NMR STUDIES OF BACTERIAL TYPE III SECRETION APPARATUS NEEDLE AND TIP PROTEINS AND THE NMR STRUCTURE OF THE HANTAVIRUS NUCLEOCAPSID COILED-COIL DOMAIN

Abstract

Many Gram-negative bacterial pathogens utilize type III secretion systems (TTSSs) for subverting the normal cellular functions of their target eukaryotic cells. The type III secretion apparatus (TTSA) functions like a syringe to inject proteins through an external needle and into a target cell's membrane and cytosol. The TTSA basal body spans the bacterial inner and outer membranes, and the external needle is topped with a tip complex that controls the secretion and delivery of translocator and effector proteins. The needle is formed by the polymerization of ~120 copies of a small acidic protein that is conserved among diverse pathogens. At the tip of the needle, a tip complex is assembled by tip proteins into a ring-like structure which serves as a platform for the assembly of the translocon by translocator proteins. We use NMR spectroscopy to understand how the needle is assembled and how the tip complex is assembled on top of the needle. We determined the solution structures of the BsaL needle monomer from Burkholderia pseudomallei and the PrgI needle monomer from Salmonella typhimurium. We characterized PrgI monomer-monomer interaction using NMR chemical shift mapping; and multiple contacts were found to be involved in Salmonella needle assembly. The tip complex is assembled by SipD, the tip protein in Salmonella, and BipD, the tip protein in Burkholderia. We also characterized PrgI-SipD and BipD-BsaL interactions by NMR. Despite weak binding affinities we learned that distinct binding sites of PrgI were involved in the PrgI-PrgI and PrgI-SipD interactions. Tip proteins were also reported to interact with deoxycholate (DOC), a small molecule component of bile acids. We also characterized the SipD-DOC interactions by NMR. Based on data described in this dissertation, we conclude that electrostatic contacts are important in needle assembly and needle-packing interactions may be different among these bacteria. With respect to PrgI the binding sites involved in the PrgI-PrgI and PrgI-SipD interactions are also distinct. In addition, SipD-PrgI and SipD-DOC interactions provide valuable structural information to understand the activation mechanism of type III secretion. The hantaviruses are emerging infectious viruses that in humans can cause a cardiopulmonary syndrome or a hemorrhagic fever with renal syndrome. The nucleocapsid (N) is the most abundant viral protein, and during viral assembly, the N protein forms trimers and packages the viral RNA genome. We determined the NMR structure of the N-terminal domain (residues 1-74, called N1-74) of the Andes hantavirus N protein. N1-74 forms two long helices (alpha 1 and alpha 2) that intertwine into a coiled coil domain. The conserved hydrophobic residues at the helix alpha 1-alpha 2 interface stabilize the coiled coil; however, there are many conserved surface residues whose function is not known. Site-directed mutagenesis, CD spectroscopy, and immunocytochemistry reveal that a point mutation in the conserved basic surface formed by Arg22 or Lys26 lead to antibody recognition based on the subcellular localization of the N protein. Thus, Arg22 and Lys26 are likely involved in a conformational change or molecular recognition when the N protein is trafficked from the cytoplasm to the Golgi, the site of viral assembly and maturation.