Biomaterial Database

Ligand-induced conformational rearrangements promote interaction between the Escherichia coli enterobactin biosynthetic proteins EntE and EntB. 2009

Sofia Khalil, and Peter D Pawelek

Department of Chemistry and Biochemistry, Concordia University, 7141 Sherbrooke Street, West, Montreal, Quebec, Canada.

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.

UI	MeSH Term	Description	Entries
D007536	Isomerism	The phenomenon whereby certain chemical compounds have structures that are different although the compounds possess the same elemental composition. (From McGraw-Hill Dictionary of Scientific and Technical Terms, 5th ed)	Isomerisms
D008024	Ligands	A molecule that binds to another molecule, used especially to refer to a small molecule that binds specifically to a larger molecule, e.g., an antigen binding to an antibody, a hormone or neurotransmitter binding to a receptor, or a substrate or allosteric effector binding to an enzyme. Ligands are also molecules that donate or accept a pair of electrons to form a coordinate covalent bond with the central metal atom of a coordination complex. (From Dorland, 27th ed)	Ligand
D008025	Ligases	A class of enzymes that catalyze the formation of a bond between two substrate molecules, coupled with the hydrolysis of a pyrophosphate bond in ATP or a similar energy donor. (Dorland, 28th ed) EC 6.	Ligase,Synthetases,Synthetase
D008958	Models, Molecular	Models used experimentally or theoretically to study molecular shape, electronic properties, or interactions; includes analogous molecules, computer-generated graphics, and mechanical structures.	Molecular Models,Model, Molecular,Molecular Model
D008969	Molecular Sequence Data	Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories.	Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D010455	Peptides	Members of the class of compounds composed of AMINO ACIDS joined together by peptide bonds between adjacent amino acids into linear, branched or cyclical structures. OLIGOPEPTIDES are composed of approximately 2-12 amino acids. Polypeptides are composed of approximately 13 or more amino acids. PROTEINS are considered to be larger versions of peptides that can form into complex structures such as ENZYMES and RECEPTORS.	Peptide,Polypeptide,Polypeptides
D011485	Protein Binding	The process in which substances, either endogenous or exogenous, bind to proteins, peptides, enzymes, protein precursors, or allied compounds. Specific protein-binding measures are often used as assays in diagnostic assessments.	Plasma Protein Binding Capacity,Binding, Protein
D011487	Protein Conformation	The characteristic 3-dimensional shape of a protein, including the secondary, supersecondary (motifs), tertiary (domains) and quaternary structure of the peptide chain. PROTEIN STRUCTURE, QUATERNARY describes the conformation assumed by multimeric proteins (aggregates of more than one polypeptide chain).	Conformation, Protein,Conformations, Protein,Protein Conformations
D002151	Calorimetry	The measurement of the quantity of heat involved in various processes, such as chemical reactions, changes of state, and formations of solutions, or in the determination of the heat capacities of substances. The fundamental unit of measurement is the joule or the calorie (4.184 joules). (McGraw-Hill Dictionary of Scientific and Technical Terms, 4th ed)
D002876	Chromosomes, Bacterial	Structures within the nucleus of bacterial cells consisting of or containing DNA, which carry genetic information essential to the cell.	Bacterial Chromosome,Bacterial Chromosomes,Chromosome, Bacterial