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Combining Structure-Based Design with Phage Display to Create New Cys His Zinc Finger Dimers

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Combining Structure-Based Design with Phage Display to Create New Cys His Zinc Finger Dimers st8703.qxd 07/03/2000 09:38 Page 739 Research Article 739 Combining structure-based design with phage display to create new Cys2His2 zinc finger dimers Scot A Wolfe, Elizabeth I Ramm and Carl O Pabo* Background: Several strategies have been reported for the design and Address: Howard Hughes Medical Institute and selection of novel DNA-binding proteins. Most of these studies have used Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Cys2His2 zinc finger proteins as a framework, and have focused on constructs that bind DNA in a manner similar to Zif268, with neighboring fingers *Corresponding author. connected by a canonical (Krüppel-type) linker. This linker does not seem ideal E-mail: [email protected] for larger constructs because only modest improvements in affinity are Key words: dimerization, DNA recognition, leucine observed when more than three fingers are connected in this manner. Two zippers, phage display, structure-based design, strategies have been described that allow the productive assembly of more zinc fingers than three canonically linked fingers on a DNA site: connecting sets of fingers using linkers (covalent), or assembling sets of fingers using dimerization Received: 13 March 2000 28 April 2000 domains (non-covalent). Accepted: Published: 21 June 2000 Results: Using a combination of structure-based design and phage display, we Structure 2000, 8:739–750 have developed a new dimerization system for Cys2His2 zinc fingers that allows the assembly of more than three fingers on a desired target site. Zinc finger 0969-2126/00/$ – see front matter constructs employing this new dimerization system have high affinity and good © 2000 Elsevier Science Ltd. All rights reserved. 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 Cys2His2 zinc fingers to be productively assembled on a DNA-binding site. Dimerization may offer certain advantages over covalent linkage for the CORE recognition of large DNA sequences. Our results also illustrate the power of Metadata, citation and similar papers at core.ac.uk Provided by Elsevier - Publishercombining Connector structure-based design with phage display in a strategy that assimilates the best features of each method. Introduction created both by design [5–8] and by selection via phage Cys2His2 zinc fingers function as recognition domains that display [9–12]. Recurring patterns observed in the DNA facilitate protein–DNA, protein–RNA, and protein–protein contacts by zinc fingers have led to proposals for a zinc interactions [1]. Each zinc finger is a minidomain of finger ‘recognition code’, but recognition is sufficiently approximately 30 amino acids that folds into a ββα motif in complicated that selection via phage display remains the the presence of zinc. Tandem repeats of fingers are usually most reliable method for the generation of fingers with employed for DNA or RNA recognition, and DNA recog- novel specificities [1,13]. The assembly of designed or nition is mediated by the α helix of each finger. The selected fingers into functional proteins also is compli- α helix inserts into the major groove such that the i, i + 3 cated by the partial overlap in the recognition sites of ridge of the helix is aligned with neighboring bases along neighboring fingers. Methods that help overcome this one strand of the DNA [2]. Fingers that bind in a canonical problem with multifinger proteins have recently been manner (i.e., like Zif268) recognize overlapping four-base- described [14–16]. pair subsites using amino acids at positions –1, 2, 3 and 6 of the α helix (Figure 1) [1]. Binding sites for individual Many linkers found between DNA-binding zinc fingers fingers overlap because the amino acid at position 2 in the conform to the consensus sequence TGEKP (in single- recognition helix from one finger often contacts the same letter amino acid code; Figure 1b). These canonical base pair as the amino acid at position 6 in the recognition linkers are well ordered in the structures of zinc helix from the neighboring N-terminal finger [3]. finger–DNA complexes [3,4,17,18], and mutagenesis studies indicate that they play an important role in DNA Cys2His2 zinc finger proteins have proven to be a versa- recognition [19,20]. Because Zif268 (and to a lesser extent tile motif for the recognition of a variety of different SP1) served as the prototype for the design and selection DNA sequences. Using the structure of Zif268 [4] as a of fingers with novel specificities, TGEKP linkers have framework, zinc fingers with novel specificities have been been used almost exclusively in the development of st8703.qxd 07/03/2000 09:38 Page 740 740 Structure 2000, Vol 8 No 7 Figure 1 sequence larger than ten base pairs (i.e., proteins with more than three fingers) may be required to obtain effi- (a) 3′ 5′ cacy as a gene therapy construct. Two approaches have C G R 6 been used to make proteins that specifically recognize 2 E 3 G C3 D 2 longer sites. These involve either insertion of a flexible R -1 Finger 3 linker between two sets of canonically linked fingers [25], C G4 T 6 or attachment of a dimerization domain that allows coop- A T5 C G H 3 erative assembly of the zinc finger proteins on the DNA 6 D 2 C G R -1 [26]. Femtomolar dissociation constants have been 7 Finger 2 observed for a six-finger construct (268//NRE) that has C G R 6 8 two three-finger proteins connected by a flexible linker G C E 3 9 D 2 [25]. Improvements in affinity and specificity observed C G R –1 10 Finger 1 with this six-finger construct are encouraging, but they A T11 5′ 3′ are still far below the values that would be (b) anticipated — based on the chelate effect — for a con- struct containing two linked three-finger proteins. –1123456 789 Finger 1 PYACPVESCDRRFSRSDELTRHIRIHTGQK Finger 2 PFQCRI--CMRNFSRSDHLTTHIRTHTGEK Many naturally occurring transcription factors use dimer- Finger 3 PFACDI--CGRKFARSDERKRHTKIHLRQK ization to enhance their affinity and specificity, and het- =DNA recognition = Zinc coordination = Canonical linker erodimerization is sometimes used in natural regulatory Structure systems to provide an additional level of transcriptional control. In principle, dimerization of zinc fingers on DNA Wild-type Zif268 recognizes its binding site using three Cys2His2 zinc could offer a number of advantages for the assembly of fingers. (a) Overall arrangement seen in the 1.6 Å structure of Zif268 bound to DNA [3]. Each finger inserts its α helix into the major groove, four or more fingers [27,28]. Cooperative recognition of a and DNA recognition is mediated by amino acids at positions –1, 2, 3 binding site provides a sharper transition between the and 6 from this helix, with the majority of contacts made to one strand bound and unbound states as a function of protein concen- of the DNA (primary strand). Interactions that are mediated by tration, thus providing tighter control of the ‘on’ and ‘off’ hydrogen bonds between the fingers and DNA are indicated by arrows regulatory states. In the presence of a large number of in the panel on the right; hydrophobic interactions are indicated by non-cognate binding sites, cooperative binding to the open circles. (b) Amino acid sequence of the three Cys2His2 zinc fingers from Zif268 [43]. Adjacent fingers in this structure are DNA may also provide faster equilibration rates than pos- connected by ‘canonical’ linkers (TGQKP and TGEKP, respectively; sible with a covalent fusion of the same number of fingers. indicated in avocado) [1]. The residues from each finger involved in defining DNA specificity are indicated in blue; residues involved in zinc coordination are indicated in purple. The secondary structure elements Our group recently described a prototype dimerization are indicated above the sequence with arrows indicating β strands and system for zinc fingers in which the coiled-coil dimeriza- the cylinder denoting α helices. tion domain of Gal4 is fused via a long linker to the C ter- minus of two fingers from Zif268 [26]. This fusion protein (ZFGD1) bound its DNA sequence with a reasonable –18 2 multifinger proteins with novel specificities. Several affinity (Kd =10 M ) and can accommodate zinc fingers groups have noted that as the number of TGEKP-linked with different sequence specificities, thus allowing recog- fingers increases from one to two to three, there is an nition of asymmetric binding sites. Building on our experi- accompanying increase in DNA-binding affinity. ence with ZFGD1, we developed an improved (Roughly speaking, the affinity increases from millimolar dimerization system for zinc fingers. Computer modeling to micromolar to nanomolar.) However, the attachment of (structure-based design) suggested that it should be possi- additional fingers (four or more in total) using the ble to create a pseudo-continuous α helix joining a leucine TGEKP linker provides only modest additional improve- zipper dimerization domain directly to the DNA recogni- ment in affinity [21–23]. The accompanying structural tion helix of a zinc finger. This fusion should give a much and energetic problems arising from the presence of four more rigid dimer interface than with Gal4 and, conse- or more fingers are not entirely clear, but these difficul- quently, help to define a preferred half-site spacing for the ties may originate from the distortion of the DNA that complex. This new design also juxtaposes the binding zinc fingers elicit upon binding in the major groove [24]. sites for each monomer, allowing recognition of a contigu- (In the resulting complex, DNA assumes a variant B-form ous DNA sequence.
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