Structure-based design of an RNA-binding zinc finger

Abstract

A structure-based approach was used to design RNA-binding zinc fingers that recognize the HIV-1 Rev response element (RRE). An arginine-rich alpha-helix from HIV-1 Rev was engineered into the zinc finger framework, and the designed fingers were shown to bind specifically to the RRE with high affinity and in a zinc-dependent manner, and display cobalt absorption and CD spectra characteristic of properly folded fingers. The results indicate that a monomeric zinc finger can recognize a specific nucleic acid site and that the alpha-helix of a finger can be used to recognize the major groove of RNA as well as DNA. The RRE-binding zinc fingers demonstrate how structure-based approaches may be used in the design of potential RNA-binding therapeutics and provide a framework for selecting RNA-binding fingers with desired specifications.

Figure 1

Design of ZF-Rev peptides. (A) The sequence of Rev14 (24) was aligned with the α-helix of Zif268 zinc finger 2 (boxed) such that the histidines required for metal binding and the amino acids required for RRE recognition (shown in bold and indicated by lines) did not overlap. The numbering above the Zif268 α-helix indicates positions (−1, +2, +3, and +6, relative to the start of the helix) typically used for DNA binding, and the β-strands of the finger are indicated by open triangles. The putative α-helix in the hybrid ZF2-Rev is boxed. Positions of cysteine-to-serine mutations and an asparagine-to-alanine mutation are shown. A similar alignment was used to construct a fusion to Zif zinc finger 1 (ZF1-Rev; not shown). (B) Overlap between the α-helical segment of Zif finger 2 (magenta) (3) and residues 34–47 of the Rev peptide (yellow) (26). Backbone heavy atoms were superimposed from Ser-47 to Thr-58 of the finger and from Arg-35 to Arg-46 of the Rev peptide by using insight ii software (Biosym Technologies, San Diego) on a Silicon Graphics workstation. The rms deviation between all 36 superimposed atoms was 0.84 Å, and a similar rms deviation (0.93 Å) was obtained by using Zif finger 1 (not shown). The metal-coordinating cysteine and histidine residues and zinc ion in the finger are shown.

Figure 2

(A) Proposed orientation of a zinc finger bound to RRE IIB. The α-helices of Rev (yellow) and Zif finger 2 (magenta) were superimposed and the aligned zinc finger was docked against the RNA. The view down the α-helical axes shows that the β-sheet portion of the finger emerges from the major groove and can accommodate the peptide backbone and side chains without steric clashes to the RNA. (B) Comparison of the Rev peptide- and ZF2-Rev-RRE complexes. The α-helix of Zif finger 2 was replaced by residues 34–47 of Rev, with two histidine substitutions (yellow). The β-sheet portion of the finger and the two cysteine ligands are shown in magenta and the zinc ion in white.

Figure 3

Cobalt absorption spectroscopy and metal-dependent peptide folding. (A) Absorption spectra of ZF2-Rev (140 μM) in the presence of stoichiometric CoCl₂ (●), or in the presence of 210 μM CoCl₂ plus 70 μM (○) or 140 μM (■) ZnCl₂ competitor. The spectra shown are characteristic of tetrahedral metal coordination in a monomeric zinc finger; red-shifted spectra have been observed with misfolded zinc finger dimers (29) and we observed similar shifts at low metal stoichiometries (data not shown), suggesting that incorrect complexes may be populated initially. (B) CD spectra of ZF2-Rev and a double-cysteine mutant (C5,8S): reduced ZF2-Rev in the absence of metal (●), ZF2-Rev in the presence of stoichiometric ZnCl₂ (○), ZF2-Rev (C5,8S) in the absence of metal (■), and ZF2-Rev (C5,8S) in the presence of stoichiometric ZnCl₂ (□). Spectra were recorded at 4°C with peptide concentrations of 30–60 μM. (C) Difference CD spectra of ZF2-Rev (●) and ZF2-Rev (C5,8S) (□), calculated by subtracting spectra in the absence of zinc from spectra in the presence of zinc (from B). The difference spectrum of ZF2-Rev shows two minima (at 208 and 222 nm) characteristic of α-helix formation whereas the spectrum of the cysteine mutant (see B) indicates little structure and no metal-dependent folding. Because ZF2-Rev showed some helix formation in the absence of zinc and had a higher helical content than ZF2-Rev (C5,8S), we performed several experiments to rule out the presence of adventitious zinc ions. We observed little change in the CD spectra by using buffers extensively treated with Chelex 100 chelating resin or in the presence of excess chelators (EDTA or 1,10-phenanthroline). Furthermore, careful titrations with cobalt and zinc showed no change in the visible absorption spectra after addition of stoichiometric CoCl₂ and competition with stoichiometric amounts of ZnCl₂, suggesting that little or no zinc was prebound to the peptide. In addition, 1D and 2D proton NMR spectra indicate that the peptide is unstructured in the absence of ZnCl₂ and becomes ordered upon titration of stoichiometric ZnCl₂ (W. Gmeiner, D.J.M., and A.D.F., unpublished data).

Figure 4

RRE IIB binding by ZF2-Rev in vitro. (A) Gel shift assays of ZF2-Rev and Rev14_ala peptides with wild-type RRE IIB RNA and a C46-G74 mutant (that reverses a base pair critical for recognition; refs. , , and 27) at the peptide concentrations (nM) indicated. ZF2-Rev binds with a similar affinity and specificity as the helical Rev peptide; apparent binding constants are substantially tighter in the absence of competitor tRNA (21, 33). (B, Left) Zinc-dependent RRE IIB RNA binding: ZF2-Rev in the presence of 25 μM ZnCl₂ (■), in the absence of zinc and with addition of 0.5 mM EDTA (□), and ZF2-Rev (C5,8S) in the presence of 25 μM ZnCl₂ (●). (Right) Binding curves quantitated by PhosphorImager analyses and apparent dissociation constants (nM) estimated by fitting to standard binding equations.

Figure 5

ZF-Rev binding to RRE IIB in vivo. (A) ZF-Rev peptides were fused to the activation domain of HIV-1 Tat, and activation of an HIV-1 LTR-CAT reporter containing RRE IIB (21) was measured in HeLa cells (bars). ZF-Rev peptides also were fused to the bacteriophage λ N protein and transcriptional antitermination of an RRE IIB-lacZ reporter (25) was scored in E. coli (+ or − above bars). For CAT assays, 10 ng of each Tat-fusion plasmid was cotransfected with 50 ng of IIB reporter plasmid, and fold activation refers to the ratio of activities generated by the Tat fusion proteins to the activity in the absence of Tat. For β-galactosidase scoring, ++++ represents the darkest blue colonies and − represents white colonies. (B) Activity of ZF2-Rev was determined by CAT assays as in A by using reporter plasmids containing the wild-type IIB RRE site, a C46-G74 mutant that reverses a base pair critical for recognition (21, 26, 27), or the bovine immunodeficiency virus (BIV) or HIV-1 TAR hairpins.