Structural definition of hSP-D recognition of Salmonella enterica LPS inner core oligosaccharides reveals alternative binding modes for the same LPS.

Jamie R Littlejohn,Trevor J Greenhough,William A Neale,Ian Burns,Ruben F Da Silva,Carrie C Smallcombe,Rose-Marie A Mackay,Jens Madsen,Annette K Shrive,Howard W Clark,Derek W Hood,Alastair S Watson

doi:10.1371/journal.pone.0199175

Abstract

The crystal structures of a biologically and therapeutically active recombinant homotrimeric fragment of native human SP-D (hSP-D) complexed with the inner core oligosaccharide of the Salmonella enterica sv Minnesota rough strains R5 and R7 (rough mutant chemotypes Rc and Rd1) have been determined. The structures reveal that hSP-D specifically and preferentially targets the LPS inner core via the innermost conserved Hep-Kdo pair with the flexibility for alternative recognition when this preferred epitope is not available for binding. Hep-Kdo binding is achieved through calcium dependent recognition of the heptose dihydroxyethyl side chain coupled with specific interactions between the Kdo and the binding site flanking residues Arg343 and Asp325 with evidence for an extended binding site for LPS inner cores containing multiple Kdo residues. In one subunit of the R5-bound structure this preferred mode of binding is precluded by the crystal lattice and oligosaccharide is bound through the terminal inner core glucose. The structures presented here thus provide unique multiple insights into the recognition and binding of bacterial LPS by hSP-D. Not only is it demonstrated that hSP-D targets the highly conserved LPS proximal inner core Hep-Kdo motif, but also that hSP-D can recognise either terminal or non-terminal sugars and has the flexibility and versatility to adopt alternative strategies for bacterial recognition, utilising alternative LPS epitopes when the preferred inner core Hep-Kdo disaccharide is not available for binding.

Highlights

The innate immune protein human surfactant protein D is a collagenous C-type lectin prototypically associated with the lung surfactant but identified throughout the human body [1]
A number of studies of gram-negative bacteria, including Escherichia coli, Salmonella enterica serovar Minnesota and Haemophilus influenzae [19,20] have reported that lipopolysaccharide (LPS) on the bacterial surface mediates human surfactant protein D (hSP-D) binding with LPS recognition initially thought to be via terminal carbohydrate residues through carbohydrate recognition domain Ca-dependent binding of a mannose-type equatorial hydroxyl pair on the terminal monosaccharide [14,15,16]
Our recent structural definition of the recognition of H. influenzae Eagan strains inner core LPS by a recombinant fragment of native hSP-D [6,7,21] revealed that recognition occurs by calcium-dependent binding of the non-terminal heptose in the proximal inner core, with the Kdo positioned to interact with the binding site flanking residues Asp325 and Arg343 [15,16]

Summary

Introduction

The innate immune protein human surfactant protein D (hSP-D) is a collagenous C-type lectin prototypically associated with the lung surfactant but identified throughout the human body [1]. A number of studies of gram-negative bacteria, including Escherichia coli, Salmonella enterica serovar Minnesota and Haemophilus influenzae [19,20] have reported that lipopolysaccharide (LPS) on the bacterial surface mediates hSP-D binding with LPS recognition initially thought to be via terminal carbohydrate residues through carbohydrate recognition domain Ca-dependent binding of a mannose-type equatorial hydroxyl pair on the terminal monosaccharide [14,15,16]. Our recent structural definition of the recognition of H. influenzae Eagan strains inner core LPS by a recombinant fragment of native hSP-D (rfhSP-D) [6,7,21] revealed that recognition occurs by calcium-dependent binding of the non-terminal heptose in the proximal inner core, with the Kdo positioned to interact with the binding site flanking residues Asp325 and Arg343 [15,16]. A recent study by Reinhardt and colleagues has identified that a number of synthetic oligosaccharide cores can be bound by wildtype hSP-D with increased affinities for heptose-rich cores and a clear preference for a terminal Hep-Kdo motif compared to Hep alone [22]

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Results

Conclusion