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A transcription blocker isolated from a designed repeat protein combinatorial library by in vivo functional screen.

Tikhonova EB, Ethayathulla AS, Su Y, Hariharan P, Xie S, Guan L - Sci Rep (2015)

Bottom Line: Functional tests suggest that the activity of MelR, a DNA-binding transcription activator and a member of AraC family of transcription factors, is inhibited by ANK-N5C-281 protein.All ANK-N5C proteins are expected to have a concave binding area with negative surface potential, suggesting that the designed ANK-N5C library proteins may facilitate the discovery of binders recognizing structural motifs with positive surface potential, like in DNA-binding proteins.Overall, our results show that the established library is a useful tool for the discovery of novel bioactive reagents.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Physiology &Molecular Biophysics, Center for Membrane Protein Research, Texas Tech University Health Sciences Center, Lubbock, Texas 79430.

ABSTRACT
A highly diverse DNA library coding for ankyrin seven-repeat proteins (ANK-N5C) was designed and constructed by a PCR-based combinatorial assembly strategy. A bacterial melibiose fermentation assay was adapted for in vivo functional screen. We isolated a transcription blocker that completely inhibits the melibiose-dependent expression of α-galactosidase (MelA) and melibiose permease (MelB) of Escherichia coli by specifically preventing activation of the melAB operon. High-resolution crystal structural determination reveals that the designed ANK-N5C protein has a typical ankyrin fold, and the specific transcription blocker, ANK-N5C-281, forms a domain-swapped dimer. Functional tests suggest that the activity of MelR, a DNA-binding transcription activator and a member of AraC family of transcription factors, is inhibited by ANK-N5C-281 protein. All ANK-N5C proteins are expected to have a concave binding area with negative surface potential, suggesting that the designed ANK-N5C library proteins may facilitate the discovery of binders recognizing structural motifs with positive surface potential, like in DNA-binding proteins. Overall, our results show that the established library is a useful tool for the discovery of novel bioactive reagents.

No MeSH data available.


Related in: MedlinePlus

Dimer interface in ANK-N5C-281.The two swapped monomers in ANK-N5C-281 crystal structure are colored in green and cyan. The structure of ANK-N5C-317 is colored in blue. (a), The 2Fo-Fc electro density map (contour at 1.2 σ) shows the hinge loop linking the N-terminal five repeats with C-terminal two repeats. (b), Superposition of ANK-N5C-317 with ANK-N5C-281. Salt-bridge interactions between Glu133 (position-33 of internal repeat III) and Arg199 (position-33 of internal repeat V) within one ANK-N5C-218 monomer are shown by dotted lines. The “hybrid monomer” is indicated. (c), Interactions between the two hinge loops in the domain-swapped dimer. Dotted lines indicate H-bonding interactions. The random residues are underlined. Helices from the internal repeats-IV and -V are indicated, respectively. The β-turn-1 of the internal repeat V of ANK-N5C-317 is colored as blue and indicated by arrow. Partial sequence alignment of ANK-N5C-62, ANK-N5C-317, and ANK-N5C-281 are shown in the box underneath. x, randomized position; *, consensus position. Amino acid positioning in protein and in repeat are indicated. Pro171 and the charge pair Glu133/Arg199 in ANK-N5C-281 are colored in blue and red, respectively. (d), BN-15%PAGE. ANK-N5C proteins (5 μg each) and the NativeMarkTM unstained protein standard were loaded on each well. (e). Site-directed mutagenesis of ANK-N5C-281. Effect of ANK-N5C-281/P171Q or F mutant on melibiose fermentation was carried out as described in the legend to Fig. 3.
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f6: Dimer interface in ANK-N5C-281.The two swapped monomers in ANK-N5C-281 crystal structure are colored in green and cyan. The structure of ANK-N5C-317 is colored in blue. (a), The 2Fo-Fc electro density map (contour at 1.2 σ) shows the hinge loop linking the N-terminal five repeats with C-terminal two repeats. (b), Superposition of ANK-N5C-317 with ANK-N5C-281. Salt-bridge interactions between Glu133 (position-33 of internal repeat III) and Arg199 (position-33 of internal repeat V) within one ANK-N5C-218 monomer are shown by dotted lines. The “hybrid monomer” is indicated. (c), Interactions between the two hinge loops in the domain-swapped dimer. Dotted lines indicate H-bonding interactions. The random residues are underlined. Helices from the internal repeats-IV and -V are indicated, respectively. The β-turn-1 of the internal repeat V of ANK-N5C-317 is colored as blue and indicated by arrow. Partial sequence alignment of ANK-N5C-62, ANK-N5C-317, and ANK-N5C-281 are shown in the box underneath. x, randomized position; *, consensus position. Amino acid positioning in protein and in repeat are indicated. Pro171 and the charge pair Glu133/Arg199 in ANK-N5C-281 are colored in blue and red, respectively. (d), BN-15%PAGE. ANK-N5C proteins (5 μg each) and the NativeMarkTM unstained protein standard were loaded on each well. (e). Site-directed mutagenesis of ANK-N5C-281. Effect of ANK-N5C-281/P171Q or F mutant on melibiose fermentation was carried out as described in the legend to Fig. 3.

Mentions: In ANK-N5C-281, the refined model reveals that two molecules exchange their identical C-terminal two repeats, forming a domain-swapped dimer (Fig. 5e–f, Fig. 6a–c). The helical packing between the internal repeats IV and V of two swapped molecules is similar to that in ANK-N5C-317. The overall fold of the “hybrid monomer” in ANK-N5C-281, which consists of five N-terminal repeats with two C-terminal repeats from another molecule, also superimposes well with ANK-N5C-317 (Fig. 6b).


A transcription blocker isolated from a designed repeat protein combinatorial library by in vivo functional screen.

Tikhonova EB, Ethayathulla AS, Su Y, Hariharan P, Xie S, Guan L - Sci Rep (2015)

Dimer interface in ANK-N5C-281.The two swapped monomers in ANK-N5C-281 crystal structure are colored in green and cyan. The structure of ANK-N5C-317 is colored in blue. (a), The 2Fo-Fc electro density map (contour at 1.2 σ) shows the hinge loop linking the N-terminal five repeats with C-terminal two repeats. (b), Superposition of ANK-N5C-317 with ANK-N5C-281. Salt-bridge interactions between Glu133 (position-33 of internal repeat III) and Arg199 (position-33 of internal repeat V) within one ANK-N5C-218 monomer are shown by dotted lines. The “hybrid monomer” is indicated. (c), Interactions between the two hinge loops in the domain-swapped dimer. Dotted lines indicate H-bonding interactions. The random residues are underlined. Helices from the internal repeats-IV and -V are indicated, respectively. The β-turn-1 of the internal repeat V of ANK-N5C-317 is colored as blue and indicated by arrow. Partial sequence alignment of ANK-N5C-62, ANK-N5C-317, and ANK-N5C-281 are shown in the box underneath. x, randomized position; *, consensus position. Amino acid positioning in protein and in repeat are indicated. Pro171 and the charge pair Glu133/Arg199 in ANK-N5C-281 are colored in blue and red, respectively. (d), BN-15%PAGE. ANK-N5C proteins (5 μg each) and the NativeMarkTM unstained protein standard were loaded on each well. (e). Site-directed mutagenesis of ANK-N5C-281. Effect of ANK-N5C-281/P171Q or F mutant on melibiose fermentation was carried out as described in the legend to Fig. 3.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4308713&req=5

f6: Dimer interface in ANK-N5C-281.The two swapped monomers in ANK-N5C-281 crystal structure are colored in green and cyan. The structure of ANK-N5C-317 is colored in blue. (a), The 2Fo-Fc electro density map (contour at 1.2 σ) shows the hinge loop linking the N-terminal five repeats with C-terminal two repeats. (b), Superposition of ANK-N5C-317 with ANK-N5C-281. Salt-bridge interactions between Glu133 (position-33 of internal repeat III) and Arg199 (position-33 of internal repeat V) within one ANK-N5C-218 monomer are shown by dotted lines. The “hybrid monomer” is indicated. (c), Interactions between the two hinge loops in the domain-swapped dimer. Dotted lines indicate H-bonding interactions. The random residues are underlined. Helices from the internal repeats-IV and -V are indicated, respectively. The β-turn-1 of the internal repeat V of ANK-N5C-317 is colored as blue and indicated by arrow. Partial sequence alignment of ANK-N5C-62, ANK-N5C-317, and ANK-N5C-281 are shown in the box underneath. x, randomized position; *, consensus position. Amino acid positioning in protein and in repeat are indicated. Pro171 and the charge pair Glu133/Arg199 in ANK-N5C-281 are colored in blue and red, respectively. (d), BN-15%PAGE. ANK-N5C proteins (5 μg each) and the NativeMarkTM unstained protein standard were loaded on each well. (e). Site-directed mutagenesis of ANK-N5C-281. Effect of ANK-N5C-281/P171Q or F mutant on melibiose fermentation was carried out as described in the legend to Fig. 3.
Mentions: In ANK-N5C-281, the refined model reveals that two molecules exchange their identical C-terminal two repeats, forming a domain-swapped dimer (Fig. 5e–f, Fig. 6a–c). The helical packing between the internal repeats IV and V of two swapped molecules is similar to that in ANK-N5C-317. The overall fold of the “hybrid monomer” in ANK-N5C-281, which consists of five N-terminal repeats with two C-terminal repeats from another molecule, also superimposes well with ANK-N5C-317 (Fig. 6b).

Bottom Line: Functional tests suggest that the activity of MelR, a DNA-binding transcription activator and a member of AraC family of transcription factors, is inhibited by ANK-N5C-281 protein.All ANK-N5C proteins are expected to have a concave binding area with negative surface potential, suggesting that the designed ANK-N5C library proteins may facilitate the discovery of binders recognizing structural motifs with positive surface potential, like in DNA-binding proteins.Overall, our results show that the established library is a useful tool for the discovery of novel bioactive reagents.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Physiology &Molecular Biophysics, Center for Membrane Protein Research, Texas Tech University Health Sciences Center, Lubbock, Texas 79430.

ABSTRACT
A highly diverse DNA library coding for ankyrin seven-repeat proteins (ANK-N5C) was designed and constructed by a PCR-based combinatorial assembly strategy. A bacterial melibiose fermentation assay was adapted for in vivo functional screen. We isolated a transcription blocker that completely inhibits the melibiose-dependent expression of α-galactosidase (MelA) and melibiose permease (MelB) of Escherichia coli by specifically preventing activation of the melAB operon. High-resolution crystal structural determination reveals that the designed ANK-N5C protein has a typical ankyrin fold, and the specific transcription blocker, ANK-N5C-281, forms a domain-swapped dimer. Functional tests suggest that the activity of MelR, a DNA-binding transcription activator and a member of AraC family of transcription factors, is inhibited by ANK-N5C-281 protein. All ANK-N5C proteins are expected to have a concave binding area with negative surface potential, suggesting that the designed ANK-N5C library proteins may facilitate the discovery of binders recognizing structural motifs with positive surface potential, like in DNA-binding proteins. Overall, our results show that the established library is a useful tool for the discovery of novel bioactive reagents.

No MeSH data available.


Related in: MedlinePlus