NMR Investigations of Bacterial Periplasmic Binding Proteins Involved in Ferric-Siderophore and Amino Acid Transport

Abstract

Periplasmic binding proteins (PBPs) can be found in the periplasmic space between the outer membrane and the cytoplasmic membrane (CM) of Gram-negative bacteria. PBPs play an important role in shuttling essential nutrients from the periplasm to their cognate CM ABC transporters for import into the cytoplasm. Understanding the mechanism by which PBPs can bind and release their ligand has implications with respect to the development of therapeutic compounds that target these PBP mediated pathways. This dissertation examines the structure, function and dynamics of PBPs involved in ferric-siderophore and amino acid transport in E. coli. I solved the first nuclear magnetic resonance (NMR) solution structures of FepB, a PBP involved in ferric-enterobactin transport whose large size (34 kDa) was technically challenging and required the development and use of specialized NMR methodologies. Changes in the structural dynamics of FepB upon ligand binding reveals a novel mechanism by which this class of PBPs is able to function. A second PBP, the histidine binding protein HisJ was also studied by NMR and the solution conformation of its apo-state was determined. HisJ is a bilobal protein which differs structurally from FepB in that it has two linkers connecting its domains as opposed to one. Comparison of the structure, function and dynamics of HisJ and its individually isolated domain proteins reveals differences that shed light on the ligand binding mechanism of this class of PBPs. In addition, the conformational dynamics associated with HisJ were explored with molecular dynamics (MD) simulations. HisJ and other structurally related proteins are known to undergo large-scale opening and closing motions upon ligand binding. My MD simulations show that the apo-open conformation of HisJ is able to spontaneously sample a closed conformation in the absence of ligand. Finally, interaction studies were performed between PBPs and the TonB protein, which is involved in energy transduction to the outer membrane in Gram-negative bacteria. Complex formation between TonB and FepB or FhuD could be detected in NMR and ITC experiments; and a PBP interaction surface on TonB was identified by NMR chemical shift perturbations. Our results also suggest that TonB-PBP interactions do not form in the presence of the OM transporter. In this dissertation, I have investigated the solution structures of two different PBPs by NMR. Although they are classified as structurally related, FepB and HisJ are shown to be distinct with respect to how they bind their ligands and what role protein dynamics plays in this process.