title: 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.
authors.affiliation: Department of Chemistry and Biochemistry, Concordia University, 7141 Sherbrooke Street, West, Montreal, Quebec, Canada.
authors.forename: Sofia
authors.initials: S
authors.lastname: Khalil
authors.forename: Peter D
authors.initials: PD
authors.lastname: Pawelek
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D055162:Biocatalysis; D002151:Calorimetry; D002876:Chromosomes, Bacterial / Q000378:metabolism; D002942:Circular Dichroism; D004758:Enterobactin / Q000096:biosynthesis*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D006867:Hydrolases / Q000737:chemistry* / Q000378:metabolism*; D062385:Hydroxybenzoates / Q000737:chemistry; D007536:Isomerism; D008024:Ligands; D008025:Ligases / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D040681:Structural Homology, Protein; D013379:Substrate Specificity
chemical_list: D029968:Escherichia coli Proteins; D062385:Hydroxybenzoates; D008024:Ligands; D010455:Peptides; D004758:Enterobactin; C009135:2,3-dihydroxybenzoic acid; D006867:Hydrolases; C064012:isochorismatase; D008025:Ligases; C061746:2,3-dihydroxybenzoate-AMP ligase, E coli