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PubMed2003

Structural basis for DNA recognition by the basic region leucine zipper transcription factor CCAAT/enhancer-binding protein alpha.

CCAAT/enhancer-binding proteins (C/EBPs) are basic region leucine zipper (bZIP) transcription factors that regulate cell differentiation, growth, survival, and inflammation. To understand the molecular basis of DNA recognition by the C/EBP family we determined the x-ray structure of a C/EBPalpha bZIP polypeptide bound to its cognate DNA site (A(-5)T(-4)T(-3)G(-2)C(-1)G(1)C(2)A(3)A(4)T(5)) and characterized several basic region mutants. Binding specificity is provided by interactions of basic region residues Arg(289), Asn(292), Ala(295), Val(296), Ser(299), and Arg(300) with DNA bases. A striking feature of the C/EBPalpha protein-DNA interface that distinguishes it from known bZIP-DNA complexes is the central role of Arg(289), which is hydrogen-bonded to base A(3), phosphate, Asn(292) (invariant in bZIPs), and Asn(293). The conformation of Arg(289) is also restricted by Tyr(285). In accordance with the structural model, mutation of Arg(289) or a pair of its interacting partners (Tyr(285) and Asn(293)) abolished C/EBPalpha binding activity. Val(296) (Ala in most other bZIPs) contributes to C/EBPalpha specificity by discriminating against purines at position -3 and imposing steric restraints on the invariant Arg(300). Mutating Val(296) to Ala strongly enhanced C/EBPalpha binding to cAMP response element (CRE) sites while retaining affinity for C/EBP sites. Thus, Arg(289) is essential for formation of the complementary protein-DNA interface, whereas Val(296) functions primarily to restrict interactions with related sequences such as CRE sites rather than specifying binding to C/EBP sites. Our studies also help to explain the phenotypes of mice carrying targeted mutations in the C/EBPalpha bZIP region.

Amino Acid Sequence↗
PubMed1998

DNA recognition by quinoxaline antibiotics: use of base-modified DNA molecules to investigate determinants of sequence-specific binding of triostin A and TANDEM.

The methodology of DNAase I footprinting has been adapted to investigate the sequence-specific binding of two quinoxaline drugs to DNA fragments containing natural and modified bases. In order to help comprehend the molecular origin of selectivity in the bis-intercalation of triostin A and TANDEM at CpG and TpA sites respectively, we have specifically examined the effect of the 2-amino group of guanine on their sequence specificity by using DNA in which that group has been either removed from guanine, added to adenine or both. Previous studies suggested that the recognition of particular nucleotide sequences by these drugs might be dependent upon the placement of the purine 2-amino group, serving as a positive or a negative effector for triostin A and TANDEM respectively. However, the footprinting data reported here indicate that this is not entirely correct, since they show that the 2-amino group of guanine is important for the binding of triostin A to DNA but has absolutely no influence on the interaction of TANDEM with TpA steps. Apparently the binding of triostin A to CpG sites is primarily due to hydrogen bonding interaction between the cyclic peptide of the antibiotic and the 2-amino group of guanine residues, whereas the selective binding of TANDEM to TpA sites is not hydrogen-bond driven and probably originates mainly from steric and/or hydrophobic interactions, perhaps involving indirect recognition of a suitable minor groove structure.

Anti-Bacterial Agents↗
PubMed2004

Specific DNA recognition by the Antp homeodomain: MD simulations of specific and nonspecific complexes.

Four molecular dynamics simulation trajectories of complexes between the wild-type or a mutant Antennapedia homeodomain and 2 DNA sequences were generated in order to probe the mechanisms governing the specificity of DNA recognition. The starting point was published affinity measurements showing that a single protein mutation combined with a replacement of 2 base pairs yields a new high-affinity complex, whereas the other combinations, with changes on only 1 macromolecule, exhibited lower affinity. The simulations of the 4 complexes yielded fluctuating networks of interaction. On average, these networks differ significantly, explaining the switch of affinity caused by the alterations in the macromolecules. The network of mostly hydrogen-bonding interactions involving several water molecules, which was suggested both by X-ray and NMR structures of the wild-type homeodomain and its DNA operator sequence, could be reproduced in the trajectory. More interestingly, the high-affinity complex with alterations in both the protein and the DNA yielded again a dynamic but very tight network of intermolecular interactions, however, attributing a significantly stronger role to direct hydrophobic interactions at the expense of water bridges. The other 2 homeodomain-DNA complexes, with only 1 molecule altered, show on average over the trajectories a clearly reduced number of protein-DNA interactions. The observations from these simulations suggest specific experiments and thus close the circle formed by biochemical, structural, and computational studies. The shift from a water-dominated to a more "dry" interface may prove important in the design of proteins binding DNA in a specific manner.

Antennapedia Homeodomain Protein↗
PubMed1990

The binding of transformed aromatic hydrocarbon (Ah) receptor to its DNA recognition site is not affected by metal depletion.

The biological effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin), a potent environmental contaminant, are mediated by a soluble intracellular protein, the aromatic hydrocarbon (Ah) receptor (AhR). TCDD:AhR complexes activate gene transcription by binding to specific DNA sequences termed dioxin-responsive elements adjacent to TCDD-responsive genes. Analogies between the AhR and receptors for steroid hormones imply similarities in their mechanism of action. The presence of chelatable, protein-bound metal(s), presumably zinc, is required for DNA binding of several proteins, including steroid hormone receptors and the transcription factor SP1. Utilizing gel retardation and DNA-cellulose binding assays we have investigated the importance of metal in DNA binding of transformed TCDD:AhR complexes. Here, we report that although 1,10-phenanthroline, a metal ion chelating agent, inhibited the DNA binding of SP1 and transformed glucocorticoid receptor, no inhibition of transformed AhR was observed. EDTA was similarly ineffective in inhibiting DNA binding of transformed AhR. Our findings suggest that the AhR, although similar to steroid receptors, appears not to require metals for binding to its specific DNA recognition sequence.

Animals↗
PubMed2007

DNA recognition mechanism of the ONECUT homeodomain of transcription factor HNF-6.

Hepatocyte nuclear factor-6 (HNF-6), a liver-enriched transcription factor, controls the development of various tissues, such as the pancreas and liver, and regulates the expression of several hepatic genes. This protein belongs to the ONECUT class of homeodomain proteins and contains a bipartite DNA-binding domain composed of a single cut domain and a characteristic homeodomain. This transcription factor has two distinct modes of DNA binding and transcriptional activation that use different coactivators depending on the target gene. The crystal structure of the bipartite DNA-binding domain of HNF-6alpha complexed with the HNF-6-binding site of the TTR promoter revealed the DNA recognition mechanism of this protein. Comparing our structure with the DNA-free structure of HNF-6 or the structure of Oct-1, we discuss characteristic features associated with DNA binding and the structural basis for the dual mode of action of this protein, and we suggest a strategy for variability of transcriptional activation of the target gene.

Amino Acid Sequence↗
PubMed1998

Mutational analysis of human DNase I at the DNA binding interface: implications for DNA recognition, catalysis, and metal ion dependence.

Human deoxyribonuclease I (DNase I), an enzyme used to treat cystic fibrosis patients, has been systematically analyzed by site-directed mutagenesis of residues at the DNA binding interface. Crystal structures of bovine DNase I complexed with two different oligonucleotides have implicated the participation of over 20 amino acids in catalysis or DNA recognition. These residues have been classified into four groups based on the characterization of over 80 human DNase I variants. Mutations at any of the four catalytic amino acids His 134, His 252, Glu 78, and Asp 212 drastically reduced the hydrolytic activity of DNase I. Replacing the three putative divalent metal ion-coordinating residues Glu 39, Asp 168, or Asp 251 led to inactive variants. Amino acids Gln 9, Arg 41, Tyr 76, Arg 111, Asn 170, Tyr 175, and Tyr 211 were also critical for activity, presumably because of their close proximity to the active site, while more peripheral DNA interactions stemming from 13 other positions were of minimal significance. The relative importance of these 27 positions is consistent with evolutionary relationships among DNase I across different species, DNase I-like proteins, and bacterial sphingomyelinases, suggesting a fingerprint for a family of DNase I-like proteins. Furthermore, we found no evidence for a second active site that had been previously implicated in Mn2+-dependent DNA degradation. Finally, we correlated our mutational analysis of human DNase I to that of bovine DNase I with respect to their specific activity and dependence on divalent metal ions.

Animals↗
PubMed1992

Application of L-DNA to the study of the specific DNA recognition mechanism of bleomycin.

The hexadeoxynucleotide analog, L-d(CGCGCG) composed of L-deoxyribose was synthesized and clearly shown to have the same conformation and dynamic properties with natural D-d(CGCGCG) except for chirality with CD spectra. This unnatural hexanucleotide was not cleaved by bleomycin, an antitumor DNA cleaving drug, but was able to bind to the DNA binding domain of bleomycin to a similar extent with the natural one. These results strongly suggest the importance of the other moiety than the DNA binding domain for the specific DNA recognition of bleomycin. Thus, L-oligonucleotides are useful for the study of DNA-drug interactions.

Binding Sites↗
PubMed2000

Thermodynamic and kinetic analyses for understanding sequence-specific DNA recognition.

Thermodynamic and kinetic analyses of biomolecular interactions reveal details of the energetic and dynamic features of molecular recognition processes, and complement structural analyses of the free and complexed conformations. The recent improvements in both isothermal titration calorimetry and surface plasmon resonance sensoring provide powerful tools for analysing biomolecular interactions in thermodynamic and kinetic approaches. The thermodynamic and kinetic parameters obtained for binding between protein and DNA indicate the mechanism of specific DNA recognition, in the high-resolution structures of the protein-DNA complexes. The effects of temperature and ionic strength reflect the conformational changes of the protein and DNA molecules upon complex formation, including important contributions of water and solutes. When combined with mutational studies, the interactions can be reduced to several energetic contributions from individual contacts. These studies should be useful to determine general features of protein functions in genetic regulation.

Animals↗
PubMed1996

In the TTF-1 homeodomain the contribution of several amino acids to DNA recognition depends on the bound sequence.

The thyroid transcription factor-1 homeodomain (TTF-1HD) shows a peculiar DNA binding specificity, preferentially recognizing sequences containing the 5'-CAAG-3' core motif. Most other homeodomains instead recognize sites containing the 5'-TAAT-3' core motif. Here, we show that TTF-1HD efficiently recognizes another sequence, called D1, devoid of the 5'-CAAG-3' core motif. Different experimental approaches indicate that TTF-1HD contacts the D1 sequence in a manner which is different to that used to interact with sequences containing the 5'-CAAG-3' core motif. The binding activities that mutants of TTF-1HD display with the D1 sequence or with the sequence containing the 5'-CAAG-3' core motif indicate that the role of several DNA-contacting amino acids is different. In particular, during recognition of the D1 sequence, backbone-interacting amino acids not relevant in binding to sequences containing the 5'-CAAG-3' core motif play an important role. In the TTF-1HD, therefore, the contribution of several amino acids to DNA recognition depends on the bound sequence. These data indicate that although a common bonding network exists in all of the HD/DNA complexes, peculiarities important for DNA recognition may occur in single cases.

Amino Acids↗
PubMed2026

VIPR RNA-guided DNA recognition by noncontiguous geometric triplex formation.

Viral interference programmable repeat (VIPR) systems use a noncontiguous code for RNA-guided transcriptional silencing. How the Vipr protein and a VIPR RNA (vrRNA) comprising alternating GGY and NN segments achieve precise DNA targeting is unknown. Here, we present 21 cryo-electron microscopy structures that help explain the mechanism of target engagement. Vipr protomers oligomerize along the vrRNA to form a right-handed helical filament, sequestering each GGY motif and positioning the adjacent NN bases for target base pairing. DNA binding, in which every third nucleotide is skipped, results in a gapped vrRNA-DNA hybrid helix that encircles the nontarget DNA strand to form a geometric triplex. These findings suggest that triplex-mediated target-strand handoff could enable noncontiguous and programmable RNA-guided DNA recognition in VIPR systems.

DNA↗
PubMed1995

Role of the N terminus in DNA recognition by the v-erb A protein, an oncogenic derivative of a thyroid hormone receptor.

The v-erb A oncogene is a mutated derivative of a normal cellular locus (c-erb A alpha) encoding a thyroid hormone receptor. Although both the v-erb A protein and thyroid hormone receptor bind to DNA, the DNA sequence specificity of the viral oncoprotein is altered from that of the normal cellular receptor. Intriguingly, amino acid differences in both the zinc-finger domain and in a less-characterized N-terminal region of the v- and c-erb A polypeptides are jointly responsible for these differences in DNA specificity. We demonstrate here that this newly recognized N-terminal determinant of DNA specificity appears to function by restricting the DNA sequence repertoire manifested by the zinc-finger domain itself. The unique presence of a tyrosine in the N terminus of the thyroid hormone receptor acts to abrogate this restriction, thereby permitting this receptor to utilize response elements containing nonconsensus half-sites. The ability of the N terminus to modulate the DNA recognition properties of nuclear hormone receptors extends to the retinoic acid receptor and contributes to the distinct DNA specificities displayed by the retinoic acid and thyroid hormone receptors.

Amino Acid Sequence↗
PubMed2002

Tandem DNA recognition by PhoB, a two-component signal transduction transcriptional activator.

PhoB is a signal transduction response regulator that activates nearly 40 genes in phosphate depletion conditions in E. coli and closely related bacteria. The structure of the PhoB effector domain in complex with its target DNA sequence, or pho box, reveals a novel tandem arrangement in which several monomers bind head to tail to successive 11-base pair direct-repeat sequences, coating one face of a smoothly bent double helix. The protein has a winged helix fold in which the DNA recognition elements comprise helix alpha 3, penetrating the major groove, and a beta hairpin wing interacting with a compressed minor groove via Arg219, tightly sandwiched between the DNA sugar backbones. The transactivation loops protrude laterally in an appropriate orientation to interact with the RNA polymerase sigma(70) subunit, which triggers transcription initiation.

Amino Acid Sequence↗
PubMed2000

DNA recognition of a 24-mer peptide derived from RecA protein.

A novel 24-residue peptide (L2-G), Ile-Arg-Met-Lys-Ile-Gly-Val-Met-Phe-Gly-Asn-Pro-Glu-Thr-Thr-Thr-Gly-Gly-Asn-Ala-Leu-Lys-Phe-Tyr, derived from RecA can discriminate a single-stranded DNA (ssDNA) from a double-stranded DNA (dsDNA) and a new developed support with this peptide recognizes not dsDNA but ssDNA. The 24-mer peptide with L2 and helix G amino acids of Escherichia coli RecA protein showed the ssDNA binding property with more than 1000 times affinity difference for the dsDNA. However, truncated 15-mer peptide showed no ssDNA binding activity. In the ssDNA binding, L2-G changed its conformation with the perturbation of an alpha-helix structure. The ssDNA binding and the DNA discrimination property of this peptide were due to almost all L2 and helix G amino acids, respectively. This result is useful to design synthetic peptides as functional materials for DNA recognition.

Amino Acid Sequence↗
PubMed2006

DNA recognition by the brinker repressor--an extreme case of coupling between binding and folding.

The Brinker (Brk) nuclear repressor is a major element of the Drosophila Decapentaplegic morphogen signaling pathway. Its N-terminal part has weak homology to the Antennapedia homeodomain and binds to GC-rich DNA sequences. We have investigated the conformation and dynamics of the N-terminal 101 amino acid residues of Brk in the absence and in the presence of cognate DNA by solution NMR spectroscopy. In the absence of DNA, Brk is unfolded and highly flexible throughout the entire backbone. Addition of cognate DNA induces the formation of a well-folded structure for residues R46 to R95. This structure consists of four helices forming a helix-turn-helix motif that differs from homeodomains, but has similarities to the Tc3 transposase, the Pax-6 Paired domain, and the human centromere-binding protein. The GC-rich DNA recognition can be explained by specific major groove hydrogen bonds from the N-terminal end of helix alpha3. The transition from a highly flexible, completely unfolded conformation in the absence of DNA to a well-formed structure in the complex presents a very extreme case of the "coupling of binding and folding" phenomenon.

Amino Acid Sequence↗
PubMed1991

Beyond zinc fingers: steroid hormone receptors have a novel structural motif for DNA recognition.

The highly conserved DNA-binding domain of the steroid hormone receptors contains two 'zinc finger'-like sequence motifs. The three-dimensional structure in solution has been determined using two-dimensional 1H nuclear magnetic resonance (NMR) spectroscopy and shows that the two 'zinc finger'-like motifs fold to form a single structural domain. The combination of this structural information and mutagenesis data reveals how this family of transcriptional regulators bind to DNA.

Amino Acid Sequence↗
PubMed1991

Metal binding and DNA recognition in transcription factor Sp1.

DNA binding domain of Sp1 encompassing three Cys2His2-type Zn-finger motifs is cloned and expressed in E.coli. The Sp1 fragment shows metal-dependent folding and DNA binding. The Zn(II)-induced folding of the three fingers is probably cooperative. Release of one equivalent of Zn decreases but does not abolish DNA binding activity of Sp1.

Amino Acid Sequence↗
PubMed2001

Studies on the molecular mechanism of the interactions between the cyanobacterial transcription factor, SmtB, and its recognition DNA sequences.

Tolerance to the heavy-metal ion stress in cyanobacterial cell is regulated by the transcriptional repressor SmtB that senses the Zn/Cd concentration in the cell. SmtB regulated the transcriptional level of class II metallothionein, SmtA. There are two recognition DNA sequences (Bbs1 and Bbs2) in the operator/promoter region of smtA gene. To clear the functional meaning of the presence of these two sequences, we have compared the affinities of native and point-mutated SmtBs to these two sites. We also have compared the sizes of the protein-DNA complexes being formed with these two sites. SmtB forms protein-DNA complex in an unique size with Bbs1, in three different sizes with Bbs2. We have further designed the modified Bbs1 and Bbs2 sequences, and the results obtained with these sequences indicate that the differences observed between the cases of Bbs1 and Bbs2 are caused by the presence of direct repeat sequence and the differences in the linker sequences.

Bacterial Proteins↗
PubMed2003

AINTEGUMENTA utilizes a mode of DNA recognition distinct from that used by proteins containing a single AP2 domain.

The Arabidopsis protein AINTEGUMENTA (ANT) is an important regulator of organ growth during flower development. ANT is a member of the AP2 subclass of the AP2/ERF family of plant-specific transcription factors. These proteins contain either one or two copies of a DNA-binding domain called the AP2 domain. Here, it is shown that ANT can act as a transcriptional activator in yeast through binding to a consensus ANT-binding site. This activity was used as the basis for a genetic screen to identify amino acids that are critical for the DNA binding ability of ANT. Mutants that showed reduced or no activation of a reporter gene under the control of ANT-binding sites were identified. The mutations identified in the screen as well as additional site-directed mutations suggest that the mode of DNA recognition by members of the AP2 subfamily is distinct from that of ERF proteins. Surprisingly, it appears that each AP2 domain of ANT uses different amino acids to contact DNA. Identification of several linker mutations argues that this sequence acts in the positioning of each AP2 domain on the DNA or makes direct DNA contacts.

Amino Acid Sequence↗