Molecular basis for peptidoglycan recognition by a bactericidal lectin

Abstract

RegIII proteins are secreted C-type lectins that kill Gram-positive bacteria and play a vital role in antimicrobial protection of the mammalian gut. RegIII proteins bind their bacterial targets via interactions with cell wall peptidoglycan but lack the canonical sequences that support calcium-dependent carbohydrate binding in other C-type lectins. Here, we use NMR spectroscopy to determine the molecular basis for peptidoglycan recognition by HIP/PAP, a human RegIII lectin. We show that HIP/PAP recognizes the peptidoglycan carbohydrate backbone in a calcium-independent manner via a conserved "EPN" motif that is critical for bacterial killing. While EPN sequences govern calcium-dependent carbohydrate recognition in other C-type lectins, the unusual location and calcium-independent functionality of the HIP/PAP EPN motif suggest that this sequence is a versatile functional module that can support both calcium-dependent and calcium-independent carbohydrate binding. Further, we show HIP/PAP binding affinity for carbohydrate ligands depends on carbohydrate chain length, supporting a binding model in which HIP/PAP molecules "bind and jump" along the extended polysaccharide chains of peptidoglycan, reducing dissociation rates and increasing binding affinity. We propose that dynamic recognition of highly clustered carbohydrate epitopes in native peptidoglycan is an essential mechanism governing high-affinity interactions between HIP/PAP and the bacterial cell wall.

Fig. 1.

HIP/PAP lacks canonical C-type lectin-carbohydrate binding motifs. (A) NMR structure of HIP/PAP (PDB code 2GO0.pdb) (22), with Loops 1 and 2 of the long loop region in Red and the β4 strand in Blue. (B) Alignment of the long loop region of RegIII family members with other C-type lectin family members. MBP-C and DC-SIGN harbor Loop 2 EPN motifs that govern sugar ligand binding and confer selectivity for mannose (Man) and GlcNAc (5). Asialoglycoprotein receptor (ASGP-R) and aggrecan contain Loop 2 QPD motifs that confer selectivity for Gal and GalNAc. Despite their selective binding to GlcNAc and Man polysaccharides (1), RegIII lectins lack the Loop 2 EPN motif.

Fig. 2.

Peptidoglycan induces HIP/PAP Loop 1 conformational changes that encompass a conserved EPN motif. (A) Quantification of HIP/PAP chemical shift changes from the ¹⁵N/¹H HSQC in the presence of 1.3 mM solubilized Staphylococcus aureus peptidoglycan (sPGN). The Red Dotted Line indicates chemical shift changes greater than the mean +2σ (0.029 ppm). (B) ¹⁵N/¹H HSQC spectra of 100 μM ¹⁵N-HIP/PAP titrated with increasing concentrations of sPGN. Nδ2, side-chain amide. (C) E114 is part of a HIP/PAP Loop 1 EPN motif that is conserved in RegIIIγ but is lacking in Ca²⁺-dependent C-type lectins. (D) ¹⁵N/¹H-HSQC spectra of 100 µM ¹⁵N-HIP/PAP in the absence and presence of 1 mM CaCl₂. The lack of chemical shift perturbations indicates no isomerization of the E-P peptide bond in the presence of Ca²⁺. (E) C(CO)NH TOCSY strip of ¹³C/¹⁵N-HIP/PAP P115. The separation between the Cγ and Cβ peaks is 5.64 ppm, which is within the expected range for a trans peptide bond (10). Dashed Circles indicate the cross-peak locations predicted for a cis peptide bond.

Fig. 3.

HIP/PAP E114 is essential for peptidoglycan binding and bactericidal activity. (A) Staphylococcus aureus sPGN titrated into 100 μM ¹⁵N-HIP/PAP Loop 1 mutants. HIP/PAP-E114Q has a reduced affinity for sPGN, as evidenced by a decrease in the dose-dependent chemical cross-peak perturbation of T113 in the E114Q mutant. K_ds were derived from chemical shift changes at eight Loop 1 residues and averaged (values ± SEM are also reported in Table 1). (B) Point mutations in HIP/PAP Loop 1 attenuate bactericidal activity. Listeria monocytogenes was incubated with purified wild-type or mutant HIP/PAP for 2 h at 37 °C and quantified by dilution plating. Assays were done in triplicate. Mean ± SEM is plotted.

Fig. 4.

Carbohydrate recognition by the HIP/PAP Loop 1 EPN motif depends on saccharide chain length. (A) Quantification of HIP/PAP chemical shift changes from the ¹⁵N/¹H HSQC recorded in the presence of 20 mM chitopentaose. The Red Dotted Line indicates chemical shift perturbations greater than the mean +2σ (0.024 ppm). (B) Overlay of residue E114 backbone amide and N116 side-chain amide chemical shifts with addition of 20 mM GlcNAc, chitobiose, chitotriose, or chitopentaose. (C) Mutation of HIP/PAP E114 abolishes chitopentaose binding. Superimposed ¹⁵N/¹H HSQC spectra of ¹⁵N-labeled HIP/PAP-E114Q with titrated chitopentaose, showing an absence of chemical shift perturbations in the mutant protein.

Fig. 5.

Carbohydrate is the principal determinant of the HIP/PAP-peptidoglycan interaction. ¹⁵N/¹H HSQC spectra overlay highlighting the opposing trajectories of E114 backbone amide and N116 side-chain amide chemical shift perturbations in response to titration with chitopentaose or GMDP. Type I and II chemical shift trajectories are indicated. sPGN induces only Type I trajectories (Fig. 2B), indicating that carbohydrate is the principal determinant of the HIP/PAP interaction with soluble native peptidoglycan.

Fig. 6.

The presence of a Loop 1 EPN motif predicts peptidoglycan binding in other RegIII family members. (A) Alignments of human and mouse RegIII family members showing a conserved EPN sequence in Loop 1 of RegIIIγ and RegIIIβ but not RegIIIα. (B) RegIIIβ but not RegIIIα binds peptidoglycan. Ten μg of recombinant RegIIIβ or RegIIIα was added to 50 μg insoluble Bacillus subtilis peptidoglycan and pelleted. Pellet and supernatant (Supe) fractions were analyzed by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie blue staining. The lower molecular weight form of RegIIIβ in the pellet results from cleavage at an N-terminal trypsin site by a peptidoglycan-associated proteolytic activity (1, 4). (C) RegIIIβ selectively binds to GlcNAc and mannose polysaccharides. RegIIIβ was bound to immobilized polysaccharide using a previously described assay (1). After washing, bound proteins were released by boiling and analyzed by SDS-PAGE and Coomassie blue staining. Unconj., unconjugated Sepharose.