Chemical shift as a probe of molecular interfaces: NMR studies of DNA binding by the three amino-terminal zinc finger domains from transcription factor IIIA

We report the NMR resonance assignments for a macromolecular protein/DNA complex containing the three amino-terminal zinc fingers (92 amino acid residues) of Xenopus laevis TFIIIA (termed zf1-3) bound to the physiological DNA target (15 base pairs), and for the free DNA. Comparisons are made of the chemical shifts of protein backbone 1HN, 15N, 13C alpha and 13C beta and DNA base and sugar protons of the free and bound species. Chemical shift changes are analyzed in the context of the structures of the zf1-3/DNA complex to assess the utility of chemical shift change as a probe of molecular interfaces. Chemical shift perturbations that occur upon binding in the zf1-3/DNA complex do not correspond directly to the structural interface, but rather arise from a number of direct and indirect structural and dynamic effects.     

Structural determinants in 5S RNA and TFIIIA for 7S RNP formation

C2H2-type zinc-finger modules define a unique structural motif, which is capable of forming specific complexes with both DNA and RNA. While the principles governing DNA binding have been defined in great detail, the mode of RNA recognition remains only poorly understood. In the absence of information from three-dimensional structural analysis of a zinc-finger/RNA complex, we have performed a number of biochemical studies to gain further insight into the molecular details of the interaction of 5S ribosomal RNA with the zinc-finger protein TFIIIA. Previous work had indicated that zinc finger 6 of TFIIIA contacts 5S RNA in close proximity or directly in the loop-A region (nucleotides 10-13). Permutation analysis of this sequence reveals that three of the four nucleotides are of vital importance for RNA recognition. Exchange of unusual and therefore characteristic aromatic residues in finger 6 against aliphatic or other aromatic amino acids reveals that the aromatic character of tryptophan 177 is essential for RNA recognition. Association with helix V in 5S RNA appears to involve specific contacts with the phosphate backbone, as evidenced by ethylation-interference assays. Introduction of multiple internal and 3'-terminal as well as 5'-terminal deletions accompanied by stabilizing sequence substitutions defines a minimal RNA fragment that is sufficient for TFIIIA binding. This RNA molecule includes a truncated/mutated helix I, helix II and helix V, as well as structurally intact loops A and E. Permutation analysis of the loop-E region emphasizes its importance for TFIIIA recognition.     

Toward a code for the interactions of zinc fingers with DNA: selection of randomized fingers displayed on phage

We have used two selection techniques to study sequence-specific DNA recognition by the zinc finger, a small, modular DNA-binding minidomain. We have chosen zinc fingers because they bind as independent modules and so can be linked together in a peptide designed to bind a predetermined DNA site. In this paper, we describe how a library of zinc fingers displayed on the surface of bacteriophage enables selection of fingers capable of binding to given DNA triplets. The amino acid sequences of selected fingers which bind the same triplet are compared to examine how sequence-specific DNA recognition occurs. Our results can be rationalized in terms of coded interactions between zinc fingers and DNA, involving base contacts from a few alpha-helical positions. In the paper following this one, we describe a complementary technique which confirms the identity of amino acids capable of DNA sequence discrimination from these positions.     

A mutation outside the two zinc fingers of ADR1 can suppress defects in either finger

A second-site mutation that restored DNA binding to ADR1 mutants altered at different positions in the two zinc fingers was identified. This mutation (called IS1) was a conservative change of arginine 91 to lysine in a region amino terminal to the two zinc fingers and known from previous experiments to be necessary for DNA binding. IS1 increased binding to the UAS1 sequence two- to sevenfold for various ADR1 mutants and twofold for wild-type ADR1. The change of arginine 91 to glycine decreased binding twofold, suggesting that this arginine is involved in DNA binding in the wild-type protein. The increase in binding by IS1 did not involve protein-protein interactions between the two ADR1 monomers, nor did it require the presence of the sequences flanking UAS1. However, the effect of IS1 was influenced by the sequence of the first finger, suggesting that interactions between the region amino terminal to the fingers and the fingers themselves could exist. A model for the role of the amino-terminal region based on these results and sequence homologies with other DNA-binding motifs is proposed.     

Polyomavirus large T antigen binds cooperatively to its multiple binding sites in the viral origin of DNA replication

Polyomavirus large T antigen binds to multiple 5'-G(A/G)GGC-3' pentanucleotide sequences in sites 1/2, A, B, and C within and adjacent to the origin of viral DNA replication on the polyomavirus genome. We asked whether the binding of large T antigen to one of these sites could influence binding to other sites. We discovered that binding to origin DNA is substantially stronger at pH 6 to 7 than at pH 7.4 to 7.8, a range often used in DNA binding assays. Large T antigen-DNA complexes formed at pH 6 to 7 were stable, but a fraction of these complexes dissociated at pH 7.6 and above upon dilution or during electrophoresis. Increased binding at low pH is therefore due at least in part to increased stability of protein-DNA complexes, and binding at higher pH values is reversible. Binding to fragments of origin DNA in which one or more sites were deleted or inactivated by point mutations was measured by nitrocellulose filter binding and DNase I footprinting. The results showed that large T antigen binds cooperatively to its four binding sites in viral DNA, suggesting that the binding of this protein to one of these sites stabilizes its binding to other sites via protein-protein contacts. Sites A, B, and C may therefore augment DNA replication by facilitating the binding of large T antigen to site 1/2 at the replication origin. ATP stabilized large T antigen-DNA complexes against dissociation in the presence, but not the absence, of site 1/2, and ATP specifically enhanced protection against DNase I digestion in the central 10 to 12 bp of site 1/2, at which hexamers are believed to form and begin unwinding DNA. We propose that large T antigen molecules bound to these multiple sites on origin DNA interact with each other to form a compact protein-DNA complex and, furthermore, that ATP stimulates their assembly into hexamers at site 1/2 by a "handover" mechanism mediated by these protein-protein contacts.     

Cooperative assembly of the bovine papilloma virus E1 and E2 proteins on the replication origin requires an intact E2 binding site

Using quantitative gel retardation assays the properties of the bovine papilloma virus (BPV) origin recognition protein E1 and the effect of the viral E2 protein on the binding of E1 to BPV origin DNA were examined. As reported previously (Seo, Y.S., Mueller, F., Lusky, M., Gibbs, E., Kim, H.-Y., Phillips, B. and J. Hurwitz (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 2865-2869), the E1 protein binds specifically to DNA sequences within the BPV origin (ori+) of replication. We also show that the presence of MgCl2 and ATP could stabilize the E1 ori+ DNA complex. At low levels of E1, ori+ DNA binding was greatly stimulated by the viral E2 protein when the intact E2 binding site 12 was present on the DNA. In addition DNA-protein complexes formed in the presence of both E1 and E2 were more stable than those formed with E1 alone. In the absence of an E2 binding site the E2 protein inhibited the binding of E1 to the BPV origin. Spacing of 0 or 9 base pairs between the E1 binding site and the E2 binding site 12 abolished the stimulation of E1-DNA binding by E2, whereas spacing of 6 base pairs between the two binding sites allowed for efficient stimulation. The data presented account for a direct role of E2 in BPV DNA replication. We propose that the cooperative binding of both the E1 and E2 proteins to BPV ori+ DNA is mediated by protein-protein interactions and by protein-DNA interactions, which include the formation of specific contacts of E2 with DNA.     

An analysis of the relationship between hydration and protein-DNA interactions

Eleven protein-DNA crystal structures were analyzed to test the hypothesis that hydration sites predicted in the first hydration shell of DNA mark the positions where protein residues hydrogen-bond to DNA. For nine of those structures, protein atoms, which form hydrogen bonds to DNA bases, were found within 1.5 A of the predicted hydration positions in 86% of the interactions. The correspondence of the predicted hydration sites with the hydrogen-bonded protein side chains was significantly higher for bases inside the conserved DNA recognition sequences than outside those regions. In two CAP-DNA complexes, predicted base hydration sites correctly marked 71% (within 1.5 A) of protein atoms, which form hydrogen bonds to DNA bases. Phosphate hydration was compared to actual protein binding sites in one CAP-DNA complex with 78% marked contacts within 2.0 A. These data suggest that hydration sites mark the binding sites at protein-DNA interfaces.     

Quantitative parameters for amino acid-base interaction: implications for prediction of protein-DNA binding sites

Inspection of the amino acid-base interactions in protein-DNA complexes is essential to the understanding of specific recognition of DNA target sites by regulatory proteins. The accumulation of information on protein-DNA co-crystals challenges the derivation of quantitative parameters for amino acid-base interaction based on these data. Here we use the coordinates of 53 solved protein-DNA complexes to extract all non-homologous pairs of amino acid-base that are in close contact, including hydrogen bonds and hydrophobic interactions. By comparing the frequency distribution of the different pairs to a theoretical distribution and calculating the log odds, a quantitative measure that expresses the likelihood of interaction for each pair of amino acid-base could be extracted. A score that reflects the compatibility between a protein and its DNA target can be calculated by summing up the individual measures of the pairs of amino acid-base involved in the complex, assuming additivity in their contributions to binding. This score enables ranking of different DNA binding sites given a protein binding site and vice versa and can be used in molecular design protocols. We demonstrate its validity by comparing the predictions using this score with experimental binding results of sequence variants of zif268 zinc fingers and their DNA binding sites.     

Structure of a histidine-X4-histidine zinc finger domain: insights into ADR1-UAS1 protein-DNA recognition

The solution structure for a mutant zinc finger peptide based on the sequence of the C-terminal ADR1 finger has been determined by two-dimensional NMR spectroscopy. The mutant peptide, called PAPA, has both proline residues from the wild-type sequence replaced with alanines. A nonessential cysteine was also replaced with alanine. The behavior of PAPA in solution implicates the prolines in the conformational heterogeneity reported earlier for the wild-type peptide [Xu, R. X., Horvath, S. J., & Klevit, R. E. (1991) Biochemistry 30, 3365-3371]. The solution structure of PAPA reveals several interesting features of the zinc finger motif. The residue immediately following the second cysteine ligand adopts a positive phi angle, which we propose is a common feature of this class of zinc fingers, regardless of whether this residue is a glycine. The NMR spectrum and resulting solution structure of PAPA suggest that a side-chain to side-chain hydrogen bond involving an arginine and an aspartic acid analogous to one observed in the Zif268 protein-DNA cocrystal structure exists in solution in the absence of DNA [Pavletich, N. P., & Pabo, C. O. (1991) Science 252, 809-817]. A model for the interaction between the two ADR1 zinc fingers and their DNA binding sites was built by superpositioning the refined solution structures of PAPA and ADR1b onto the Zif268 structure. This model offers structural explanations for a variety of mutations to the ADR1 zinc finger domains that have been shown to affect DNA-binding affinity or specificity.