Abstract

Protein complexes are essential components of many biological processes. Therefore, protein-protein interactions are crucial for many essential cellular functions and are considered good targets for the development of novel therapeutics.
Siderophore biosynthesis is one of the biological processes that has an absolute requirement for protein-protein interactions. Siderophores are small iron-scavenging molecules that are synthesized and secreted by iron-starved bacteria to chelate ferric iron (Fe3+) from the environment. Ferric iron, which is essential for survival and growth of most bacteria, is insoluble at neutral pH, or is bound to host iron storage proteins such as transferrin. By taking up Fe3+-siderophore complexes, such bacteria can survive and proliferate in low-iron environments.
Enterobactin is a catecholate type siderophore of E. coli that is synthesized in its cytoplasm by seven enzymes, EntA-F and EntH. These sequentially-related enzymes function together to produce enterobactin, which is a cyclic trimer of 2,3-dihydroxy-N-benzoyl-L-serine. Enterobactin biosynthetic enzymes are organized in two functional modules: the DHB module (EntCBA) and the non-ribosomal peptide synthesis (NRPS) module (EntBDEF). Interactions between EntBDEF in the NRPS module have been previously reported. Our research group has since reported in vitro evidence of an interaction between EntA and EntE, the enzymes at the interface of the DHB and NRPS modules.
The research presented here is focused on the identification of novel protein-protein interactions in the DHB module as well as the study of subunit orientation in the Ent complexes.
The first research chapter is centered on the subunit orientation in the intracellular EntA-EntE complex. In this study Chrome Azurol S (CAS) assays and bacterial adenylate cyclase two-hybrid (BACTH) assays were employed to study the EntA-EntE complexation in vivo. CAS assays were used to validate the functionality of EntA and EntE BACTH constructs. BACTH experiments were then performed to identify the intracellular complexation of EntA and EntE and to determine the orientation of EntA relative to EntE in the complex. BACTH results were further validated by automated docking simulations.
The second research chapter focuses on the construction of two Fur-controlled bidirectional protein expression vectors. Ferric Uptake Regulator (Fur) is a protein involved in iron homeostasis in E. coli. When intracellular iron is abundant, Fur forms a complex with Fe2+. This complex binds to the Fur box and inhibits the transcription of iron responsive genes such as ent genes. The Fur box is the consensus sequence that is located near or within the promoter region of iron responsive genes. The novel expression vectors are derivatives of low copy number plasmids pACYC184 and pBR322 and contain a bidirectional promoter, FLAG or HA tags, TEV cleavage site and a multiple cloning site (MCS) compatible with the MCS of BACTH vectors.
The third research chapter involves the identification of a novel protein-protein interaction between two enzymes in the DHB module, EntA and EntB. Furthermore, ternary complex formation between EntA, EntB and EntE was investigated in this chapter. BACTH was employed as the primary method for the detection of protein-protein interactions between EntA and EntB. Functionality of all the constructs used in the BACTH was confirmed using the CAS assay and growth studies. Automated docking simulations were also used to generate a model for an EntA-EntB-EntE ternary complex. The EntE-EntB interaction interface in the generated model was in accordance with the published crystal structure for the EntE-EntB complex and therefore supported our experimental results.