PDBsum entry 4o60

De novo protein

PDB id: 4o60

Contents

Protein chains

230 a.a.

Waters ×55

PDB id:

4o60

Name:

De novo protein

Title:

Structure of ankyrin repeat protein

Structure:

Ank-n5c-317. Chain: a, b. Engineered: yes

Source:

Synthetic: yes. Synthetic construct. Organism_taxid: 32630. Other_details: synthetic construct

Resolution:

2.52Å R-factor: 0.192 R-free: 0.248

Authors:

A.S.Ethayathulla,L.Guan

Key ref:

E.B.Tikhonova et al. (2015). A transcription blocker isolated from a designed repeat protein combinatorial library by in vivo functional screen. Sci Rep, 5, 8070. PubMed id: 25627011 DOI: 10.1038/srep08070

Date:

20-Dec-13 Release date: 25-Mar-15

Protein chains

No UniProt id for this chain

Struc: 230 a.a.

DOI no: 10.1038/srep08070

Sci Rep 5:8070 (2015)

PubMed id: 25627011

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

E.B.Tikhonova, A.S.Ethayathulla, Y.Su, P.Hariharan, S.Xie, L.Guan.

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.