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Characterization of structure, dynamics, function and interactions of components from the type IV secretion system of Xanthomonas citri by solution nuclear magnetic resonance

Oliveira, Luciana Coutinho De

Biblioteca Digital de Teses e Dissertações da USP; Universidade de São Paulo; Instituto de Química 2016-02-01

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  • Título:
    Characterization of structure, dynamics, function and interactions of components from the type IV secretion system of Xanthomonas citri by solution nuclear magnetic resonance
  • Autor: Oliveira, Luciana Coutinho De
  • Orientador: Salinas, Roberto Kopke
  • Assuntos: Biologia Estrutural; Ressonância Magnética Nuclear; Sistema De Secreção Do Tipo Iv; Xanthomonas Citri; Nuclear Magnetic Resonance; Structural Biology; Type Iv Secretion System
  • Notas: Tese (Doutorado)
  • Descrição: Bacteria use specialized systems, called secretion systems, in order to translocate substrates to the environment or to other cells, or even to uptake molecules from the exterior environment. Six different secretion systems have been described in Gram-negative bacteria. The Type IV Secretion System (T4SS) is involved in translocation of virulence factors, bacterial conjugation, uptake and release of DNA, and in the secretion of antibacterial toxins. The T4SS channel corresponds to a toroidal upramolecular complex consisting of 14 repetitions of the VirB7-VirB9-VirB10 heterotrimer. This channel, also called \"core complex\", is divided in two layers, an outer layer consisting of the VirB7 lipoprotein in complex with the C-terminal domains of VirB9 (VirB9CT) and VirB10 (VirB10CT), and an inner layer composed by the N-terminal domains of VirB9 (VirB9NT) and VirB10 (VirB10NT). Xanthomonas citri pv. citri (Xac) is a gram-negative bacterium that infects citrus plants causing a disease called \"citrus canker\". Although not directly involved in causing the disease, the chromosomally encoded T4SS is responsible for the secretion of toxins, working as a bacterial killing machine (Souza et al., 2015). The three-dimensional structure of Xac\'s VirB7 obtained by Nuclear Magnetic Resonance (NMR) spectroscopy (PDB 2L4W) revealed that, unlike the canonical VirB7, Xac\'s VirB7 consists of a flexible N-terminal domain followed by a C-terminal globular domain. The flexible N-terminal tail is involved in interaction with VirB9CT. In this thesis, the NMR structure of the complex formed between VirB9CT and a peptide derived from the N-terminal tail of Xac-VirB7 (VirB7NT) was solved. This complex is stabilized by hydrophobic interactions involving the side chains of particular amino acid residues such as Phe30, Trp34 and Val37 in VirB7, and Arg250, Tyr167 and Tyr169 in VirB9. Mutations of such amino acids affect not only the stability of the VirB9:VirB7 complex in vitro, but also reduce the T4SS activity and impairs its assembly in vivo. Furthermore, the ability of forming VirB7:VirB7 oligomers is essential for a functional T4SS, although it is not required for assembling the complex. The structural propensity and flexibility of a fragment derived from the proline-rich region (PRR) of the N-terminal tail of VirB10 (VirB10NT - residues 85 to 182) were studied. Measurements of the {1H}-15N heteronuclear NOE showed that VirB10NT is highly flexible on a sub-nanosecond time scale. Analysis of chemical shifts and NOEs showed that the ensemble and time average conformation of VirB10NT consists of a short alpha helix between residues 151-163, and that this helix is involved in interactions with VirB9NT. These findings provide the first compelling evidence for the interaction between the N-terminal domains of VirB9 and VirB10, and for the existence of significant flexibility within Xacs T4SS.
  • DOI: 10.11606/T.46.2016.tde-15092016-084423
  • Editor: Biblioteca Digital de Teses e Dissertações da USP; Universidade de São Paulo; Instituto de Química
  • Data de criação/publicação: 2016-02-01
  • Formato: Adobe PDF
  • Idioma: Inglês
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  • Realização: ABCD-USP USP   Logos de Redes Sociais: Freepik

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