Structural studies of the BAM complex, OmpU outer membrane protein and lipoprotein N-acyl transferase in Gram-negative bacteria

Li, Huanyu (2017) Structural studies of the BAM complex, OmpU outer membrane protein and lipoprotein N-acyl transferase in Gram-negative bacteria. Doctoral thesis, University of East Anglia.

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Abstract

Structural studies of membrane proteins represent a significant challenge in the field owing to their hydrophobic nature, unstable property and resistance to be crystallized. In Gramnegative bacteria, membrane proteins contribute to the characteristic membranous architecture composed of an asymmetric layer of outer membrane (OM) and a symmetric inner cytoplasmic membrane (IM).

Outer membrane proteins (OMPs) play essential roles in nutrient uptake, protein transport, outer membrane assembly, and pathogenesis of Gram-negative bacteria. In Escherichia coli, nearly all the outer membrane proteins are inserted into the outer membrane by the β-barrel assembly machinery (BAM), which contains one conserved membrane protein BamA and four lipoproteins BamBCDE. The individual protein structures of the BAM complex have been reported, but the mechanism of OMP assembly by the BAM complex is halted by a lack of structure of the whole complex. During the course of the collaborative BAM complex project, I participated in structural studies of the BAM complex and generated high resolution crystallographic diffraction data that contributes to one of the two determined structures of the BAM complex, and the structural insights have enlightened understanding of the in vivo insertion mechanism.
Of diverse types of β-barrel OMPs that are inserted into the OM by the BAM complex, an outer membrane protein called OmpU from Vibrio cholerae is a potential virulence factor in addition to its porin identity with undefined atomic structure. I determined the crystal structure of this OMP, in which the long and flexible extracellular loop L4 and a novel Nterminal coil in the pore lumen provide direct structural evidence underlying its particular functions.

The symmetric lipid bilayer of IM accommodates an even more diverse array of IMPs composed of the contrasting dominance of α-helices. Three IMPs are responsible for conducting post-translational modifications of lipoproteins in Gram-negative bacteria, a class of proteins destined to reside on the periplasmic side of either the IM or the OM via acyl chains post-translationally linked to the N-terminal cysteine residues, and they are called phosphatidylglycerol:prolipoprotein diacylglyceryl transferase (Lgt), Prolipoprotein signal peptidase (Lsp) and apolipoprotein N-acyltransferase (Lnt). Structural studies were carried out on Lnt but unsuccessful in determining the atomic structure. Recent structures of Lnt reported during the course of this project are consistent with earlier biochemical studies that piloted the understanding of its function and further elucidate the molecular mechanisms of its primary acyl-transfer function.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Medicine and Health Sciences
Depositing User: Jennifer Whitaker
Date Deposited: 31 Oct 2018 13:02
Last Modified: 31 Oct 2018 13:02
URI: https://ueaeprints.uea.ac.uk/id/eprint/68718
DOI:

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