Login | Register

Subunit Orientation in the Escherichia coli Enterobactin Biosynthetic EntA-EntE Complex Revealed by a Two-Hybrid Approach

Title:

Subunit Orientation in the Escherichia coli Enterobactin Biosynthetic EntA-EntE Complex Revealed by a Two-Hybrid Approach

Pakarian, Paknoosh and Pawelek, Peter D. (2016) Subunit Orientation in the Escherichia coli Enterobactin Biosynthetic EntA-EntE Complex Revealed by a Two-Hybrid Approach. Biochimie, 127 . pp. 1-9.

[img]
Preview
Text (application/pdf)
MS006_EA_BACTH_BIOCHIMIE_FINAL_REVISION_SUBMIT.pdf
13MB

Official URL: http://www.sciencedirect.com/science/article/pii/S...

Abstract

The siderophore enterobactin is synthesized by the enzymes EntA-F and EntH in the E. coli cytoplasm. We previously reported in vitro evidence of an interaction between tetrameric EntA and monomeric EntE. Here we used bacterial adenylate cyclase two-hybrid (BACTH) assays to demonstrate that the E. coli EntA-EntE interaction occurs intracellularly. Furthermore, to obtain information on subunit orientation in the EntA-EntE complex, we fused BACTH reporter fragments T18 and T25 to EntA and EntE in both N-terminal and C-terminal orientations. To validate functionality of our fusion proteins, we performed Chrome Azurol S (CAS) assays using E. coli entE- and entA- knockout strains transformed with our BACTH constructs. We found that transformants expressing N-terminal and C-terminal T18/T25 fusions to EntE exhibited CAS signals, indicating that these constructs could rescue the entE- phenotype. While expression of EntA with N-terminal T18/T25 fusions exhibited CAS signals, C-terminal fusions did not, presumably due to disruption of the EntA tetramer in vivo. Bacterial growth assays supported our CAS findings. Co-transformation of functional T18/T25 fusions into cya- E. coli BTH101 cells resulted in positive BACTH signals only when T18/T25 fragments were fused to the N-termini of both EntA and EntE. Co-expression of N-terminally fused EntA with C-terminally fused EntE resulted in no detectable BACTH signal. Analysis of protein expression by Western blotting confirmed that the loss of BACTH signal was not due to impaired expression of fusion proteins. Based on our results, we propose that the N-termini of EntA and EntE are proximal in the intracellular complex, while the EntA N-terminus and EntE C-terminus are distal. A protein-protein docking simulation using SwarmDock was in agreement with our experimental observations.

Divisions:Concordia University > Faculty of Arts and Science > Chemistry and Biochemistry
Item Type:Article
Refereed:Yes
Authors:Pakarian, Paknoosh and Pawelek, Peter D.
Journal or Publication:Biochimie
Date:August 2016
Funders:
  • NSERC
ID Code:981276
Deposited By: PETER PAWELEK
Deposited On:19 May 2016 17:53
Last Modified:18 Jan 2018 17:52
All items in Spectrum are protected by copyright, with all rights reserved. The use of items is governed by Spectrum's terms of access.

Repository Staff Only: item control page

Downloads per month over past year

Back to top Back to top