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Ligand-Induced Conformational Rearrangements Promote Interaction between the Escherichia coli Enterobactin Biosynthetic Proteins EntE and EntB

Title:

Ligand-Induced Conformational Rearrangements Promote Interaction between the Escherichia coli Enterobactin Biosynthetic Proteins EntE and EntB

Khalil, Sofia and Pawelek, Peter D. (2009) Ligand-Induced Conformational Rearrangements Promote Interaction between the Escherichia coli Enterobactin Biosynthetic Proteins EntE and EntB. Journal of Molecular Biology, 393 (3). 658-671 . ISSN 0022-2836

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Official URL: http://dx.doi.org/10.1016/j.jmb.2009.08.036

Abstract

Siderophores are small-molecule iron chelators that many bacteria synthesize and secrete in order to survive in iron-depleted environments. Biosynthesis of enterobactin, the Escherichia coli catecholate siderophore, requires adenylation of 2,3-dihydroxybenzoic acid (2,3-DHB) by the cytoplasmic enzyme EntE. The DHB-AMP product is then transferred to the active site of holo-EntB subsequent to formation of an EntE–EntB complex. Here we investigate the binding of 2,3-DHB to EntE and how DHB binding affects EntE–EntB interaction. We overexpressed and purified recombinant forms of EntE and EntB with N-terminal hexahistidine tags (H6-EntE and H6-EntB). Isothermal titration calorimetry showed that 2,3-DHB binds to H6-EntE with a 1:1 stoichiometry and a Kd of 7.4 μM. Fluorescence spectra revealed enhanced 2,3-DHB emission at 440 nm (λex = 280 nm) when bound to H6-EntE due to fluorescence resonance energy transfer (FRET) between EntE intrinsic fluorophore donors and bound 2,3-DHB acceptor. A FRET signal was not observed when H6-EntE was mixed with either 2,5-dihydroxybenzoic acid or 3,5-dihydroxybenzoic acid. The H6-EntE–2,3-DHB FRET signal was quenched by H6-EntB in a concentration-dependent manner. From these data, we were able to determine the EC50 of EntE–EntB interaction to be approximately 1.5 μM. We also found by fluorescence and CD measurements that H6-EntB can bind 2,3-DHB, resulting in conformational changes in the protein. Additional alterations in H6-EntB near-UV and far-UV CD spectra were observed upon mixture with H6-EntE and 2,3-DHB, suggesting that further conformational rearrangements occur in EntB upon interaction with substrate-loaded EntE. We also found that H6-EntB as a bait protein pulled down a higher concentration of chromosomally expressed EntE in the presence of exogenous 2,3-DHB. Taken together, our results show that binding of 2,3-DHB to EntE and EntB primes these proteins for efficient complexation, thus facilitating direct channeling of the siderophore precursor 2,3-DHB-AMP.

Divisions:Concordia University > Faculty of Arts and Science > Chemistry and Biochemistry
Item Type:Article
Refereed:Yes
Authors:Khalil, Sofia and Pawelek, Peter D.
Journal or Publication:Journal of Molecular Biology
Date:October 2009
Funders:
  • NSERC
ID Code:6535
Deposited By:PETER PAWELEK
Deposited On:24 Mar 2010 13:25
Last Modified:08 Dec 2010 18:38
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