Bohl, Thomas2018-08-142018-08-142018-05https://hdl.handle.net/11299/198993University of Minnesota Ph.D. dissertation.May 2018. Major: Biochemistry, Molecular Bio, and Biophysics. Advisor: Hideki Aihara. 1 computer file (PDF); xii, 164 pages.Gram-negative bacteria are distinguished from Gram-positive bacteria by the secondary membrane that surrounds their peptidoglycan cell wall. The outer leaflet of this membrane is primarily composed of the glycolipid lipopolysaccharide (LPS), which has lipid A, core oligosaccharide, and O-antigen portions. LPS helps protect Gram-negative bacteria from hydrophobic toxins and, in pathogenic bacteria, from the host immune system. The membrane anchor portion of LPS (lipid A) is responsible for stimulation of the inflammatory response of the mammalian immune system by LPS via activation of the Toll-like receptor 4/myeloid differentiation factor 2 complex. In systemic infections, overstimulation of this receptor causes acute inflammation, which can cause septic shock. Modifications to the LPS, particularly to the lipid A portion, can help bacteria evade the host immune system by disguising the bacteria, modulating the inflammatory response, and inhibiting interactions with antimicrobial host factors. Lipid A is synthesized in the well characterized and largely conserved Raetz pathway in the cytosol and at the cytosolic face of the inner membrane. The non-repeating core oligosaccharide is synthesized on lipid A the cytoplasmic face of the inner membrane, and the repeating O-antigen polysaccharide is attached to the core-lipid A molecule at the periplasmic face of the inner membrane. The completed LPS molecules are then transported to the extracellular leaflet of the outer membrane. Structures of proteins involved in LPS synthesis have proved critical to our understanding of LPS synthesis and transport. Moreover, these structures provide targets for rational design of antibiotics targeting Gram-negative bacteria. As the Raetz pathway is the most conserved part of LPS synthesis, the enzymes of the Raetz pathway provide particularly promising targets for development of broad spectrum antibiotics, such as those needed to treat sepsis. Therefore, I studied the structures of two enzymes in the Raetz pathway (LpxH and LpxB). LpxH was crystallized with the α-helical substrate-binding cap domain in a displaced conformation, suggesting that this domain is highly mobile. The structural dynamics of this domain and their relevance to substrate binding were further explored by hydrogen-deuterium exchange mass spectrometry, molecular dynamics simulations, and activity assays. These data supported a model in which a loop in the core hydrolase domain acts as a wedge to promote opening of the capping helices and allow facile substrate binding between these helices. In addition, the first structure of LpxB was determined showing a Glycosyltransferase B superfamily (GT-B) fold modified by the formation of a novel C-terminally swapped dimer wherein the last 87 residues of one subunit complete the GT-B fold of the other subunit. Furthermore, the binding site of the sugar-donor substrate was identified by a structure of LpxB with the UDP product bound. Activity assays supported the formation of this C-terminally swapped dimer in solution and showed that a surface-exposed hydrophobic patch is critical for LpxB activity, which suggested this patch allows productive membrane association required for substrate binding. Thus, the present research has expanded our understanding of two enzymes important in Gram-negative bacterial physiology that are potential targets for antibiotic development.englycosyltransferaseGram-negative bacterialipopolysaccharideprotein structurepyrophosphatasex-ray crystallographyThesis or Dissertation