Stereochemical basis of DNA recognition by Zn fingers.

DNA-recognition rules for Zn fingers are discussed in terms of crystal structures. The rules can explain the DNA-binding characteristics of a number of Zn finger proteins for which there are no crystal structures. The rules have two parts: chemical rules, which list the possible pairings between the 4 DNA bases and the 20 amino acid residues, and stereochemical rules, which describe the specific base positions contacted by several amino acid positions in the Zn finger. It is discussed that to maintain the correct binding geometry, in which the N-terminus of the recognition helix is closer to the DNA than the C-terminus, the residues facing the DNA on the helix must be larger near the C-terminus, and that two different types of fingers (A and B) bind to DNA in distinctly different ways and cover different numbers of base pairs.     

Protein-DNA interactions: amino acid conservation and the effects of mutations on binding specificity

We investigate the conservation of amino acid residue sequences in 21 DNA-binding protein families and study the effects that mutations have on DNA-sequence recognition. The observations are best understood by assigning each protein family to one of three classes: (i) non-specific, where binding is independent of DNA sequence; (ii) highly specific, where binding is specific and all members of the family target the same DNA sequence; and (iii) multi-specific, where binding is also specific, but individual family members target different DNA sequences. Overall, protein residues in contact with the DNA are better conserved than the rest of the protein surface, but there is a complex underlying trend of conservation for individual residue positions. Amino acid residues that interact with the DNA backbone are well conserved across all protein families and provide a core of stabilising contacts for homologous protein-DNA complexes. In contrast, amino acid residues that interact with DNA bases have variable levels of conservation depending on the family classification. In non-specific families, base-contacting residues are well conserved and interactions are always found in the minor groove where there is little discrimination between base types. In highly specific families, base-contacting residues are highly conserved and allow member proteins to recognise the same target sequence. In multi-specific families, base-contacting residues undergo frequent mutations and enable different proteins to recognise distinct target sequences. Finally, we report that interactions with bases in the target sequence often follow (though not always) a universal code of amino acid-base recognition and the effects of amino acid mutations can be most easily understood for these interactions.     

Interconversion between serine and aspartic acid in the alpha helix of the N-terminal zinc finger of Sp1: implication for general recognition code and for design of novel zinc finger peptide recognizing complementary strand

In the typical base recognition mode of the C(2)H(2)-type zinc finger, the amino acid residues at alpha-helical positions -1, 3, and 6 make a contact with the base in one strand (the primary strand), and the residue at position 2 interacts with the base in a complementary strand (the secondary strand). The N-terminal zinc finger of the three-zinc-finger domain of Sp1 has inherently a unique five-base-pair binding mode in which the guanine bases are recognized in both strands. To clarify the effect of the amino acid at position 2 on DNA binding affinity and base specificity, we have created a library of the mutants by the interconversion between serine and aspartic acid in the N-terminal zinc finger of Sp1 and recombinant variants of finger order. Gel mobility shift and methylation interference assays showed that the combination of arginine and serine at positions -1 and 2, respectively, provides a newly strong guanine contact in the secondary strand and a higher binding affinity than that of wild-type Sp1. Of special interest are the facts that the mutant with lysine and aspartic acid at positions -1 and 2 in the alpha helix predominantly recognizes the bases in the secondary strand and that its DNA binding affinity is higher than that of the wild-type. The aspartic acid or serine at position 2 independently contributes to the DNA binding affinity and base specificity. The present results provide useful information for the design of a novel zinc finger protein with priority for the bases in the secondary strand.     

Recognition of the mRNA AU-rich element by the zinc finger domain of TIS11d

The tandem zinc finger (TZF) domain of the protein TIS11d binds to the class II AU-rich element (ARE) in the 3' untranslated region (3' UTR) of target mRNAs and promotes their deadenylation and degradation. The NMR structure of the TIS11d TZF domain bound to the RNA sequence 5'-UUAUUUAUU-3' comprises a pair of novel CCCH fingers of type CX(8)CX(5)CX(3)H separated by an 18-residue linker. The two TIS11d zinc fingers bind in a symmetrical fashion to adjacent 5'-UAUU-3' subsites on the single-stranded RNA via a combination of electrostatic and hydrogen-bonding interactions, with intercalative stacking between conserved aromatic side chains and the RNA bases. Sequence specificity in RNA recognition is achieved by a network of intermolecular hydrogen bonds, mostly between TIS11d main-chain functional groups and the Watson-Crick edges of the bases. The TIS11d structure provides insights into the RNA-binding functions of this large family of CCCH zinc finger proteins.     

High-resolution solution structure of the double Cys2His2 zinc finger from the human enhancer binding protein MBP-1.

The high-resolution three-dimensional structure of a synthetic 57-residue peptide comprising the double zinc finger of the human enhancer binding protein MBP-1 has been determined in solution by nuclear magnetic resonance spectroscopy on the basis of 1280 experimental restraints. A total of 30 simulated annealing structures were calculated. The backbone atomic root-mean-square distributions about the mean coordinate positions are 0.32 and 0.33 A for the N- and C-terminal fingers, respectively, and the corresponding values for all atoms, excluding disordered surface side chains, are 0.36 and 0.40 A. Each finger comprises an irregular antiparallel sheet and a helix, with the zinc tetrahedrally coordinated to two cysteines and two histidines. The overall structure is nonglobular in nature, and the angle between the long axes of the helices is 47 +/- 5 degrees. The long axis of the antiparallel sheet in the N-terminal finger is approximately parallel to that of the helix in the C-terminal finger. Comparison of this structure with the X-ray structure of the Zif-268 triple finger complexed with DNA indicates that the relative orientation of the individual zinc fingers is clearly distinct in the two cases. This difference can be attributed to the presence of a long Lys side chain in the C-terminal finger of MBP-1 at position 40, instead of a short Ala or Ser side chain at the equivalent position in Zif-268. This finding suggests that different contacts may be involved in the binding of the zinc fingers of MBP-1 and Zif-268 to DNA, consistent with the findings from methylation interference experiments that the two fingers of MBP-1 contact 10 base pairs, while the three fingers of Zif-268 contact only 9 base pairs.     

Finger-positional change in three zinc finger protein Sp1: influence of terminal finger in DNA recognition

The connection of functional modules is effective for the design of DNA binding molecules with the desired sequence specificity. C(2)H(2)-type zinc finger proteins have a tandemly repeated array structure consisting of independent finger modules and are expected to recognize any DNA sequences by permutation, multi-connection, and the substitution of various sets of zinc fingers. To investigate the effects of the replacement of the terminal finger on the DNA recognition by other fingers, we have constructed the three zinc finger peptides with finger substitution at the N- or C-terminus, Sp1(zf223), Sp1(zf323), and Sp1(zf321). From the results of gel mobility shift assays, each mutant peptide binds preferentially to the target sequence that is predicted if the fingers act in a modular fashion. The methylation interference analyses demonstrate that in the cases of the N-terminal finger substitution mutants, Sp1(zf223) and Sp1(zf323), the N-terminal finger recognizes bases to different extents from that of the wild-type peptide, Sp1(zf123). Of special interest is the fact that the N-terminal finger of the C-terminal finger substitution mutant, Sp1(zf321), shows a distinct base recognition from those of Sp1(zf123) and Sp1(zf323). DNase I footprinting analyses indicate that the C-terminal finger (active finger) induces a conformational change in the DNA in the region for the binding of the N-terminal finger (passive finger). The present results strongly suggest that the extent of base recognition of the N-terminal finger is dominated by the binding of the C-terminal finger. This information provides an important clue for the creation of a zinc finger peptide with the desired specificity, which is applicable to the design of novel drugs and biological tools.     

Solution structures of two CCHC zinc fingers from the FOG family protein U-shaped that mediate protein-protein interactions

BACKGROUND: Zinc finger domains have traditionally been regarded as sequence-specific DNA binding motifs. However, recent evidence indicates that many zinc fingers mediate specific protein-protein interactions. For instance, several zinc fingers from FOG family proteins have been shown to interact with the N-terminal zinc finger of GATA-1. RESULTS: We have used NMR spectroscopy to determine the first structures of two FOG family zinc fingers that are involved in protein-protein interactions: fingers 1 and 9 from U-shaped. These fingers resemble classical TFIIIA-like zinc fingers, with the exception of an unusual extended portion of the polypeptide backbone prior to the fourth zinc ligand. [15N,(1)H]-HSQC titrations have been used to define the GATA binding surface of USH-F1, and comparison with other FOG family proteins indicates that the recognition mechanism is conserved across species. The surface of FOG-type fingers that interacts with GATA-1 overlaps substantially with the surface through which classical fingers typically recognize DNA. This suggests that these fingers could not contact both GATA and DNA simultaneously. In addition, results from NMR, gel filtration, and sedimentation equilibrium experiments suggest that the interactions are of moderate affinity. CONCLUSIONS: Our results demonstrate unequivocally that zinc fingers comprising the classical betabetaalpha fold are capable of mediating specific contacts between proteins. The existence of this alternative function has implications for the prediction of protein function from sequence data and for the evolution of protein function.     

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

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

Structure of the Wilms tumor suppressor protein zinc finger domain bound to DNA

The zinc finger domain of the Wilms tumor suppressor protein (WT1) contains four canonical Cys(2)His(2) zinc fingers. WT1 binds preferentially to DNA sequences that are closely related to the EGR-1 consensus site. We report the structure determination by both X-ray crystallography and NMR spectroscopy of the WT1 zinc finger domain in complex with DNA. The X-ray structure was determined for the complex with a cognate 14 base-pair oligonucleotide, and composite X-ray/NMR structures were determined for complexes with both the 14 base-pair and an extended 17 base-pair DNA. This combined approach allowed unambiguous determination of the position of the first zinc finger, which is influenced by lattice contacts in the crystal structure. The crystal structure shows the second, third and fourth zinc finger domains inserted deep into the major groove of the DNA where they make base-specific interactions. The DNA duplex is distorted in the vicinity of the first zinc finger, with a cytidine twisted and tilted out of the base stack to pack against finger 1 and the tip of finger 2. By contrast, the composite X-ray/NMR structures show that finger 1 continues to follow the major groove in the solution complexes. However, the orientation of the helix is non-canonical, and the fingertip and the N terminus of the helix project out of the major groove; as a consequence, the zinc finger side-chains that are commonly involved in base recognition make no contact with the DNA. We conclude that finger 1 helps to anchor WT1 to the DNA by amplifying the binding affinity although it does not contribute significantly to binding specificity. The structures provide molecular level insights into the potential consequences of mutations in zinc fingers 2 and 3 that are associated with Denys-Drash syndrome and nephritic syndrome. The mutations are of two types, and either destabilize the zinc finger structure or replace key base contact residues.     

The maize ID1 flowering time regulator is a zinc finger protein with novel DNA binding properties

The INDETERMINATE protein, ID1, plays a key role in regulating the transition to flowering in maize. ID1 is the founding member of a plant-specific zinc finger protein family that is defined by a highly conserved amino sequence called the ID domain. The ID domain includes a cluster of three different types of zinc fingers separated from a fourth C2H2 finger by a long spacer; ID1 is distinct from other ID domain proteins by having a much longer spacer. In vitro DNA selection and amplification binding assays and DNA binding experiments showed that ID1 binds selectively to an 11 bp consensus motif via the ID domain. Unexpectedly, site-directed mutagenesis of the ID1 protein showed that zinc fingers located at each end of the ID domain are not required for binding to the consensus motif despite the fact that one of these zinc fingers is a canonical C2H2 DNA binding domain. In addition, an ID1 in vitro deletion mutant that lacks the extra spacer between zinc fingers binds the same 11 bp motif as normal ID1, suggesting that all ID domain-containing proteins recognize the same DNA target sequence. Our results demonstrate that maize ID1 and ID domain proteins have novel zinc finger configurations with unique DNA binding properties.     

Effects of length and position of an extended linker on sequence-selective DNA recognition of zinc finger peptides

Engineered zinc finger proteins revealed that a linker sequence connecting zinc finger units has a significant effect on the DNA binding property of the protein. The recognition for a noncontiguous DNA target beyond the current recognition code of zinc finger proteins has never been determined because of the limitation of a zinc finger framework. DNA recognition of zinc finger proteins is limited only to a contiguous subset of three base pairs. We propose the recognition for a noncontiguous DNA target by inserting amino acids into the canonical linker between zinc finger units. The sequence selectivity of the new zinc finger peptides was evaluated by gel mobility shift assays. DNase I footprinting analyses clearly showed different DNA binding of various linker-extended zinc finger peptides. The application of a SPR measurement also revealed a DNA sequence selectivity of peptides. Insertion of three amino acids is enough for recognition of a noncontiguous DNA target with sequence selectivity. An extended linker will be useful for expansion of the recognition code of zinc finger proteins and for development of a new role for linker sequences in DNA binding of zinc finger proteins.