialized motif involved in inter-zinc finger interactions.
... In these studies, complexes formed by maC2H2 type ZnFs (with several consecutive ZnFs) were interpreted as "higher order structures" or dimers using yeast two-hybrid systems, co-transfection of isoforms and gel shift assays, but no exact stoichiometry was established (Sun et al, 1996;Tsai & Reed, 1998). Multimerization is usually mediated via ZnFs not participating in DNA recognition using two different modes: hydrophobic interactions through the ZnF surface (Wang et al, 2001), as it was shown for proteins like GL1 (Pavletich & Pabo, 1993) and SW15 (Dutnall et al, 1996), or ZnF-ZnF interactions mediated by the same amino acids conferring the DNA sequence specificity (Wolfe et al, 2000;Mccarty et al, 2003). It has been argued that this multimerization serves to increase the binding affinity and efficiency to target DNA sequences (reviewed in Iuchi (2001)). ...
PRDM9 is a trans-acting factor directing meiotic recombination to specific DNA-binding sites by its zinc finger (ZnF) array. It was suggested that PRDM9 is a multimer; however, we do not know the stoichiometry or the components inducing PRDM9 multimerization. In this work, we used in vitro binding studies and characterized with electrophoretic mobility shift assays, mass spectrometry, and fluorescence correlation spectroscopy the stoichiometry of the PRDM9 multimer of two different murine PRDM9 alleles carrying different tags and domains produced with different expression systems. Based on the migration distance of the PRDM9–DNA complex, we show that PRDM9 forms a trimer. Moreover, this stoichiometry is adapted already by the free, soluble protein with little exchange between protein monomers. The variable ZnF array of PRDM9 is sufficient for multimerization, and at least five ZnFs form already a functional trimer. Finally, we also show that only one ZnF array within the PRDM9 oligomer binds to the DNA, whereas the remaining two ZnF arrays likely maintain the trimer by ZnF–ZnF interactions.
... We fused VP64, a tetrameric repeat of the 11-aa activation region of VP16, a potent transactivation domain from the herpes simplex virus to the C terminus to the zinc fingers ( Fig. 1A) (20). Although a variety of zinc finger-based libraries have been described before (21)(22)(23)(24)(25)(26)(27), a distinguishing and important feature of our ATF design is inclusion of a 15-aa peptide that serves as an ID, allowing dimerization of the ATF with another ATF through the hydrophobic surface of the first zinc finger of EGR1 (28). The inclusion of the ID in our ATF design adds a layer of control to the ATF library by allowing the ATFs to harness cooperative binding and synergistic activation (16). ...
Significance
The ability to convert cells into desired cell types enables tissue engineering, disease modeling, and regenerative medicine; however, methods to generate desired cell types remain difficult, uncertain, and laborious. We developed a strategy to screen gene regulatory elements on a genome scale to discover paths that trigger cell fate changes. The proteins used in this study cooperatively bind DNA and activate genes in a synergistic manner. Subsequent identification of transcriptional networks does not depend on prior knowledge of specific regulators important in the biological system being tested. This powerful forward genetic approach enables direct cell state conversions as well as other challenging manipulations of cell fate.
... phage display [133,134], coiled-coiled interactions characterized by protein microarrays [135], as well as small molecules [136], can be attached to ATFs to recruit other natural factors or ATFs. Even short rationally designed dipeptides, such as the 'WM' motif used to recruit the Exd (extradenticle) homeodomain, function as IDs ( Figures 2B and 2C) [99,114,137]. ...
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.
... Although the majority of classical zinc fingers are known to bind nucleic acids, a number of these proteins containing these motifs have been identified to function by mediating protein-protein interactions using their zinc fingers. For instance, Roaz, GL1, SW15, Ikaros, TRPS-1 and Zac form homodimers using their classical zinc fingers (Mackay and Crossley, 1998;McCarty et al., 2003;Sun et al., 1996;Tsai and Reed, 1998;Wang et al., 2001). GL1 forms a homodimer through the hydrophobic surface of its first zinc finger that is not involved in DNA-binding. ...
... Previous reports have described various frameworks for creating dimeric ZF proteins. In all of these studies, elements derived from naturally occurring TFs (Pomerantz et al., 1998;Wolfe et al., 2000) or ones selected from combinatorial peptide libraries (Wang et al., 2001;Wang and Pabo, 1999) were used to dimerize two-finger units. A disadvantage of this strategy is that a dimerized two-finger complex would have a maximum specificity of 12 bps (assuming that each of the four fingers in the dimer specifies 3 bps). ...
Eukaryotic transcription factors (TFs) perform complex and combinatorial functions within transcriptional networks. Here, we present a synthetic framework for systematically constructing eukaryotic transcription functions using artificial zinc fingers, modular DNA-binding domains found within many eukaryotic TFs. Utilizing this platform, we construct a library of orthogonal synthetic transcription factors (sTFs) and use these to wire synthetic transcriptional circuits in yeast. We engineer complex functions, such as tunable output strength and transcriptional cooperativity, by rationally adjusting a decomposed set of key component properties, e.g., DNA specificity, affinity, promoter design, protein-protein interactions. We show that subtle perturbations to these properties can transform an individual sTF between distinct roles (activator, cooperative factor, inhibitory factor) within a transcriptional complex, thus drastically altering the signal processing behavior of multi-input systems. This platform provides new genetic components for synthetic biology and enables bottom-up approaches to understanding the design principles of eukaryotic transcriptional complexes and networks.
... Several structural studies suggested that zinc finger domains in the multi-finger protein could interact with bases outside of their triplet subsites [14, 28]. This " target site overlap " occurs when certain amino acid residues at position 2 in the recognition helix interact with the first base of the neighboring DNA subsite. ...
Expression of the genome is primarily regulated at the level of transcription by gene-specific transcription factors, which recognize specific DNA sequences to activate or inhibit transcription. The ability to control gene expression at will would provide scientists with a powerful tool for biotechnology and drug-discovery research. Over the last decade or so, researchers have made great strides in our understanding of the structures and mechanisms of action of naturally occurring transcription factors. Such research has revealed that members of the Cys2-His2 zinc finger family of transcription factors consist of functional modules that recognize a wide variety of DNA sequences. This review describes recent advances in the development of novel methods to design and construct artificial transcription factors to control gene expression at will. The applications of artificial transcription factors in the areas of medicine and biotechnology are discussed.
... The structure of this dimer revealed that the 15 amino acid peptide mediates dimerisation by interacting with a hydrophobic patch on the opposite ZFP (Figure 4). 67 It remains The type of linker used determines the binding mode of the peptide, with short canonical peptides such as -TGEKP-directing binding to contiguous sequences, 58,59 longer flexible linkers allowing binding across a range of non-bound DNA stretches 60,61 and structured linkers, which show a preference for a particular span of non-bound DNA. 61 (B) Connecting two-finger domains to create six-finger peptides which bind 18 bp of DNA. ...
Within the last 20 years, the understanding of the biology of the ‘classical’ or Cys2His2 zinc finger domain has progressed rapidly from the initial identification of the zinc finger as a repetitive zinc-binding
motif in transcription factors to its use in biotechnology. The domain is the most abundant DNA-binding motif in the human
genome and is a component of many key eukaryotic transcription factors involved in growth and development. Numerous structures
now exist for this domain and its mode of action is known in a variety of zinc finger–DNA complexes. Application of this knowledge
has led to the development of ‘designer’ transcription factors where zinc fingers have been engineered to bind desired DNA
sequences. Recently, advances have been made in this field that potentially allow the targeting of any DNA site. Consideration
of chromatin structure and the use of effector domains in these ‘designer’ transcription factors have made possible the regulation
of a number of endogenous genes. These advances in the customised regulation of genes will be discussed in detail, as well
as the potential to use these proteins in functional genomics and gene therapy applications.
Transcriptional regulators select their targets from a large pool of similar genomic sites. The binding of the Drosophila dosage compensation complex (DCC) exclusively to the male X chromosome provides insight into binding site selectivity rules. Previous studies showed that the male-specific organizer of the complex, MSL2, and ubiquitous DNA-binding protein CLAMP directly interact and play an important role in the specificity of X chromosome binding. Here, we studied the highly specific interaction between the intrinsically disordered region of MSL2 and the N-terminal zinc-finger C2H2-type (C2H2) domain of CLAMP. We obtained the NMR structure of the CLAMP N-terminal C2H2 zinc finger, which has a classic C2H2 zinc-finger fold with a rather unusual distribution of residues typically used in DNA recognition. Substitutions of residues in this C2H2 domain had the same effect on the viability of males and females, suggesting that it plays a general role in CLAMP activity. The N-terminal C2H2 domain of CLAMP is highly conserved in insects. However, the MSL2 region involved in the interaction is conserved only within the Drosophila genus, suggesting that this interaction emerged during the evolution of a mechanism for the specific recruitment of the DCC on the male X chromosome in Drosophilidae.
Transcriptional regulators select their targets from a large pool of similar genomic sites. The binding of the Drosophila dosage compensation complex (DCC) exclusively to the male X chromosome provides insight into binding site selectivity rules. Previous studies showed that the male-specific organizer of the complex, MSL2, and ubiquitous DNA-binding protein CLAMP directly interact and play an important role in the specificity of X chromosome binding. Here we studied the highly specific interaction between the intrinsically disordered region of MSL2 and the N-terminal zinc-finger C2H2-type (C2H2) domain of CLAMP. We obtained the NMR structure of the CLAMP N-terminal C2H2 zinc finger, which has a classic C2H2 zinc-finger fold with a rather unusual distribution of residues typically used in DNA recognition. Substitutions of residues in this C2H2 domain had the same effect on the viability of males and females, suggesting that it plays a general role in CLAMP activity. The N-terminal C2H2 domain of CLAMP is highly conserved in insects. However, the MSL2 region involved in the interaction is conserved only within the Drosophila genus, suggesting that this interaction emerged during the evolution of a mechanism for the specific recruitment of the DCC on the male X chromosome in Drosophilidae.
PRDM9 has been identified as a meiosis-specific protein that plays a key role in determining the location of meiotic recombination hotspots. Although it is well-established that PRDM9 is a trans-acting factor directing the double strand break machinery necessary for recombination to its DNA binding site, the details of PRDM9 binding and complex formation are not well known. It has been suggested in several instances that PRDM9 acts as a multimer in vivo; however, there is little understanding about the protein stoichiometry or the components inducing PRDM9 multimerization. In this work, we used in vitro binding studies and mass spectrometry to characterize the size of the PRDM9 multimer within the active DNA-protein complex of two different murine PRDM9 alleles, PRDM9Cst and PRDM9Dom2. For this purpose, we developed a strategy to infer the molecular weight of the PRDM9-DNA complex from native gel electrophoresis based on gel shift assays (EMSAs). Our results show that PRDM9 binds as a trimer with the DNA. This multimerization is catalysed by the long ZnF array (ZnF) at the C-terminus of the protein and 11, 10, 7 or 5 ZnFs are already sufficient to form a functional trimer. Finally, we also show that only one ZnF-array within the PRDM9 trimer actively binds to the DNA, while the remaining two ZnF-arrays likely maintain the multimer by ZnF-ZnF interactions. Our results have important implications in terms of PRDM9 dosage, which determines the number of active hotspots in meiotic cells, and contribute to elucidate the molecular interactions of PRDM9 with other components of the meiotic initiation machinery.
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.
Transcription factor; Cys 2 His 2 (C2H2) zinc finger motifs are a class of transcription regulatory proteins that control a wide range of cellular processes such as differentiation, development, and tumor suppression. They are one of the major structural motifs involved in eukaryotic nucleic acid binding. 1–7 Functional specificity of individual C2H2 zinc finger–containing transcription factors is achieved through both the transactivation and C2H2 DNA binding domains. Each individual finger contains 22 to 30 amino acids (AAs) and recognizes a specific DNA sequence spanning 3 to 5 bp. 7 Tandem repeats of the C2H2 motif connected by highly conserved peptide linker sequences are found in most transcription factors of this class. Repetition of the zinc finger domain is often accompanied by altered DNA-site preferences at different fingers. Thus, transcriptional control of individual genes within a large gene pool is achieved through sequence-specific DNA recognition and high-affinity binding by a number of small repetitive C2H2 domains. Functions of the C2H2 zinc finger motif extend beyond DNA binding to protein–RNA and protein–protein interactions. The number of repeated C2H2 motifs found in a single protein ranges from 1 up to 37. Each finger contains one Zn(II) atom coordinated by two cysteines and two histidines at the N-terminal and C-terminal regions of the protein respectively. The general sequence is of the form X 2-Cys-X 2−4-Cys-X 9−12-His-X 2−6-His-L, where X is any amino acid, and L is the peptide linker connecting adjacent fingers. 3D Structure Schematic representation of the solution structure of the C2H2 zinc finger from Xfin (PDB code 1ZNF; model 1).
A great number of C2H2 zinc finger proteins selectively bind to specific DNA sequences and play a critical role in controlling transcription of
genes. The specific binding is achieved by zinc finger domains with ββα structure that is formed by tetrahedral binding of
Zn2+ ion to the canonical cysteine and histidine residues. Two to three tandem zinc fingers are necessary and sufficient for the
specific binding without participation of any other domains. Zinc fingers bind in the major groove of the DNA, wrapping around
the strands, with specificity conferred by side chains of several amino acid on the α helices. Some zinc finger proteins undergo
homodimerization by hydrophobic interactions or by finger-finger binding and reinforce the specific binding to DNA. Conserved
linkers between tandem fingers are necessary for stabilizing the DNA complex. Regulatory mechanisms of zinc finger binding
to DNA are emerging. Some cellular factors are found to acetylate and phosphorylate zinc fingers and the linkers of a few
proteins. These modifications alter the binding activity of the zinc finger proteins and hence control expression of their
target genes. Other factors can methylate promoter regions of genes. This modification alters affinity of zinc finger proteins
for the DNA segments and hence controls expression of their target genes.
The possibility of using designed transcription factors to control gene expression is highly appealing in view of a wide range
of promising applications in research and biomedicine. In the last decade, the efforts of several research groups have clarified
the molecular interactions between zinc finger proteins and DNA, generating a “recognition code” that relates amino acids
in specific positions of the finger domain to its DNA target sequence. This chapter describes the methods to design artificial
zinc finger transcription factors using the “code” and recent novel selection approaches. Efficacy and possible applications
of artificial transcription factors are discussed.
Artificial transcription factors based on modified zinc-finger DNA-binding domains have been shown to activate or repress the transcription of endogenous genes in multiple organisms. Advances in both the construction of novel zinc-finger proteins and our understanding of the characteristics of a productive regulatory site have fueled these achievements.
Electron holes are known to migrate along the DNA or RNA duplexes and to localize preferentially on successive guanines. The stationary point conformations of Gua pairs that can trap or let pass these holes have been characterized by quantum chemistry calculations. Here we show their recurrent occurrence in DNA and RNA X-ray structures, often in quadruplex conformations or in interaction with proteins, ligands or metal ions. These findings give support to the biological, possibly regulatory, roles of charge migration in cell functioning.
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.
Regulation of endosomal trafficking by Rab GTPases depends on selective interactions with multivalent effectors, including EEA1 and Rabenosyn-5, which facilitate endosome tethering, sorting, and fusion. Both EEA1 and Rabenosyn-5 contain a distinctive N-terminal C(2)H(2) zinc finger that binds Rab5. How these C(2)H(2) zinc fingers recognize Rab GTPases remains unknown. Here, we report the crystal structure of Rab5A in complex with the EEA1 C(2)H(2) zinc finger. The binding interface involves all elements of the zinc finger as well as a short N-terminal extension but is restricted to the switch and interswitch regions of Rab5. High selectivity for Rab5 and, to a lesser extent Rab22, is observed in quantitative profiles of binding to Rab family GTPases. Although critical determinants are identified in both switch regions, Rab4-to-Rab5 conversion-of-specificity mutants reveal an essential requirement for additional substitutions in the proximal protein core that are predicted to indirectly influence recognition through affects on the structure and conformational stability of the switch regions.
Eukaryotic transcription factors are composed of interchangeable modules. This has led to the design of a wide variety of modular artificial transcription factors (ATFs) that can stimulate or inhibit the expression of targeted genes. The ability to regulate the expression of any targeted gene using a 'programmable' ATF offers a powerful tool for functional genomics and bears tremendous promise in developing the field of transcription-based therapeutics.
Zinc-finger proteins offer a versatile and effective framework for the recognition of DNA binding sites. By connecting multiple fingers together with canonical TGEKP linkers, a protein may be designed to recognize almost any desired target DNA sequence. However, proteins containing more than three zinc-fingers do not bind as tightly as one might predict, and it appears that some type of strain is introduced when a six-finger protein is constructed with canonical linkers. In an attempt to understand the sources of this strain, we have solved the 2.2A resolution X-ray crystallographic structure of a complex that has two copies of the three-finger Zif268 protein bound to adjacent sites on one duplex DNA. Conceptually, this is equivalent to a six-finger protein in which the central linker has been removed and the complex has been allowed to "relax" to its most stable conformation. As in other Zif268-DNA complexes, the DNA is approximately linear and is slightly underwound. Surprisingly, the structure of the complex is similar (within 0.5A) to an arrangement that would allow a canonical linker at the center of the complex, and it seems possible that entropic effects (involving the librational degrees of freedom in the complex) could be important in determining optimal linker length.
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.
The classical Zn finger contains a phenylalanine at the crux of its three architectural elements: a beta-hairpin, an alpha-helix, and a Zn(2+)-binding site. Surprisingly, phenylalanine is not required for high-affinity Zn2+ binding, but instead contributes to the specification of a precise DNA-binding surface. Substitution of phenylalanine by leucine leads to a floppy but native-like structure whose Zn affinity is maintained by marked entropy-enthalpy compensation (DeltaDeltaH -8.3 kcal/mol and -TDeltaDeltaS 7.7 kcal/mol). Phenylalanine and leucine differ in shape, size, and aromaticity. To distinguish which features correlate with dynamic stability, we have investigated a nonstandard finger containing cyclohexanylalanine at this site. The structure of the nonstandard finger is similar to that of the native domain. The cyclohexanyl ring assumes a chair conformation, and conformational fluctuations characteristic of the leucine variant are damped. Although the nonstandard finger exhibits a lower affinity for Zn2+ than does the native domain (DeltaDeltaG -1.2 kcal/mol), leucine-associated perturbations in enthalpy and entropy are almost completely attenuated (DeltaDeltaH -0.7 kcal/mol and -TDeltaDeltaS -0.5 kcal/mol). Strikingly, global changes in entropy (as inferred from calorimetry) are in each case opposite in sign from changes in configurational entropy (as inferred from NMR). This seeming paradox suggests that enthalpy-entropy compensation is dominated by solvent reorganization rather than nominal molecular properties. Together, these results demonstrate that dynamic and thermodynamic perturbations correlate with formation or repair of a solvated packing defect rather than type of physical interaction (aromatic or aliphatic) within the core.
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.
Zac encodes a zinc finger protein that promotes apoptosis and cell cycle arrest and is maternally imprinted. Here, we show that Zac contains transactivation and repressor activities and that these transcriptional activities are differentially controlled by DNA binding. Zac transactivation mapped to two distinct domains. One of these contained multiple repeats of the peptide PLE, which behaved as an autonomous activation unit. More importantly, we identified two related high-affinity DNA-binding sites which were differentially bound by seven Zac C(2)H(2) zinc fingers. Zac bound as a monomer through zinc fingers 6 and 7 to the palindromic DNA element to confer transactivation. In contrast, binding as a monomer to one half-site of the repeat element turned Zac into a repressor. Conversely, Zac dimerization at properly spaced direct and reverse repeat elements enabled transactivation, which strictly correlated with DNA-dependent and -independent contacts of key residues within the recognition helix of zinc finger 7. The later ones support specific functional connections between Zac DNA binding and transcriptional-regulatory surfaces. Both classes of DNA elements were identified in a new Zac target gene and confirmed that the zinc fingers communicate with the transactivation function. Together, our data demonstrate a role for Zac as a transcription factor in addition to its role as coactivator for nuclear receptors and p53.
Hox homeodomain proteins are developmental regulators that determine body plan in a variety of organisms. A majority of the vertebrate Hox proteins bind DNA as heterodimers with the Pbx1 homeodomain protein. We report here the 2.35 Å structure of a ternary complex containing a human HoxB1–Pbx1 heterodimer bound to DNA. Heterodimer contacts are mediated by the hexapeptide of HoxB1, which binds in a pocket in the Pbx1 protein formed in part by a three–amino acid insertion in the Pbx1 homeodomain. The Pbx1 DNA-binding domain is larger than the canonical homeodomain, containing an additional α helix that appears to contribute to binding of the HoxB1 hexapeptide and to stable binding of Pbx1 to DNA. The structure suggests a model for modulation of Hox DNA binding activity by Pbx1 and related proteins.
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.
The Saccharomyces cerevisiae MATa1 and MATα2 homeodomain proteins, which play a role in determining yeast cell type, form a heterodimer that binds DNA
and represses transcription in a cell type-specific manner. Whereas the α2 and a1 proteins on their own have only modest affinity
for DNA, the a1/α2 heterodimer binds DNA with high specificity and affinity. The three-dimensional crystal structure of the
a1/α2 homeodomain heterodimer bound to DNA was determined at a resolution of 2.5 Å. The a1 and α2 homeo- domains bind in a
head-to-tail orientation, with heterodimer contacts mediated by a 16-residue tail located carboxyl-terminal to the α2 homeodomain.
This tail becomes ordered in the presence of a1, part of it forming a short amphipathic helix that packs against the a1 homeodomain
between helices 1 and 2. A pronounced 60° bend is induced in the DNA, which makes possible protein-protein and protein-DNA
contacts that could not take place in a straight DNA fragment. Complex formation mediated by flexible protein-recognition
peptides attached to stably folded DNA binding domains may prove to be a general feature of the architecture of other classes
of eukaryotic transcriptional regulators.
Obtaining an electron-density map from X-ray diffraction data can be difficult and time-consuming even after the data have been collected, largely because MIR and MAD structure determinations currently require many subjective evaluations of the qualities of trial heavy-atom partial structures before a correct heavy-atom solution is obtained. A set of criteria for evaluating the quality of heavy-atom partial solutions in macromolecular crystallography have been developed. These have allowed the conversion of the crystal structure-solution process into an optimization problem and have allowed its automation. The SOLVE software has been used to solve MAD data sets with as many as 52 selenium sites in the asymmetric unit. The automated structure-solution process developed is a major step towards the fully automated structure-determination, model-building and refinement procedure which is needed for genomic scale structure determinations.
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.
Reduction of model bias in macromolecular crystallography through various omit-map techniques has been investigated. The two cases studied were the p21 protein complexed with GDP at 2.25 angstrom resolution and the AN02 Fab fragment of an anti-dinitrophenyl-spin-label murine monoclonal antibody complexed with its hapten at 2.9 angstrom resolution. In the former case, the correct model was compared to a partially incorrect model consisting of an exchanged pair of beta strands along with rearrangement of the connecting loops whereas, in the latter case, the correct placement of an active-site tryptophan side chain was compared to an incorrect rotamer conformation. Partial structures were created by omission of spherical regions around the incorrect region. Omit maps without refinement of the partial structure showed a large degree of model bias. Model bias could be reduced significantly by refinement of the partial structure. Simulated-annealing refinement of the partial structure showed the best results, followed by conjugate-gradient minimization with or without prior randomization of the partial structure. To avoid compensation for missing atoms during simulated-annealing refinement of the partial structure, a suitable 'boundary' region was restrained to the starting coordinates. Model bias removal by iterative density modification was not successful in that it reduced density for both the correct and incorrect conformations.
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.
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 zinc finger DNA-binding motif occurs in many proteins that regulate eukaryotic gene expression. The crystal structure
of a complex containing the three zinc fingers from Zif268 (a mouse immediate early protein) and a consensus DNA-binding site
has been determined at 2.1 angstroms resolution and refined to a crystallographic R factor of 18.2 percent. In this complex,
the zinc fingers bind in the major groove of B-DNA and wrap part way around the double helix. Each finger has a similar relation
to the DNA and makes its primary contacts in a three-base pair subsite. Residues from the amino-terminal portion of an alpha
helix contact the bases, and most of the contracts are made with the guanine-rich strand of the DNA. This structure provides
a framework for understanding how zinc fingers recognize DNA and suggests that this motif may provide a useful basis for the
design of novel DNA-binding proteins.
We have recently identified by cDNA cloning a set of genes that are rapidly activated in mouse 3T3 cells by serum or purified growth factors. Here we report that the cDNA (clone 268) derived from one of these immediate early genes (zif/268) encodes a protein with three tandem "zinc finger" sequences typical of a class of eukaryotic transcription factors. The mRNA of zif/268 is present in many organs and tissues of the mouse and is especially abundant in the brain and thymus tissue. The 5' genomic flanking sequence of zif/268 has sequences related to binding sites for known regulatory proteins, including four sequences that resemble the core of the serum response elements (SREs) upstream of the c-fos and actin genes. The SRE-like sequences could be responsible for the coordinate activation of zif/268 and fos after serum stimulation of 3T3 cells.
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 finger proteins, of the type first discovered in transcription factor IIIA (TFIIIA), are one of the largest and most
important families of DNA-binding proteins. The crystal structure of a complex containing the five Zn fingers from the human
GLI oncogene and a high-affinity DNA binding site has been determined at 2.6 A resolution. Finger one does not contact the
DNA. Fingers two through five bind in the major groove and wrap around the DNA, but lack the simple, strictly periodic arrangement
observed in the Zif268 complex. Fingers four and five of GLI make extensive base contacts in a conserved nine base-pair region,
and this section of the DNA has a conformation intermediate between B-DNA and A-DNA. Analyzing the GLI complex and comparing
it with Zif268 offers new perspectives on Zn finger-DNA recognition.
The functional mimicry of a protein by an unrelated small molecule has been a formidable challenge. Now, however, the biological activity of a 166-residue hematopoietic growth hormone, erythropoietin (EPO), with its class 1 cytokine receptor has been mimicked by a 20-residue cyclic peptide unrelated in sequence to the natural ligand. The crystal structure at 2.8 A resolution of a complex of this agonist peptide with the extracellular domain of EPO receptor reveals that a peptide dimer induces an almost perfect twofold dimerization of the receptor. The dimer assembly differs from that of the human growth hormone (hGH) receptor complex and suggests that more than one mode of dimerization may be able to induce signal transduction and cell proliferation. The EPO receptor binding site, defined by peptide interaction, corresponds to the smaller functional epitope identified for hGH receptor. Similarly, the EPO mimetic peptide ligand can be considered as a minimal hormone, and suggests the design of nonpeptidic small molecule mimetics for EPO and other cytokines may indeed be achievable.
The 2Cys-2His (C2-H2) zinc finger is a protein domain commonly used for sequence-specific DNA recognition. The zinc fingers of the yeast transcription factors SWI5 and ACE2 share strong sequence homology, which extends into a region N-terminal to the first finger, suggesting that the DNA-binding domains of these two proteins include additional structural elements.
Structural analysis of the zinc fingers of SWI5 reveals that a 15 residue region N-terminal to the finger motifs forms part of the structure of the first finger domain, adding a beta strand and a helix not previously observed in other zinc finger structures. Sequence analysis suggests that other zinc finger proteins may also have this structure. Biochemical studies show that this additional structure increases DNA-binding affinity.
The structural analysis presented reveals a novel zinc finger structure in which additional structural elements have been added to the C2-H2 zinc finger fold. This additional structure may enhance stability and has implications for DNA recognition by extending the potential DNA-binding surface of a single zinc finger domain.
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.
Twinning is a crystal growth anomaly in which a specimen is composed of separate crystal domains whose orientations differ in a specific way. Multiple-crystal growth disorders are common, but twinning refers to special cases where some or all of the lattice directions in separate domains are parallel. This leads to either partial or complete coincidence among the lattices of the distinct domains. Twinning is fairly common in crystals of inorganic and some organic compounds. Several different categories of twinning can be defined according to whether the separate lattices coincide in fewer than three dimensions, in all three dimensions, only approximately in three dimensions, or only over a sublattice of points in three dimensions. Merohedral twinning describes the cases in which lattices of two or more distinct domains coincide exactly in three dimensions. It is more sinister than the other forms because it is more difficult to recognize and leads to more fundamental crystallographic problems. Higher forms of merohedral twinning are sometimes seen in crystals of small molecules; the only kind that has been reported for macromolecules is hemihedral in which only two distinct orientations are assumed by the individual twin domains. The chapter focuses on the problems of detecting and overcoming hemihedral twinning in macromolecular crystals.
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.
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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.
Peptides that inhibit binding of vascular endothelial growth factor (VEGF) to its receptors, KDR and Flt-1, have been produced using phage display. Libraries of short disulfide-constrained peptides yielded three distinct classes of peptides that bind to the receptor-binding domain of VEGF with micromolar affinities. The highest affinity peptide was also shown to antagonize VEGF-induced proliferation of primary human umbilical vascular endothelial cells. The peptides bind to a region of VEGF known to contain the contact surface for Flt-1 and the functional determinants for KDR binding. This suggests that the receptor-binding region of VEGF is a binding "hot spot" that is readily targeted by selected peptides and supports earlier assertions that phage-derived peptides frequently target protein-protein interaction sites. Such peptides may lead to the development of pharmacologically useful VEGF antagonists.
Hox homeodomain proteins are developmental regulators that determine body plan in a variety of organisms. A majority of the vertebrate Hox proteins bind DNA as heterodimers with the Pbx1 homeodomain protein. We report here the 2.35 A structure of a ternary complex containing a human HoxB1-Pbx1 heterodimer bound to DNA. Heterodimer contacts are mediated by the hexapeptide of HoxB1, which binds in a pocket in the Pbx1 protein formed in part by a three-amino acid insertion in the Pbx1 homeodomain. The Pbx1 DNA-binding domain is larger than the canonical homeodomain, containing an additional alpha helix that appears to contribute to binding of the HoxB1 hexapeptide and to stable binding of Pbx1 to DNA. The structure suggests a model for modulation of Hox DNA binding activity by Pbx1 and related proteins.
During the development of multicellular organisms, gene expression must be tightly regulated, both spatially and temporally. One set of transcription factors that are important in animal development is encoded by the homeotic (Hox) genes, which govern the choice between alternative developmental pathways along the anterior-posterior axis. Hox proteins, such as Drosophila Ultrabithorax, have low DNA-binding specificity by themselves but gain affinity and specificity when they bind together with the homeoprotein Extradenticle (or Pbxl in mammals). To understand the structural basis of Hox-Extradenticle pairing, we determine here the crystal structure of an Ultrabithorax-Extradenticle-DNA complex at 2.4 A resolution, using the minimal polypeptides that form a cooperative heterodimer. The Ultrabithorax and Extradenticle homeodomains bind opposite faces of the DNA, with their DNA-recognition helices almost touching each other. However, most of the cooperative interactions arise from the YPWM amino-acid motif of Ultrabithorax-located amino-terminally to its homeodomain-which forms a reverse turn and inserts into a hydrophobic pocket on the Extradenticle homeodomain surface. Together, these protein-DNA and protein-protein interactions define the general principles by which homeotic proteins interact with Extradenticle (or Pbx1) to affect development along the anterior-posterior axis of animals.
Furin is a secretory pathway endoprotease that catalyses the maturation of a strikingly diverse group of proprotein substrates, ranging from growth factors and receptors to pathogen proteins, in multiple compartments within the trans-Golgi network (TGN)/endosomal system. This review focuses on recent developments in the biochemistry and cell biology of the endoprotease, including the mechanism of TGN localization, phosphorylation-dependent regulation of protein traffic, and novel insights into early embryogenesis, extracellular matrix formation and pathogen virulence.
Transcription in eukaryotes is frequently regulated by a mechanism termed combinatorial control, whereby several different proteins must bind DNA in concert to achieve appropriate regulation of the downstream gene. X-ray crystallographic studies of multiprotein complexes bound to DNA have been carried out to investigate the molecular determinants of complex assembly and DNA binding. This work has provided important insights into the specific protein-protein and protein-DNA interactions that govern the assembly of multiprotein regulatory complexes. The results of these studies are reviewed here, and the general insights into the mechanism of combinatorial gene regulation are discussed.
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.
The hinge region on the Fc fragment of human immunoglobulin G interacts with at least four different natural protein scaffolds
that bind at a common site between the CH2 and CH3domains. This “consensus” site was also dominant for binding of random peptides selected in vitro for high affinity (dissociation
constant, about 25 nanomolar) by bacteriophage display. Thus, this site appears to be preferred owing to its intrinsic physiochemical
properties, and not for biological function alone. A 2.7 angstrom crystal structure of a selected 13–amino acid peptide in
complex with Fc demonstrated that the peptide adopts a compact structure radically different from that of the other Fc binding
proteins. Nevertheless, the specific Fc binding interactions of the peptide strongly mimic those of the other proteins. Juxtaposition
of the available Fc-complex crystal structures showed that the convergent binding surface is highly accessible, adaptive,
and hydrophobic and contains relatively few sites for polar interactions. These are all properties that may promote cross-reactive
binding, which is common to protein-protein interactions and especially hormone-receptor complexes.
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.
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