Winged Helix DNA-binding Protein Research Articles

Targeting the forkhead box protein P1 pathway as a novel therapeutic approach for cardiovascular diseases.

Cardiovascular disease (CVD) is the leading cause of death worldwide and encompasses diverse diseases of the vasculature, myocardium, cardiac electrical circuit, and cardiac development. Forkhead box protein P1 (Foxp1) is a large multi-domain transcriptional regulator belonging to the Fox family with winged helix DNA-binding protein, which plays critical roles in cardiovascular homeostasis and disorders. The broad distribution of Foxp1 and alternative splicing isoforms implicate its distinct functions in diverse cardiac and vascular cells and tissue types. Foxp1 is essential for diverse biological processes and has been shown to regulate cellular proliferation, apoptosis, oxidative stress, fibrosis, angiogenesis, cardiovascular remodeling, and dysfunction. Notably, both loss-of-function and gain-of-function approaches have defined critical roles of Foxp1 in CVD. Genetic deletion of Foxp1 results in pathological cardiac remodeling, exacerbation of atherosclerotic lesion formation, prolonged occlusive thrombus formation, severe cardiac defects, and embryo death. In contrast, activation of Foxp1 performs a wide range of physiological effects, including cell growth, hypertrophy, differentiation, angiogenesis, and cardiac development. More importantly, Foxp1 exerts anti-inflammatory and anti-atherosclerotic effects in controlling coronary thrombus formation and myocardial infarction (MI). Thus, targeting for Foxp1 signaling has emerged as a pre-warning biomarker and a novel therapeutic approach against progression of CVD, and an increased understanding of cardiovascular actions of the Foxp1 signaling will help to develop effective interventions. In this review, we focus on the diverse actions and underlying mechanisms of Foxp1 highlighting its roles in CVD, including heart failure, MI, atherosclerosis, congenital heart defects, and atrial fibrillation.

Alanine substitution mutations in the DNA binding region of a global staphylococcal virulence regulator affect its structure, function, and stability

SarA, a winged-helix DNA binding protein, is a global virulence regulator in Staphylococcus aureus. The putative DNA binding region of SarA is located between amino acid residues Leu 53 and Gln 97. Previous studies have demonstrated that residues at positions 84, 88, 89, and 90 are critical for its function. To precisely understand the roles of the DNA binding residues, we have investigated nine mutants of a recombinant SarA (rSarA) along with the rSarA mutants carrying mutations at the above four positions. Of the thirteen mutants, eleven mutants show weaker DNA binding activity in vitro compared to rSarA. As noted earlier, the DNA binding affinity of rSarA was maximally affected due to the mutation at position 84 or 90. Each of the functionally-defective mutants also possesses an altered structure and stability. Additionally, the mutations at positions 84 and 90 have severely affected the formation of hydrogen (H) bonds at the interface between SarA and the cognate DNA. The mutation at position 64 also has perturbed the generation of some interface H-bonds. Therefore, the disruption of H-bonds in the protein-DNA interface and the structural alteration in the protein may be responsible for the reduced DNA binding activity of the mutants.

Determining the Roles of a Conserved α-Helix in a Global Virulence Regulator from Staphylococcus aureus.

SarA, a pleiotropic transcription regulator, is encoded by Staphylococcus aureus, a pathogenic bacterium. The expression of many virulence and non-virulence genes in S. aureus is modulated by this regulator. Structural studies have shown it to be a winged-helix DNA-binding protein carrying two monomers. Each SarA monomer is composed of five α-helices (α1-α5), three β-strands (β1-β3) and multiple loops. The putative DNA binding region of SarA is constituted with α3, α4, β2, and β3, whereas, its dimerization seems to occur using α1, α2, and α5. Interestingly, many SarA-like proteins are dimeric and use three or more helices for their dimerization. To clearly understand the roles of helix α1 in the dimerization, we have constructed and purified a SarA mutant (Δα1) that lacks helix α1. Our in-depth studies with Δα1 indicate that the helix α1 is critical for preserving the structure, DNA binding activity and thermodynamic stability of SarA. However, the helix has little affected its dimerization ability. Possible reasons for such anomaly have been discussed at length.

The crystal structure of AphB, a virulence gene activator from Vibrio cholerae, reveals residues that influence its response to oxygen and pH

Expression of the two critical virulence factors of Vibrio cholerae, toxin-coregulated pilus and cholera toxin, is initiated at the tcpPH promoter by the regulators AphA and AphB. AphA is a winged helix DNA-binding protein that enhances the ability of AphB, a LysR-type transcriptional regulator, to activate tcpPH expression. We present here the 2.2 Å X-ray crystal structure of full-length AphB. As reported for other LysR-type proteins, AphB is a tetramer with two distinct subunit conformations. Unlike other family members, AphB must undergo a significant conformational change in order to bind to DNA. We have found five independent mutations in the putative ligand-binding pocket region that allow AphB to constitutively activate tcpPH expression at the non-permissive pH of 8.5 and in the presence of oxygen. These findings indicate that AphB is responsive to intracellular pH as well as to anaerobiosis and that residues in the ligand-binding pocket of the protein influence its ability to respond to both of these signals. We have solved the structure of one of the constitutive mutants, and observe conformational changes that would allow DNA binding. Taken together, these results describe a pathway of conformational changes allowing communication between the ligand and DNA binding regions of AphB.

The Crystal Structure of AphB, a Virulence Gene Activator from Vibrio Cholerae, Reveals Residues that Influence its Response to Oxygen and pH

For Vibrio cholerae O1 to cause disease, it must express two critical virulence factors: the toxin-coregulated pilus and cholera toxin. Expression of these virulence factors is controlled by a transcriptional cascade initiated at the tcpPH promoter by the regulatory proteins AphA and AphB. AphA is a winged helix DNA-binding protein that enhances the ability of AphB, a LysR-type transcriptional regulator, to activate expression of the tcpPH promoter. We present here the 2.2 A X-ray crystal structure of full-length AphB. As reported for several other LysR-type proteins, AphB is a tetramer with two distinct subunit conformations. Unlike other family members, AphB must undergo a significant conformational change in order for DNA binding to occur. Although a natural ligand for AphB has not yet been identified, we have found five independent mutations in the putative ligand-binding pocket region that allow AphB to constitutively activate transcription of tcpPH at the non-permissive pH of 8.5 and in the presence of oxygen. These findings indicate that the activity of AphB is responsive to intracellular pH as well as to anaerobiosis and that residues in the ligand-binding pocket of the protein influence its ability to respond to both of these signals. We have solved the structure of one of the constitutive mutants, and observe conformational changes that would allow DNA binding. Taken together, these results describe a pathway of conformational changes allowing communication between the ligand and DNA binding regions of AphB, suggesting a mechanism by which ligand binding leads to activation of the AphB-tcpPH promoter complex.

Crystal Structure of the Pseudomonas aeruginosa Virulence Factor Regulator

Virulence factor regulator (Vfr) enhances Pseudomonas aeruginosa pathogenicity through its role as a global transcriptional regulator. The crystal structure of Vfr shows that it is a winged-helix DNA-binding protein like its homologue cyclic AMP receptor protein (CRP). In addition to an expected primary cyclic AMP-binding site, a second ligand-binding site is nestled between the N-terminal domain and the C-terminal helix-turn-helix domain. Unlike CRP, Vfr is a symmetric dimer in the absence of DNA. Removal of seven disordered N-terminal residues of Vfr prevents the growth of P. aeruginosa.

Evolution of Metal(loid) Binding Sites in Transcriptional Regulators

Expression of the genes for resistance to heavy metals and metalloids is transcriptionally regulated by the toxic ions themselves. Members of the ArsR/SmtB family of small metalloregulatory proteins respond to transition metals, heavy metals, and metalloids, including As(III), Sb(III), Cd(II), Pb(II), Zn(II), Co(II), and Ni(II). These homodimeric repressors bind to DNA in the absence of inducing metal(loid) ion and dissociate from the DNA when inducer is bound. The regulatory sites are often three- or four-coordinate metal binding sites composed of cysteine thiolates. Surprisingly, in two different As(III)-responsive regulators, the metalloid binding sites were in different locations in the repressor, and the Cd(II) binding sites were in two different locations in two Cd(II)-responsive regulators. We hypothesize that ArsR/SmtB repressors have a common backbone structure, that of a winged helix DNA-binding protein, but have considerable plasticity in the location of inducer binding sites. Here we show that an As(III)-responsive member of the family, CgArsR1 from Corynebacterium glutamicum, binds As(III) to a cysteine triad composed of Cys(15), Cys(16), and Cys(55). This binding site is clearly unrelated to the binding sites of other characterized ArsR/SmtB family members. This is consistent with our hypothesis that metal(loid) binding sites in DNA binding proteins evolve convergently in response to persistent environmental pressures.

The Putative DNA-Binding Protein Sto12a from the Thermoacidophilic Archaeon Sulfolobus tokodaii Contains Intrachain and Interchain Disulfide Bonds

The Sto12a protein, from the thermoacidophilic archaeon Sulfolobus tokodaii, has been identified as a small putative DNA-binding protein. Most of the proteins with a high level of amino acid sequence homology to this protein are derived from members of the Sulfolobaceae family, including a transcriptional regulator. We determined the crystal structure of Sto12a at 2.05 Å resolution by multiple-wavelength anomalous dispersion phasing from the selenomethionine-containing protein crystal. This is the first structure of a member of this family of DNA-binding proteins. The Sto12a protein forms a homodimer, and the structure is composed of an N-terminal α-helix, a winged-helix–turn–helix domain, and a C-terminal α-helix that forms an interchain antiparallel coiled coil. The two winged-helix domains are located at both ends of the coiled coil, with putative DNA-recognition helices separated by approximately 34 Å. A structural homology search indicated that the winged-helix domain shared a high level of homology with those found in B-DNA- or Z-DNA-binding proteins from various species, including archaea, bacteria, and human, despite a low level of sequence similarity. The unique structural features of the Sto12a protein include intrachain and interchain disulfide bonds, which stabilize the chain and homodimer structures. There are three cysteine residues: Cys15 and Cys16 in the N-terminal α-helix, and Cys100 in the C-terminal α-helix. Cys15 is involved in an interchain disulfide bridge with the other Cys15, and Cys16 forms an intrachain disulfide bridge with Cys100. This is a novel fold among winged-helix DNA-binding proteins. Possible DNA-binding interactions of the Sto12a protein are discussed based on the crystal structure of Sto12a and comparisons to other winged-helix DNA-binding proteins.

Fis Targets Assembly of the Xis Nucleoprotein Filament to Promote Excisive Recombination by Phage Lambda

The phage-encoded Xis protein is the major determinant controlling the direction of recombination in phage lambda. Xis is a winged-helix DNA binding protein that cooperatively binds to the attR recombination site to generate a curved microfilament, which promotes assembly of the excisive intasome but inhibits formation of an integrative intasome. We find that lambda synthesizes surprisingly high levels of Xis immediately upon prophage induction when excision rates are maximal. However, because of its low sequence-specific binding activity, exemplified by a 1.9 Å co-crystal structure of a non-specifically bound DNA complex, Xis is relatively ineffective at promoting excision in vivo in the absence of the host Fis protein. Fis binds to a segment in attR that almost entirely overlaps one of the Xis binding sites. Instead of sterically excluding Xis binding from this site, as has been previously believed, we show that Fis enhances binding of all three Xis protomers to generate the microfilament. A specific Fis–Xis interface is supported by the effects of mutations within each protein, and relaxed, but not completely sequence-neutral, binding by the central Xis protomer is supported by the effects of DNA mutations. We present a structural model for the 50 bp curved Fis–Xis cooperative complex that is assembled between the arm and core Int binding sites whose trajectory places constraints on models for the excisive intasome structure.

HucR, a Novel Uric Acid-responsive Member of the MarR Family of Transcriptional Regulators from Deinococcus radiodurans

The MarR family of transcriptional regulators comprises a subset of winged helix DNA-binding proteins and includes numerous members that function in environmental surveillance of aromatic compounds. We describe the characterization of HucR, a novel MarR homolog from Deinococcus radiodurans that demonstrates phenolic sensing capabilities. HucR binds as a homodimer to a single site within its promoter/operator region with Kd = 0.29 +/- 0.02 nM. The HucR binding site contains a pseudopalindromic sequence, composed of 8-bp half-sites separated by 2 bp. The location of the HucR binding site in the intergenic region between hucR and a putative uricase suggests a mechanism of simultaneous co-repression of these two genes. The substrate of uricase, uric acid, is an efficient antagonist of DNA binding, reducing HucR-DNA complex formation to 50% at 0.26 mM ligand, compared with 5.2 and 46 mM for the aromatic compounds salicylate and acetylsalicylate, respectively. Enhanced levels in vivo of hucR and uricase transcript and increased uricase activity under conditions of excess uric acid further indicate a novel regulatory mechanism of aromatic catabolism in D. radiodurans. Since uric acid is a scavenger of reactive oxygen species, we hypothesize that HucR is a participant in the intrinsic resistance of D. radiodurans to high levels of oxidative stress.

Crystal structure of F-93 from Sulfolobus spindle-shaped virus 1, a winged-helix DNA binding protein.

Sulfolobus spindle-shaped viruses (SSVs), or Fuselloviridae, are ubiquitous crenarchaeal viruses found in high-temperature acidic hot springs around the world (pH </=4.0; temperature of >/=70 degrees C). Because they are relatively easy to isolate, they represent the best studied of the crenarchaeal viruses. This is particularly true for the type virus, SSV1, which contains a double-stranded DNA genome of 15.5 kilobases, encoding 34 putative open reading frames. Interestingly, the genome shows little sequence similarity to organisms other than its SSV homologues. Together, sequence similarity and biochemical analyses have suggested functions for only 6 of the 34 open reading frames. Thus, even though SSV1 is the best-studied crenarchaeal virus, functions for most (28) of its open reading frames remain unknown. We have undertaken biochemical and structural studies for the gene product of open reading frame F-93. We find that F-93 exists as a homodimer in solution and that a tight dimer is also present in the 2.7-A crystal structure. Further, the crystal structure reveals a fold that is homologous to the SlyA and MarR subfamilies of winged-helix DNA binding proteins. This strongly suggests that F-93 functions as a transcription factor that recognizes a (pseudo-)palindromic DNA target sequence.

Transcriptional and DNA binding activity of the Foxp1/2/4 family is modulated by heterotypic and homotypic protein interactions.

Foxp1, Foxp2, and Foxp4 are large multidomain transcriptional regulators belonging to the family of winged-helix DNA binding proteins known as the Fox family. Foxp1 and Foxp2 have been shown to act as transcriptional repressors, while regulatory activity of the recently identified Foxp4 has not been determined. Given the importance of this Fox gene subfamily in neural and lung development, we sought to elucidate the mechanisms by which Foxp1, Foxp2, and Foxp4 repress gene transcription. We show that like Foxp1 and Foxp2, Foxp4 represses transcription. Analysis of the N-terminal repression domain in Foxp1, Foxp2, and Foxp4 shows that this region contains two separate and distinct repression subdomains that are highly homologous termed subdomain 1 and subdomain 2. However, subdomain 2 is not functional in Foxp4. Screening for proteins that interact with subdomains 1 and 2 of Foxp2 using yeast two-hybrid analysis revealed that subdomain 2 binds to C-terminal binding protein 1, which can synergistically repress transcription with Foxp1 and Foxp2, but not Foxp4. Subdomain 1 contains a highly conserved leucine zipper similar to that found in N-myc and confers homo- and heterodimerization to the Foxp1/2/4 family members. These interactions are dependent on the conserved leucine zipper motif. Finally, we show that the integrity of this subdomain is essential for DNA binding, making Foxp1, Foxp2, and Foxp4 the first Fox proteins that require dimerization for DNA binding. These data reveal a complex regulatory mechanism underlying Foxp1, Foxp2, and Foxp4 activity, demonstrating that Foxp1, Foxp2, and Foxp4 are the first Fox proteins reported whose activity is regulated by homo- and heterodimerization.

Constitutive Binding of the Transcription Factor Interleukin-2 (IL-2) Enhancer Binding Factor to the IL-2 Promoter

A positive regulatory element in the interleukin-2 (IL-2) promoter, designated the antigen receptor response element-2, is essential for the induction of IL-2 gene expression upon the binding of an inducible multiprotein complex of proteins known as nuclear factor of activated T cells. In the current study, we demonstrated that the winged-helix transcription factor IL-2 enhancer binding factor (ILF) is constitutively expressed in both resting and activated Jurkat cells and binds to two adjacent sequence motifs immediately downstream of the binding site for NFAT. One of these elements has a high degree of homology with consensus binding sites for a variety of winged-helix DNA binding proteins, and the second site functions to modulate ILF binding. Mutagenesis of each of the two sequence elements required for ILF binding decreased IL-2 promoter activity when assayed in transfection assays. Although ILF bound constitutively to the IL-2 promoter, it was not detected as a component of the NFAT complex. These results suggest that important regulatory sequences in the IL-2 promoter are bound by ILF and that this binding may be involved in the control of IL-2 gene expression.