1 Department of Chemistry and Biochemistry, Concordia University, 7141 Sherbrooke St., W., Montreal, Quebec, H4B 1R6, Canada.
2 Department of Chemistry and Biochemistry, Concordia University, 7141 Sherbrooke St., W., Montreal, Quebec, H4B 1R6, Canada. Electronic address: peter.pawelek@concordia.ca.

Siderophores are high-affinity small-molecule chelators employed by bacteria to acquire iron from the extracellular environment. The Gram-negative bacterium Escherichia coli synthesizes and secretes enterobactin, a tris-catechol siderophore. Enterobactin is synthesized by six cytoplasmic enzyme activities: EntC, EntB (isochorismatase (IC) domain), EntA, EntE, EntB (aryl carrier protein (ArCP) domain), and EntF. While various pairwise protein-protein interactions have been reported between EntB, EntA, EntE, and EntF, evidence for an interaction between EntC and EntB has remained elusive. We have employed bacterial two-hybrid assays and in vivo crosslinking to demonstrate an intracellular EntC-EntB interaction. A T18-EntC/T25-EntB co-transformant exhibited a positive two-hybrid signal compared to a control T18-EntC/T25 co-transformant. In vivo formaldehyde crosslinking of E. coli cells co-expressing HA-tagged EntB and H6-tagged EntC resulted in an observable ~80 kDa band on Western blots that cross-reacted with anti-HA and anti-H6, corresponding to one HA-EntB monomer (33 kDa) crosslinked with one H6-EntC monomer (45 kDa). This band disappeared upon sample boiling, confirming it to be a formadehyde-crosslinked species. Bands of molecular masses greater than 80 kDa that cross-reacted with both antibodies were also observed. Automated docking of the crystal structures of monomeric EntC and dimeric EntB resulted in a top-ranked candidate docked ensemble in which the active sites of EntC and EntB were oriented in apposition and connected by an electropositive surface potentially capable of channeling negatively charged isochorismate. These research outcomes provide the first reported evidence of an EntC-EntB interaction, as well as the first experimental evidence of higher-order complexes containing EntC and EntB.