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PDBsum entry 4o60
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De novo protein
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PDB id
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4o60
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References listed in PDB file
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Key reference
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Title
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A transcription blocker isolated from a designed repeat protein combinatorial library by in vivo functional screen.
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Authors
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E.B.Tikhonova,
A.S.Ethayathulla,
Y.Su,
P.Hariharan,
S.Xie,
L.Guan.
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Ref.
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Sci Rep, 2015,
5,
8070.
[DOI no: ]
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PubMed id
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Abstract
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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.
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