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Ligand-induced conformational rearrangements promote interaction between the Escherichia coli enterobactin biosynthetic proteins EntE and EntB.

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 K(d) of 7.4 microM. Fluorescence spectra revealed enhanced 2,3-DHB emission at 440 nm (lambda(ex)=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 EC(50) of EntE-EntB interaction to be approximately 1.5 microM. 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.

journal_name

J Mol Biol

journal_title

Journal of molecular biology

authors

Khalil S,Pawelek PD

doi

10.1016/j.jmb.2009.08.036

subject

Has Abstract

pub_date

2009-10-30 00:00:00

pages

658-71

issue

eissn

0022-2836

issn

1089-8638

pii

S0022-2836(09)01037-7

journal_volume

393

pub_type

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Ligand-induced conformational rearrangements promote interaction between the Escherichia coli enterobactin biosynthetic proteins EntE and EntB.