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. 2014 Jun 30:5:4269.
doi: 10.1038/ncomms5269.

Molecular basis for bacterial peptidoglycan recognition by LysM domains

Stéphane Mesnage  1 , Mariano Dellarole  2 , Nicola J Baxter  3 , Jean-Baptiste Rouget  4 , Jordan D Dimitrov  5 , Ning Wang  6 , Yukari Fujimoto  6 , Andrea M Hounslow  1 , Sébastien Lacroix-Desmazes  5 , Koichi Fukase  6 , Simon J Foster  1 , Michael P Williamson  1
Affiliations
Free PMC article

Molecular basis for bacterial peptidoglycan recognition by LysM domains

Stéphane Mesnage et al. Nat Commun. .
Free PMC article

Abstract

Carbohydrate recognition is essential for growth, cell adhesion and signalling in all living organisms. A highly conserved carbohydrate binding module, LysM, is found in proteins from viruses, bacteria, fungi, plants and mammals. LysM modules recognize polysaccharides containing N-acetylglucosamine (GlcNAc) residues including peptidoglycan, an essential component of the bacterial cell wall. However, the molecular mechanism underpinning LysM-peptidoglycan interactions remains unclear. Here we describe the molecular basis for peptidoglycan recognition by a multimodular LysM domain from AtlA, an autolysin involved in cell division in the opportunistic bacterial pathogen Enterococcus faecalis. We explore the contribution of individual modules to the binding, identify the peptidoglycan motif recognized, determine the structures of free and bound modules and reveal the residues involved in binding. Our results suggest that peptide stems modulate LysM binding to peptidoglycan. Using these results, we reveal how the LysM module recognizes the GlcNAc-X-GlcNAc motif present in polysaccharides across kingdoms.

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Figures

Figure 1
Figure 1. Contribution of LysM modules (1–6) and linker sequences (L) to the folding and binding activity of the LysM domain.
(a) Domain organization of E. faecalis glucosaminidase AtlA and LysM-derived polypeptides studied. SP, signal peptide; T,E,P-rich, N-terminal domain of unknown function rich in threonine, glutamic acid and proline residues. Amino acid numbers refer to the transition between modules. (b) Sequence alignment of the six LysM modules present in the C-terminal domain of AtlA. Numbering refers to residues corresponding to the LysM module (49 residues); linker sequences are in italics. Identical amino acids in at least four modules are in dark grey boxes, conserved amino acids are in light grey boxes. Secondary structures determined by NMR for 1LysM are indicated. (c) SDS–PAGE of purified recombinant LysM polypeptides described in a; the molecular weight of each purified polypeptide is indicated. (d) Differential scanning calorimetry (DSC) profiles of recombinant LysM polypeptides in the absence (No PG) and presence of peptidoglycan (+PG). Red and dotted blue lines are theoretical curves corresponding to a two-state or a three-state unfolding, respectively. (e) Detection of peptidoglycan binding activities of LysM domains harbouring one to six LysM modules by ELISA.
Figure 2
Figure 2. Identification of the structural motif recognized by LysM. Red rectangle is GlcNAc, pink rectangle is MurNAc, blue circles are peptide stem with (darker blue) crosslinking peptides.
The six LysM module polypeptide (L1–L6) was immobilized on a CM5 chip and binding was measured with surface plasmon resonance (SPR) using analyte mixtures corresponding to soluble Staphylococcus aureus peptidoglycan fragments generated using three enzymes with distinct cleavage specificities: (a) amidase digest, containing a mixture of peptide stems and glycan chains; (b) endopeptidase digest, containing linear (non cross-linked) peptidoglycan; (c) muramidase digest, containing disaccharides linked to peptide stems, some of which are cross-linked; (d) synthetic peptide stems. RU, resonance units (e) Affinity purification of AtlA L1–L6 LysM domain and cytochrome c with insoluble polysaccharides: 1, peptidoglycan; 2, chitin; 3, cellulose; 4, xylan. Protein remaining in the supernatant (Unbound) and associated with the pellet (Bound) were analysed by SDS–PAGE and Coomassie staining. Cytochrome c was used as a control, as this protein displays a similar isoelectric point to the LysM domain (pI=9.6 versus 10.06, respectively). No binding activity was detected with cytochrome c using any of the polysaccharides.
Figure 3
Figure 3. The 1LysM module and its interaction with carbohydrates.
(a) NMR structure of 1LysM. β-Sheets are residues T4-V8 and G42-V47; α-helices are residues L14-Y21 and V25-N32. (b) 15N HSQC NMR spectra of 1LysM, free (blue) and saturated with 44 equivalents of GlcNAc6 (red). Residues showing significant chemical shift changes are indicated by arrows, and residues broadened and not reappearing by the end of the titration are circled. (c) Summary of chemical shift changes observed for the titration of 1LysM with peptidoglycan (blue), octasaccharide (GlcNAc-MurNAc)4 (red) and GlcNAc6 (yellow). Results for GlcNAc5 and GlcNAc4 are very similar to those for GlcNAc6, whereas tetrasaccharide (GlcNAc-MurNAc)2 is similar to octasaccharide. The sugar backbone is glucosamine for all three oligosaccharides, but in octasaccharide and peptidoglycan alternate sugars are N-acetyl muramic acid (that is, bearing a lactate group at O3′), while in peptidoglycan most N-acetyl muramic acids also carry a peptide stem. Thus, effects of the lactate are seen as differences between GlcNAc6 and the other two, whereas effects of the peptide stem are seen as differences between peptidoglycan and the other two. Residues broadened in the titrations and not observed by the end of the titration are shown as bars with a normalized shift change of 100. Residues are not shown for which any shift change is smaller than the mean. (d) Representation of the data shown in c. Residues broadened beyond detection are in red, residue T13 implicated in binding the N-acetyl muramic acid lactate group is in magenta and residues implicated in binding peptide stems are in yellow.
Figure 4
Figure 4. Model of the structure of 1LysM bound to GlcNAc5.
(a) Partially transparent protein surface indicated, showing the groove along the surface, with deeper pockets to accommodate the N-acetyl groups. The O3′ atoms where the MurNAc peptide stems would be attached in a peptidoglycan ligand are shown in orange. (b) Identical view, without the protein surface. Sidechain atoms are shown for T13 and F40. The colour schemes in a and b are the same as in Fig. 3d. (c) Key interactions with MurNAc-(GlcNAc-MurNAc)2. Hydrogen bonds are shown by dashed lines. The horizontal bars indicate that the hydrophobic interaction is to the face of a sugar ring. The two pockets are indicated by green lines. Also shown in blue is the binding location involving N15 discussed in the text which may be important for distinguishing between chitin and peptidoglycan. As discussed in the text, residues I17 and A18 (which show large changes in 15N HSQC; Fig. 3) are buried and do not interact directly with the ligand, but move because of conformational rearrangement. The shift change seen for D37 HN is due to the interaction with G36 carbonyl. (d,e) Model of the interaction between 1LysM and peptidoglycan with peptide stem. The peptide stem is shown in two possible orientations, either side of the glycan backbone, interacting either with G11/K16 or with L38.
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