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

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.

Fig 1. Core LPS structure of Salmonella enterica rough mutant strains.

The LPS phenotypes (Ra, Rb1, Rb2, Rb3, Rc, Rd1, Rd2, and Re) and the mutant strains R5 and R7 are shown (modified from Mansfield and Forsythe, 2001 [29]). Glc: glucose; GlcNAc: N-acetyl-glucosamine; Gal: galactose; Hep: L-D-Heptose; P: phosphate; PEtn: phosphoethanolamine; Kdo: 3-deoxy-D-manno-oct-2-ulosonic acid.

Fig 2. Structure of the product of mild acid hydrolysis of S. enterica Minnesota rough mutants R5 and R7.

Glc: glucose; Hep: L-D-Heptose; Kdo: 3-deoxy-D-manno-oct-2-ulosonic acid.

Fig 3. Structure of the 4,7 closure furanoid derivative (anhydro Kdo) of Kdo following LPS delipidation by mild acid hydrolysis and β-elimination of the Kdo O4 substituent, reported by Shrive and co-workers (PDB ID 4E52, ligand KD5).

Anhydro Kdo is proposed to be a racemic mixture (Auzanneau et al., 1991 [43]) with the 2-oxobutanoic acid side chain at C4 both alpha and beta to the glycosidic bond (at C5). (a) Original Kdo numbering retained. (b) Numbering according to pdb entry 4E52 (alpha) and used for the structure here.

Fig 4. Electron density for the Salmonella enterica oligosaccharides bound to rfhSP-D (subunit B) with HepI in the Ca1 binding site.

The calcium ion is in green and the four calcium and ligand coordinating residues Glu321, Asn323, Glu329 and Asn341 are shown in cpk. (a) R7 oligosaccharide KdoI (anhydro)-HepI-HepII (b) R5 oligosaccharide KdoI (anhydro)-HepI-HepII-Glc1. Maps are 2mFo-DFc contoured at 1σ.

Fig 5. S. enterica R5 oligosaccharide bound to rfhSP-D (subunit C) with the inner core HepI in the Ca1 binding site.

KdoI (anhydro) interacts with both Asp325 and Arg343. Similar binding is observed in subunit B and in subunits B and C of the R7-bound structure which lacks the terminal glucose GlcI. Calcium coordinating bonds are represented by dots and protein-ligand interactions by dashes. The interactions of Glu333 are also indicated.

Fig 6. S. enterica R5 oligosaccharide bound to rfhSP-D (subunit A) with the terminal glucose GlcI in the Ca1 binding site.

HepI interacts with Arg343. No ligand was observed in subunit A of the R7-bound structure due to crystal lattice constraints. Calcium coordinating bonds are represented by dots and protein-ligand interactions by dashes. The interactions of Glu333 are also indicated.

Fig 7

(a) Superposition of subunit A (GlcI bound) in cyan and subunit B (HepI bound) in yellow of the R5 LPS-bound structure following a least-squares fit of the CRD protein main chain atoms. (b) Superposition of subunit A (GlcI bound) in cyan of the R5 LPS-bound structure and subunit A (Glc1 bound) in coral of the maltose-bound structure [14] following a least-squares fit of the CRD protein main chain atoms.

Fig 8. Model of complete S. enterica R5 oligosaccharide bound to rfhSP-D with the inner core HepI in the Ca1 binding site.

KdoI (intact) is positioned to interact with Asp325, with KdoII and KdoIII interacting with Arg343 and Glu347 respectively. (a) subunit C, R5-bound structure (b) the rfhSP-D trimer with R5 similarly placed in each subunit and the link to lipid A from KdoI O2' indicated by ★.