ch4106.qxd 02/15/2000 09:01 Page 34 34 Design of novel sequence-specific DNA-binding proteins David J Segal and Carlos F Barbas III* The design and selection of DNA-binding proteins or individual Helical domains domains capable of novel sequence recognition continues to For proteins that do not contact DNA with an organized make great strides. Recent studies have also highlighted the α-helix, success in generating novel sequence-specific pro- role of the non-DNA-contacting portions of the protein and the teins has been extremely limited. For example, the optimal assembly of the domains. For the first time, it appears base-specific hydrogen bonds between the restriction that it is possible to produce proteins capable of targeting any endonuclease EcoRV and its recognition base pairs are gene with an 18 base pair recognition domain. A variety of made through a highly cooperative set of interactions by applications are being explored, such as targeted five residues in a surface loop of the enzyme (Figure 1a; transcriptional regulation, recombination and viral integration. [1]). Through application of DNA shuffling, mutagenesis These proteins will probably find diverse applications in gene and screening, Pingoud and co-workers [2••] were able to therapy, functional genomics, and agriculture. extend recognition to include the base pairs immediately flanking the EcoRV site. Although this is an impressive Addresses study, it remains to be seen if more can be expected from The Skaggs Institute for Chemical Biology and the Department of these types of complex recognition motifs. Molecular Biology, The Scripps Research Institute, BCC-515, North Torrey Pines Road, La Jolla, CA 92037, USA A decade ago, the dimeric bZIP proteins were proposed as *e-mail: [email protected] candidates for the development of novel DNA-binding Current Opinion in Chemical Biology 2000, 4:34–39 proteins [3]. Each bZIP monomer contacts DNA with an α 1367-5931/00/$ — see front matter © 2000 Elsevier Science Ltd. -helix that is oriented and constrained by being part of All rights reserved. longer α-helix, which makes up the leucine zipper dimer- ization interface (Figure 1b; [4]). In vitro selection methods Abbreviations DBD DNA-binding domain produced mutants of the bZIP protein C/EBP that could ER estrogen receptor recognize sequences that differed in two of its five half-site PR progesterone receptor nucleotides [5]. Interestingly, some of the mutations occurred in the hinge region between the α-helix of the Introduction DNA-binding domain (DBD) and the leucine zipper The design of proteins with the capacity to recognize framework, suggesting that reorientation of the α-helix extended nucleotide sequences with high affinity and may have been required. specificity has been a goal of two decades of research; a goal that has remained elusive until the past two years. Theoretically, DBDs of bZIP heterodimers, such as Jun- Ideally, proteins could be designed to specifically regu- Fos, could be modified to recognize novel 10-nucleotide, late the transcription of a single gene in a genome as non-palindromic sites. Alternatively, tandem monomers of complex as our own four billion base pair genome. These the bZIP DBDs could potentially be used to recognize proteins would allow scientists to reach into genomes extended sequences, but these typically bind poorly to and to modulate the transcription of a gene of interest, to DNA. In an elegant design approach, the affinity of a bZIP assign its function or to change the phenotype of monomer DBD, that of GCN4, was improved 270-fold by the organism. grafting the DNA-contacting residues from the α-helix of GCN4 onto the stable and compact scaffold of the avian Specific recognition of double-stranded DNA by pro- pancreatic polypeptide [6••]. This miniature 42-amino- teins is most typically accomplished using an α-helix as acid protein, consisting of a single amphipathic α-helix the key recognition element. An α-helix provides a rigid that is stabilized through hydrophobic interactions with a scaffold from which amino-acid sidechains are presented type II polyproline helix, was able to bind a GCN4 half site to the accessible edges of the bases in the major grove of with an affinity and specificity greater than that of the DNA. This presentation is modulated by ‘framework’ parental α-helix. This study again illustrates that binding structural elements of the protein. For proteins in which can be improved by optimizing not only the DNA-contact- the α-helix is the key recognition element, the task of ing residues but also the display and stability of the creating DNA-binding proteins with novel sequence α-helix. It remains unclear if these domains can be effec- specificity consists of modifying the DNA-contacting tively linked as tandem multimers to recognize extended residues in the α-helix, then modifying the framework to sites. Chemical linkage of two mini-proteins provided a optimize the display of the α-helix. In this review, we ‘dimer’ with increased affinity but lower specificity [6••]. discuss the recent successful application of these princi- ples to create novel DNA-binding proteins, with Nuclear receptors, such as the estrogen receptor (ER) particular emphasis on the advances achieved using and progesterone receptor (PR), also bind as dimers to α Cys2-His2 zinc finger proteins. their DNA targets. Each monomer inserts an -helix of a ch4106.qxd 02/15/2000 09:01 Page 35 Design of novel sequence-specific DNA-binding proteins Segal and Barbas 35 Figure 1 DBDs that have been used to create proteins with novel sequence specificity. (a) EcoRV (a) (b) [1]. (b) bZIP of GCN4 [4]. (c) Cys4 zinc fingers of glucocorticoid receptor [8]. (d) Helix-turn-helix domains of Myb [10]. (e) Cys2–His2 zinc fingers of Zif268 [13]. Double stranded DNA is shown in orange and brown, DBD domains are shown in red, blue or green ribbon representation, and zinc ions are shown as yellow spheres. (d) (c) (e) Current Opinion in Chemical Biology Cys4-type zinc finger into the major grove, and for some By any measure, the greatest success in producing proteins receptors the DBD contains a strong dimerization inter- with novel binding specificity has been achieved with the face (Figure 1c; [7,8]). Using in vivo survival-based classic Cys2–His2 zinc-finger domains (Figure 1e). These selection of randomized libraries, Shapiro and co-workers zinc fingers are compact domains containing a single [9••] found PR-DBD mutants that lost their ability to rec- amphipathic α-helix stabilized by two β-strands and zinc ognize the PR response element (PRE) but could bind ligation [13]. Like the Myb domains, zinc-finger proteins the ER response element (ERE), which differs in two of contain multiple tandem repeats and display varying the six half-site nucleotides. The mutants bound the degrees of inter-domain cooperativity. Fortunately, a subset ERE with a 15-fold higher affinity than wild-type ER- of zinc-finger proteins, including the murine transcription DBD, albeit with a specificity considerably broader than factor Zif268 and the human protein Sp1, display only min- wild-type ER-DBD. imal — though still troublesome — cooperativity. In these proteins, each finger recognizes a three nucleotide site with From domains to proteins relative independence, which has allowed several groups to Tandemly repeated elements, although having the potential produce zinc fingers with novel specificities using rational for extended, non-palindromic recognition, introduce anoth- or combinatorial methods (reviewed in [14••]). er level of complexity: inter-domain cooperativity. One domain may make protein–protein contacts with the next Early attempts to produce zinc fingers with novel domain, affect the binding geometry of adjacent domains, or sequence recognition gave hope to the idea that there it may contact the nucleotides of another domain’s binding might be a simple 1:1 amino acid to base recognition code site. These interactions that allow for concerted recognition that could be used to build fingers that could specifically may be considered as evolutionary levers for optimizing the recognize any three nucleotide sequence [15,16]. binding of a particular protein; however, for those seeking to Unfortunately, this simplistic code is poorly predictive of design novel binding proteins these interactions present the actual specificity of these domains [17,18•]. In one additional challenges. For example, Myb domains report of a code-based three-finger protein, only five of the (Figure 1d) consist of two or three tandem repeats (desig- nine nucleotides appear to be correctly specified [17]. nated R1, R2 and R3) of a helix-turn-helix motif, similar to the motif found in the λ repressor and homeodomian pro- There are at least three reasons why a simple code is insuf- teins [10]. Myb DBDs with novel specificity have been ficient for comprehensive recognition. The first is generated by combining the R2 and R3 of different species technical. Phage display has proven to be a powerful tool [11]; however, the combination of two tandem R3 repeats for revealing the repertoire of zinc-finger–DNA interac- severely reduced binding affinity [12]. The authors con- tions. This procedure involves the display of a large cluded that cooperative interactions between the R2 and R3 (typically 107–109 member) library of randomized proteins domains were required for high affinity binding. on the surface of a filamentous bacteriophage. Because ch4106.qxd 02/15/2000 09:01 Page 36 36 Interaction, assembly and processing Table 1 two α-helices including position 2 [20•,21], or by replace- ment of the offending domain with a finger that does not Affinities of sequentially-selected and stitched three-finger contain aspartate in position 2 and is not expected to have proteins. any cooperative effects. Protein Target sequence Published Normalized ‡ § KD (nM) KD (nM) Concerns regarding cooperativity can also be addressed by the selection methodology if sequential selections are Stitched study Zif268* GCG TGG GCG 10 10 applied [22].