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Title: Structural studies of surfactant protein D in complex with bacterial lipopolysaccharide ligands
Author: da Silva, Ruben Filipe
ISNI:       0000 0004 6423 0606
Awarding Body: Keele University
Current Institution: Keele University
Date of Award: 2017
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This work is focused on the recognition of natural lipopolysaccharide (LPS) by the innate immune protein human lung surfactant protein D (hSP-D) in the form of a biologically active recombinant fragment (rfhSP-D), containing the α-helical coiled-coil and three carbohydrate recognition domains (CRD). Intact LPS from two bacterial strains, S. minnesota (R5 mutant) and H. influenzae type b Eagan (CA7 mutant), were delipidated by means of mild acid hydrolysis, leaving the purified polysaccharide (PS) to be used in X-ray diffraction studies by means of co-crystallisation with rfhSP-D. S. minnesota R7 full LPS was also investigated following development of a suitable solubilisation method which also utilised the LPS from E. coli O111:B4. The structural studies of rfhSP-D bound to H. influenzae Eagan CA7 PS (solved and refined at 2.98 Å) and to S. minnesota rough mutant LPS/PS (solved and refined at 3.3 Å) reveal that rfhSP-D binds to LPS preferentially through the non-terminal inner core heptose HepI via the O6’ and O7’ hydroxyls. rfhSP-D recognition of S. minnesota HepI shows a similar bound heptose orientation to that previously reported for heptose binding by rfhSP-D in the literature with an indication of normal Kdo in the inner core Kdo-Hep-Hep trisaccharide. rfhSP-D recognition of the HepI of H. influenzae Eagan CA7 reveals a novel bound heptose orientation, with the heptose rotated by 180° about C5-C6, resulting in the O6’ and O7’ hydroxyls being interchanged with respect to coordination to Ca1 and protein. The novel orientation of HepI is accompanied by a salt bridge being formed between the flanking residue Arg343 and Glu347, both of which adopt a previously unseen conformation. The novel binding mechanism of rfhSP-D for Eagan CA7 suggests flexibility in recognition and offers evidence to explain why this mutant binds more weakly than the Eagan 4A mutant to both rfhSP-D and hSP-D.
Supervisor: Greenhough, T. J. ; Shrive, A. K. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QH Natural history