Effects of bulkiness and hydrophobicity of an aliphatic amino acid in the recognition helix of the GAGA zinc finger on the stability of the hydrophobic core and DNA binding affinity

The GAGA factor of Drosophila melanogaster uses a single Cys 2His 2-type zinc finger for specific DNA binding. The conformation and DNA binding mode of the GAGA zinc finger are similar to those of other structurally characterized zinc fingers. In almost all Cys 2His 2-type zinc fingers, the fourth position of the DNA-recognizing helix is occupied by the Leu residue involved in the formation of the minimal hydrophobic core. However, no systematic study on the precise role of the Leu residue in the hydrophobic core formation and DNA binding function has been reported. In this study, the Leu residue is substituted with other aliphatic amino acids having different side chain lengths and hydrophobicities, namely, Ile, Val, Aib, and Ala. The metal binding properties were studied by UV-vis spectroscopy. The peptide conformations were examined by CD and NMR spectroscopies. Furthermore, the DNA binding ability was examined with a gel mobility shift assay. Though the Ile, Val, and Aib mutants exhibited conformations similar to those of the wild type, the DNA binding affinity decreased as the side chain length of the amino acid decreased. Interestingly, the Val mutant can bind to the cognate DNA, while Aib cannot, in spite of the similarity in their secondary structures based on the CD measurements. Variable-temperature NMR experiments clearly indicated differences in the stability of the hydrophobic core between the Val and Aib mutants. This study demonstrates that the bulkiness of the conserved aliphatic residue is important in the formation of the well-packed minimal hydrophobic core and proper ternary structure and that the hydrophobic core stabilization is apparently related to the DNA binding function of the GAGA zinc finger.     

Swapping of the beta-hairpin region between Sp1 and GLI zinc fingers: significant role of the beta-hairpin region in DNA binding properties of C2H2-type zinc finger peptides

The recent design strategy of zinc finger peptides has mainly focused on the alpha-helix region, which plays a direct role in DNA recognition. On the other hand, the study of non-DNA-contacting regions is extremely scarce. By swapping the beta-hairpin regions between the Sp1 and GLI zinc fingers, in this study, we investigated how the beta-hairpin region of the C(2)H(2)-type zinc finger peptides contributes to the DNA binding properties. Surprisingly, the Sp1 mutant with the GLI-type beta-hairpin had a higher DNA binding affinity than that of the wild-type Sp1. The result of the DNase I footprinting analyses also showed the change in the DNA binding pattern. In contrast, the GLI zinc finger completely lost DNA binding ability as a result of exchanging the beta-hairpin region. These results strongly indicate that the beta-hairpin region appears to function as a scaffold and has an important effect on the DNA binding properties of the C(2)H(2)-type zinc finger peptides.     

Structure-function analysis of the DNA binding domain of Saccharomyces cerevisiae ABF1.

To localize the DNA binding domain of the Saccharomyces cerevisiae Ars binding factor 1 (ABF1), a multifunctional DNA binding protein, plasmid constructs carrying point mutations and internal deletions in the ABF1 gene were generated and expressed in Escherichia coli. Normal and mutant ABF1 proteins were purified by affinity chromatography and their DNA binding activities were analyzed. The substitution of His61, Cys66 and His67 respectively, located in the zinc finger motif in the N-terminal region (amino acids 40-91), eliminated the DNA binding activity of ABF1 protein. Point mutations in the middle region of ABF1, specifically at Leu353, Leu399, Tyr403, Gly404, Phe410 and Lys434, also eliminated or reduced DNA binding activity. However, the DNA binding activity of point mutants of Ser307, Ser496 and Glu649 was the same as that of wild-type ABF1 protein and deletion mutants of amino acids 200-265, between the zinc finger region and the middle region (residues 323-496) retained DNA binding activity. As a result, we confirmed that the DNA binding domain of ABF1 appears to be bipartite and another DNA binding motif, other than the zinc finger motif, is situated between amino acid residues 323 and 496.     

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.     

Structure/function mapping of amino acids in the N-terminal zinc finger of the human immunodeficiency virus type 1 nucleocapsid protein: residues responsible for nucleic acid helix destabilizing activity

The nucleocapsid protein (NC) of HIV-1 is 55 amino acids in length and possesses two CCHC-type zinc fingers. Finger one (N-terminal) contributes significantly more to helix destabilizing activity than finger two (C-terminal). Five amino acids differ between the two zinc fingers. To determine at the amino acid level the reason for the apparent distinction between the fingers, each different residue in finger one was incrementally replaced by the one at the corresponding location in finger two. Mutants were analyzed in annealing assays with unstructured and structured substrates. Three groupings emerged: (1) those similar to wild-type levels (N17K, A25M), (2) those with diminished activity (I24Q, N27D), and (3) mutant F16W, which had substantially greater helix destabilizing activity than that of the wild type. Unlike I24Q and the other mutants, N27D was defective in DNA binding. Only I24Q and N27D showed reduced strand transfer in in vitro assays. Double and triple mutants F16W/I24Q, F16W/N27D, and F16W/I24Q/N27D all showed defects in DNA binding, strand transfer, and helix destabilization, suggesting that the I24Q and N27D mutations have a dominant negative effect and abolish the positive influence of F16W. Results show that amino acid differences at positions 24 and 27 contribute significantly to finger one's helix destabilizing activity.     

Remote homologue identification of Drosophila GAGA factor in mouse

GAGA factor (GAF) is involved in both gene activation and gene repression and plays a role in the modulation of chromatin structure. In Drosophila, Trithroax like (Trl) gene encodes the DNA binding protein called GAGA factor (GAF). Trl-GAF binds to GAGA sites through its C2H2 zinc finger domain and has an N-terminal BTB/POZ domain. Identification of Trl-GAF homologue in mouse helps in deeper understanding of the mechanism and function. Conventional alignment tools such as BLAST and FASTA cannot identify homologues in mouse genome as their sequence identity is below 30%. In the present study, various sequence and structure analyses were followed for the detection of remote homologues of Drosophila GAGA FACTOR in mouse to identify as Zbtb3. Through homology modeling and docking approach, the zinc finger region of mouse Zbtb3 showed conserved residues and favorable DNA binding sites with GAGA sites similar to that of Drosophila GAGA FACTOR.     

Functional analysis of the two zinc fingers of SRE, a GATA-type factor that negatively regulates siderophore synthesis in Neurospora crassa

Multiple GATA factors, zinc finger DNA binding proteins that recognize consensus GATA elements, exist in Neurospora crassa. One of them, SRE, is involved in controlling the iron metabolic pathway of N. crassa. In N. crassa, iron transport is mediated by a number of small cyclic peptides, known as siderophores. The siderophore synthesis pathway is negatively regulated by SRE; a loss-of-function sre mutant strain showed partial constitutive synthesis of siderophore. In the research presented here, the negative function of SRE was further confirmed by a heterokaryon test and by gene complementation. SRE was expressed as a GST fusion protein. In vitro EMSA revealed that SRE binds specifically to DNA molecules containing GATA sequence elements. Autoregulation of sre gene expression appears possible because the sre gene promoter itself contains GATA sequences. Mutations were introduced into sre that lead to amino acid substitutions in each of the zinc fingers that will disrupt their function. In vitro EMSA revealed that both N-terminal and C-terminal zinc fingers of SRE are involved in DNA binding. This feature is different from that found with the vertebrate two zinc finger GATA factors. Invivo tests, accomplished by transforming the mutant sre genes into sre rip mutant, showed that SRE with mutations in either or both zinc fingers still maintained its function under low-iron conditions. In contrast, these mutant SRE proteins fail to function under high-iron conditions. Our results predict the presence of other positive or negative regulators of the siderophore synthetic pathway.     

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.     

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.     

Cooperative, non-specific binding of a zinc finger peptide to DNA.

The DNA binding and structural properties of Xfin-31 (Lee, M.S., Gippert, G.P., Soman, K.V., Case, D.A. and Wright, P.E., 1989, Science 245, 635-637), a twenty five amino acid zinc finger peptide, in the reduced, oxidized and zinc complex forms, as well as the fourteen residue helical segment of the zinc finger (residues 12-25) have been compared using affinity coelectrophoresis (ACE) and circular dichroism (CD) spectroscopy. The zinc complex and oxidized peptides bind cooperatively to DNA although the cooperativity factor, omega, is more than 15-fold greater for the zinc complex. The reduced peptide in the absence of zinc and the helical segment do not bind cooperatively (omega = 1). Hence, the binding constant for singly contiguous sites (K omega) ranges over 100-fold for the various peptides even though the intrinsic binding constants (K) are similar. An increase in binding order and affinity for the other forms of Xfin-31 is correlated with an increasing similarity of the CD spectrum to that of the Xfin-31 zinc complex. The surprising DNA binding activity of the oxidized peptide may result from hydrophobic interactions between the amino-terminal loop formed by the Cys3-Cys6 disulfide bond and conserved hydrophobic residues in the carboxyl-terminal segment. Xfin-31 may be a particularly useful model for studying several poorly understood aspects of cooperative, non-specific DNA binding since it is small, has a stable, well-defined structure, and structures of zinc fingers bound to DNA have been determined.