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Structural studies of surfactant protein D in complex with
bacterial lipopolysaccharide ligands

da Silva, Ruben Filipe

Structural studies of surfactant protein D in complex with
bacterial lipopolysaccharide ligands Thumbnail


Authors

Ruben Filipe da Silva



Abstract

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 a-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.

Publicly Available Date Mar 28, 2024

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