scholarly journals Exploring the Multiligand Binding Specificity of Saposin B Reveals Two Binding Sites

ACS Omega ◽  
2017 ◽  
Vol 2 (10) ◽  
pp. 7141-7145 ◽  
Author(s):  
Jay Tinklepaugh ◽  
Britannia M. Smith ◽  
Etta Hanlon ◽  
Chloe Zubieta ◽  
Fadi Bou-Abdallah ◽  
...  
Keyword(s):  
Binding Sites ◽  
Binding Specificity ◽  
Saposin B

scholarly journals E2F in vivo binding specificity: Comparison of consensus versus nonconsensus binding sites

2008 ◽  
Vol 18 (11) ◽  
pp. 1763-1777 ◽  
Author(s):  
A. Rabinovich ◽  
V. X. Jin ◽  
R. Rabinovich ◽  
X. Xu ◽  
P. J. Farnham
Keyword(s):  
Binding Sites ◽  
Binding Specificity ◽  
In Vivo Binding

Nucleotides flanking a conserved TAAT core dictate the DNA binding specificity of three murine homeodomain proteins

1993 ◽  
Vol 13 (4) ◽  
pp. 2354-2365
Author(s):  
K M Catron ◽  
N Iler ◽  
C Abate
Keyword(s):  
Dna Binding ◽  
Binding Sites ◽  
Target Genes ◽  
Equilibrium Constants ◽  
Binding Specificity ◽  
Binding Activity ◽  
Selection Strategy ◽  
Homeodomain Proteins ◽  
Control Regions

Murine homeobox genes play a fundamental role in directing embryogenesis by controlling gene expression during development. The homeobox encodes a DNA binding domain (the homeodomain) which presumably mediates interactions of homeodomain proteins with specific DNA sites in the control regions of target genes. However, the bases for these selective DNA-protein interactions are not well defined. In this report, we have characterized the DNA binding specificities of three murine homeodomain proteins, Hox 7.1, Hox 1.5, and En-1. We have identified optimal DNA binding sites for each of these proteins by using a random oligonucleotide selection strategy. Comparison of the sequences of the selected binding sites predicted a common consensus site that contained the motif (C/G)TAATTG. The TAAT core was essential for DNA binding activity, and the nucleotides flanking this core directed binding specificity. Whereas variations in the nucleotides flanking the 5' side of the TAAT core produced modest alterations in binding activity for all three proteins, perturbations of the nucleotides directly 3' of the core distinguished the binding specificity of Hox 1.5 from those of Hox 7.1 and En-1. These differences in binding activity reflected differences in the dissociation rates rather than the equilibrium constants of the protein-DNA complexes. Differences in DNA binding specificities observed in vitro may contribute to selective interactions of homeodomain proteins with potential binding sites in the control regions of target genes.

scholarly journals H-NS Represses inv Transcription in Yersinia enterocolitica through Competition with RovA and Interaction with YmoA

2006 ◽  
Vol 188 (14) ◽  
pp. 5101-5112 ◽  
Author(s):  
Damon W. Ellison ◽  
Virginia L. Miller
Keyword(s):  
Yersinia Enterocolitica ◽  
Binding Sites ◽  
Yersinia Pseudotuberculosis ◽  
Binding Specificity ◽  
Peyer's Patches ◽  
Published Data ◽  
M Cells ◽  
Binding Region ◽  
Similar Region ◽  
Repression Complex

ABSTRACT Yersinia enterocolitica is able to efficiently invade Peyer's patches with the aid of invasin, an outer member protein involved in the attachment and invasion of M cells. Invasin is encoded by inv, which is positively regulated by RovA in both Y. enterocolitica and Yersinia pseudotuberculosis while negatively regulated by YmoA in Y. enterocolitica and H-NS in Y. pseudotuberculosis. In this study we present data indicating H-NS and RovA bind directly and specifically to the inv promoter of Y. enterocolitica. We also show that RovA and H-NS from Y. enterocolitica bind to a similar region of the inv promoter and suggest they compete for binding sites. This is similar to recently published data from Y. pseudotuberculosis, revealing a potentially conserved mechanism of inv regulation between Y. enterocolitica and Y. pseudotuberculosis. Furthermore, we present data suggesting H-NS and YmoA form a repression complex on the inv promoter, with H-NS providing the binding specificity and YmoA interacting with H-NS to form a repression complex. We also demonstrate that deletion of the predicted H-NS binding region relieves the requirement for RovA-dependent transcription of the inv promoter, consistent with RovA acting as a derepressor of H-NS-mediated repression. Levels of H-NS and YmoA are similar between 26°C and 37°C, suggesting that the H-NS/YmoA repression complex is present at both temperatures, while the levels of rovA transcript are low at 37°C and high at 26°C, leading to expression of inv at 26°C. Expression of RovA at 37°C results in transcription of inv and production of invasin. Data presented here support a model of inv regulation where the level of RovA within the cell governs inv expression. As RovA levels increase, RovA can successfully compete for binding to the inv promoter with the H-NS/YmoA complex, resulting in derepression of inv transcription.

scholarly journals In vitro DNA-binding profile of transcription factors: methods and new insights

2011 ◽  
Vol 210 (1) ◽  
pp. 15-27 ◽  
Author(s):  
Jinke Wang ◽  
Jie Lu ◽  
Guangming Gu ◽  
Yingxun Liu
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Dna Sequences ◽  
Binding Sites ◽  
Target Genes ◽  
Binding Specificity ◽  
Cell Physiology ◽  
Binding Profile ◽  
Dna Binding Specificity

The DNA-binding specificity of transcription factors (TFs) has broad impacts on cell physiology, cell development and in evolution. However, the DNA-binding specificity of most known TFs still remains unknown. The specificity of a TF protein is determined by its relative affinity to all possible binding sites. In recent years, the development of several in vitro techniques permits high-throughput determination of relative binding affinity of a TF to all possible k bp-long DNA sequences, thus greatly promoting the characterization of DNA-binding specificity of many known TFs. All DNA sequences that can be bound by a TF with various binding affinities form their DNA-binding profile (DBP). The DBP is important to generate an accurate DNA-binding model, identify all DNA-binding sites and target genes of TFs in the whole genome, and build transcription regulatory network. This study reviewed these techniques, especially two master techniques: double-stranded DNA microarray and systematic evolution of ligands by exponential enrichment in combination with parallel DNA sequencing techniques (SELEX-seq).

scholarly journals Structure-function analysis of SH3 domains: SH3 binding specificity altered by single amino acid substitutions.

1995 ◽  
Vol 15 (10) ◽  
pp. 5627-5634 ◽  
Author(s):  
Z Weng ◽  
R J Rickles ◽  
S Feng ◽  
S Richard ◽  
A S Shaw ◽  
...  
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Binding Proteins ◽  
Hydrophobic Interactions ◽  
Function Analysis ◽  
Binding Specificity ◽  
Sh3 Domain ◽  
Single Amino Acid ◽  
Sh3 Domains ◽  
Cell Extracts

SH3 domains mediate intracellular protein-protein interactions through the recognition of proline-rich sequence motifs on cellular proteins. Structural analysis of the Src SH3 domain (Src SH3) complexed with proline-rich peptide ligands revealed three binding sites involved in this interaction: two hydrophobic interactions (between aliphatic proline dipeptides in the SH3 ligand and highly conserved aromatic residues on the surface of the SH3 domain), and one salt bridge (between Asp-99 of Src and an Arg three residues upstream of the conserved Pro-X-X-Pro motif in the ligand). We examined the importance of the arginine binding site of SH3 domains by comparing the binding properties of wild-type Src SH3 and Abl SH3 with those of a Src SH3 mutant containing a mutated arginine binding site (D99N) and Abl SH3 mutant constructs engineered to contain an arginine binding site (T98D and T98D/F91Y). We found that the D99N mutation diminished binding to most Src SH3-binding proteins in whole cell extracts; however, there was only a moderate reduction in binding to a small subset of Src SH3-binding proteins (including the Src substrate p68). p68 was shown to contain two Arg-containing Asp-99-dependent binding sites and one Asp-99-independent binding site which lacks an Arg. Moreover, substitution of Asp for Thr-98 in Abl SH3 changed the binding specificity of this domain and conferred the ability to recognize Arg-containing ligands. These results indicate that Asp-99 is important for Src SH3 binding specificity and that Asp-99-dependent binding interactions play a dominant role in Src SH3 recognition of cellular binding proteins, and they suggest the existence of two Src SH3 binding mechanisms, one requiring Asp-99 and the other independent of this residue.

scholarly journals Nucleotides flanking a conserved TAAT core dictate the DNA binding specificity of three murine homeodomain proteins.

1993 ◽  
Vol 13 (4) ◽  
pp. 2354-2365 ◽  
Author(s):  
K M Catron ◽  
N Iler ◽  
C Abate
Keyword(s):  
Dna Binding ◽  
Binding Sites ◽  
Target Genes ◽  
Equilibrium Constants ◽  
Binding Specificity ◽  
Binding Activity ◽  
Selection Strategy ◽  
Homeodomain Proteins ◽  
Control Regions

Murine homeobox genes play a fundamental role in directing embryogenesis by controlling gene expression during development. The homeobox encodes a DNA binding domain (the homeodomain) which presumably mediates interactions of homeodomain proteins with specific DNA sites in the control regions of target genes. However, the bases for these selective DNA-protein interactions are not well defined. In this report, we have characterized the DNA binding specificities of three murine homeodomain proteins, Hox 7.1, Hox 1.5, and En-1. We have identified optimal DNA binding sites for each of these proteins by using a random oligonucleotide selection strategy. Comparison of the sequences of the selected binding sites predicted a common consensus site that contained the motif (C/G)TAATTG. The TAAT core was essential for DNA binding activity, and the nucleotides flanking this core directed binding specificity. Whereas variations in the nucleotides flanking the 5' side of the TAAT core produced modest alterations in binding activity for all three proteins, perturbations of the nucleotides directly 3' of the core distinguished the binding specificity of Hox 1.5 from those of Hox 7.1 and En-1. These differences in binding activity reflected differences in the dissociation rates rather than the equilibrium constants of the protein-DNA complexes. Differences in DNA binding specificities observed in vitro may contribute to selective interactions of homeodomain proteins with potential binding sites in the control regions of target genes.

The orphan receptor Rev-ErbA alpha activates transcription via a novel response element

1993 ◽  
Vol 13 (5) ◽  
pp. 3113-3121
Author(s):  
H P Harding ◽  
M A Lazar
Keyword(s):  
Dna Sequences ◽  
Binding Sites ◽  
Hormone Receptor ◽  
Thyroid Hormone Receptor ◽  
Specific Binding ◽  
Binding Specificity ◽  
Nuclear Hormone Receptor ◽  
Genomic Arrangement ◽  
Opposite Strand ◽  
Half Site

Rev-ErbA alpha (Rev-Erb) is a nuclear hormone receptor-related protein encoded on the opposite strand of the alpha-thyroid hormone receptor (TR) gene. This unusual genomic arrangement may have a regulatory role, but the conservation of human and rodent Rev-Erb amino acid sequences suggests that the protein itself has an important function, potentially as a sequence-specific transcriptional regulator. However, despite its relationship to the TR, Rev-Erb bound poorly to TR binding sites. To determine its DNA-binding specificity in an unbiased manner, Rev-Erb was synthesized in Escherichia coli, purified, and used to select specific binding-sites from libraries of random double-stranded DNA sequences. We found that Rev-Erb binds to a unique site consisting of a specific 5-bp A/T-rich sequence adjacent to a TR half-site. Rev-Erb contacts this entire asymmetric 11-bp sequence, which is the longest nonrepetitive element specifically recognized by a member of the thyroid/steroid hormone receptor superfamily, and mutations in either the A/T-rich or TR half-site regions abolished specific binding. The binding specificity of wild-type Rev-Erb was nearly identical to that of C- and N-terminally truncated forms. This binding was not enhanced by retinoid X receptor, TR, or other nuclear proteins, none of which formed heterodimers with Rev-Erb. Rev-Erb also appeared to bind to the selected site as a monomer. Furthermore, Rev-Erb activates transcription through this binding site even in the absence of exogenous ligand. Thus, Rev-Erb is a transcriptional activator whose properties differ dramatically from those of classical nuclear hormone receptors, including the TR encoded on the opposite strand of the same genomic locus.

scholarly journals Cloning, expression in Escherichia coli and characterization of the recombinant Neu5Acα2,6Galβ1,4GlcNAc-specific high-affinity lectin and its mutants from the mushroom Polyporus squamosus

2004 ◽  
Vol 382 (2) ◽  
pp. 667-675 ◽  
Author(s):  
Hiroaki TATENO ◽  
Harry C. WINTER ◽  
Irwin J. GOLDSTEIN
Keyword(s):  
Escherichia Coli ◽  
Binding Sites ◽  
Expression System ◽  
Binding Specificity ◽  
Single Amino Acid ◽  
Soluble Form ◽  
Carbohydrate Binding ◽  
Terminal Portion ◽  
Sugar Binding ◽  
Polyporus Squamosus

Lectin from the mushroom Polyporus squamosus (PSL) has a unique carbohydrate-binding specificity for sialylated glycoconjugates containing Neu5Acα2,6Galβ1,4Glc/GlcNAc trisaccharide sequences of asparagine-linked glycoproteins. In the present study, we elucidate the molecular basis for its binding specificity as well as establish a consistent source of this useful lectin using a bacterial expression system. cDNA cloning revealed that PSL contains a ricin B chain-like (QXW)3 domain at its N-terminus that is composed of three homologous subdomains (α, β and γ). A recombinant lectin was expressed in Escherichia coli as a fully active, soluble form. It agglutinated rabbit erythrocytes and showed the highest affinity for Neu5Acα2,6Galβ1,4GlcNAc, but not for the sialyl α2,3-linked isomer. We also investigated the structure–function relationship of PSL. A monomeric C-terminal deletion mutant lacking 40% of the lectin's molecular mass retained sugar-binding activity, indicating that the carbohydrate-binding sites are situated in the N-terminal portion of the lectin, whereas the C-terminal portion probably functions in oligomerization and structural stabilization. Mutant constructs that have single amino acid substitutions in the putative sugar-binding sites, based on sequence alignment with the ricin B-chain, indicate that the β and γ subdomains are most probably sugar-binding sites. The recombinantly expressed lectin will be a valuable reagent for the detection of the Neu5Acα2,6Galβ1,4GlcNAc sequence of asparagine-linked glycans.

Evidence for an androgen receptor in porcine Leydig cells

1987 ◽  
Vol 115 (1) ◽  
pp. 119-124 ◽  
Author(s):  
Veli Isomaa ◽  
Mauri Orava ◽  
Reijo Vihko
Keyword(s):  
Androgen Receptor ◽  
Binding Sites ◽  
Leydig Cells ◽  
Androgen Receptors ◽  
Cultured Cells ◽  
Binding Specificity ◽  
Culture Conditions ◽  
High Affinity ◽  
The Mean ◽  
Steroid Binding

Abstract. Cytosol and nuclear androgen receptor concentrations were measured in freshly prepared and cultured Leydig cells of immature pig testis with exchange assays using [3H]methyltrienolone as labelled ligand. Androgen receptors in Leydig cells had high affinity for [3H]methyltrienolone and steroid binding specificity typical of an androgen receptor. The mean receptor concentrations were 76 fmol/mg protein and 210 fmol/mg DNA for cytosol and nuclei, respectively. In sucrose gradients, cytosol androgen receptors sedimented in the 4 S region. The cells maintained androgen receptors under culture conditions. Exposure of cultured cells to [3H]methyltrienolone (10 nmol/l) resulted in accumulation of androgen receptors in the nuclei with maximal uptake by 1 h. We conclude that methyltrienolone binding sites with characteristics of androgen receptors were identified in both cytosol and nuclei of porcine Leydig cells.

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