Limits...
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

X-ray crystal structures.(a–d), ANK-N5C-317; (e), (f), ANK-N5C-281. (a), Cartoon representation of the crystal structure of ANK-N5C-317 protein (PDB ID 4O60, 2.5 Å). The N- and C-terminal CAP repeats (N-CAP ad C-CAP) and five internal repeats (IR) are indicated. (b), Superposition of repeats of ANK-N5C-317 protein. The secondary structure elements are indicated. The arrow points to the disturbed β-turn-1 of the internal repeat IV. (c), Surface representation. All 25 randomized positions are indicated by red-colored C-α positions. (d), Surface electrostatic potential map on ANK-N5C-317 structure was calculated by APBS program. (e), Overall folding of the domain-swapped dimer ANK-N5C-281 (PDB ID 4QFV, 2.0 Å). (f), Surface potential map on ANK-N5C-281 structure was calculated by APBS program.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4308713&req=5

f5: X-ray crystal structures.(a–d), ANK-N5C-317; (e), (f), ANK-N5C-281. (a), Cartoon representation of the crystal structure of ANK-N5C-317 protein (PDB ID 4O60, 2.5 Å). The N- and C-terminal CAP repeats (N-CAP ad C-CAP) and five internal repeats (IR) are indicated. (b), Superposition of repeats of ANK-N5C-317 protein. The secondary structure elements are indicated. The arrow points to the disturbed β-turn-1 of the internal repeat IV. (c), Surface representation. All 25 randomized positions are indicated by red-colored C-α positions. (d), Surface electrostatic potential map on ANK-N5C-317 structure was calculated by APBS program. (e), Overall folding of the domain-swapped dimer ANK-N5C-281 (PDB ID 4QFV, 2.0 Å). (f), Surface potential map on ANK-N5C-281 structure was calculated by APBS program.

Mentions: High-resolution crystal structures for two ANK-N5C proteins, ANK-N5C-317 and ANK-N5C-281, were resolved to 2.5 (PDB ID, 4O60) and 2.0 Å (PDB ID, 4QFV), respectively (Table 2). The purified proteins are stable and readily crystallized. The 3-D structure of ANK-N5C-317 protein reveals a typical topology for ANK proteins2124 (Fig. 5a–d); surprisingly, ANK-N5C-281 forms a domain-swapped dimer (Fig. 5e, f).


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)

X-ray crystal structures.(a–d), ANK-N5C-317; (e), (f), ANK-N5C-281. (a), Cartoon representation of the crystal structure of ANK-N5C-317 protein (PDB ID 4O60, 2.5 Å). The N- and C-terminal CAP repeats (N-CAP ad C-CAP) and five internal repeats (IR) are indicated. (b), Superposition of repeats of ANK-N5C-317 protein. The secondary structure elements are indicated. The arrow points to the disturbed β-turn-1 of the internal repeat IV. (c), Surface representation. All 25 randomized positions are indicated by red-colored C-α positions. (d), Surface electrostatic potential map on ANK-N5C-317 structure was calculated by APBS program. (e), Overall folding of the domain-swapped dimer ANK-N5C-281 (PDB ID 4QFV, 2.0 Å). (f), Surface potential map on ANK-N5C-281 structure was calculated by APBS program.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: X-ray crystal structures.(a–d), ANK-N5C-317; (e), (f), ANK-N5C-281. (a), Cartoon representation of the crystal structure of ANK-N5C-317 protein (PDB ID 4O60, 2.5 Å). The N- and C-terminal CAP repeats (N-CAP ad C-CAP) and five internal repeats (IR) are indicated. (b), Superposition of repeats of ANK-N5C-317 protein. The secondary structure elements are indicated. The arrow points to the disturbed β-turn-1 of the internal repeat IV. (c), Surface representation. All 25 randomized positions are indicated by red-colored C-α positions. (d), Surface electrostatic potential map on ANK-N5C-317 structure was calculated by APBS program. (e), Overall folding of the domain-swapped dimer ANK-N5C-281 (PDB ID 4QFV, 2.0 Å). (f), Surface potential map on ANK-N5C-281 structure was calculated by APBS program.
Mentions: High-resolution crystal structures for two ANK-N5C proteins, ANK-N5C-317 and ANK-N5C-281, were resolved to 2.5 (PDB ID, 4O60) and 2.0 Å (PDB ID, 4QFV), respectively (Table 2). The purified proteins are stable and readily crystallized. The 3-D structure of ANK-N5C-317 protein reveals a typical topology for ANK proteins2124 (Fig. 5a–d); surprisingly, ANK-N5C-281 forms a domain-swapped dimer (Fig. 5e, f).

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