Smallcombe, Caroline (2016) Structural studies of the recognition of bacterial lipopolysaccharides by human surfactant protein-D. Doctoral thesis, Keele University.

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SP-D is a large hydrophilic protein, consisting of four collagenous trimers, which is secreted by alveolar type II and non-ciliated bronchiolar epithelial cells and participates in calcium-dependant agglutination of inhaled microorganisms. Known high-resolution crystal structures of mono- and disaccharide bound recombinant human SP-D include a heptose disaccharide that mimics the inner core fragment of bacterial lipopolysaccharide. This thesis focuses on the structural characterisation of SP-D binding to lipopolysaccharides from Gram-negative bacteria. Intact LPS and several hydrolysed smooth and rough mutants were soaked into native crystals of a recombinant head and neck fragment of SP-D. Those soaked with hydrolysed Escherichia coli J5 and Salmonella minnesota R7 LPS showed electron density corresponding to ligand in the ligand-binding site. All crystals processed conformed to spacegroup P21, all with unit cells close to a= 55, b= 108, c= 55 Å, = 91°. The maximum resolution diffraction observed was 1.63 Å.

The R7-soaked structure was refined at 1.77 Å, with a final R-factor of 18.71% and R-free of 22.05%. The structure reveals a well-defined trisaccharide unit of two heptose saccharides and a single Kdo residue in protein chains B and C. The Kdo is present as a five-membered, anhydro residue known to form during mild acid hydrolysis. The third, outermost heptose of the R7 inner core is not visible in the electron density.

The refined structure demonstrates binding of LPS via the O6 and O7 hydroxyl groups of the glycerol sidechain of HepI, the innermost heptose, demonstrating for the first time structurally not only the binding of a physiologically relevant bacterially derived ligand but also the recognition of a non-terminal monosaccharides by SP-D. No direct interaction is observed between the second heptose and the protein, but two hydrogen bonds are seen between the anhydro-Kdo and the amino acids Arg343 and Asp325 which flank the SP-D ligand binding pocket.

Item Type: Thesis (Doctoral)
Subjects: Q Science > QR Microbiology
Divisions: Faculty of Natural Sciences > School of Life Sciences
Contributors: Greenhough, TJ (Thesis advisor)
Depositing User: Lisa Bailey
Date Deposited: 13 Apr 2017 08:29
Last Modified: 29 Jul 2022 15:51

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