The S-layer homology domains of Paenibacillus alvei surface protein SpaA bind to cell wall polysaccharide through the terminal monosaccharide residue

Abstract

Self-assembling (glyco)protein surface layers (S-layers) are ubiquitous prokaryotic cell-surface structures involved in structural maintenance, nutrient diffusion, host adhesion, virulence, and other processes, which makes them appealing targets for therapeutics and biotechnological applications as biosensors or drug delivery systems. However, unlocking this potential requires expanding our understanding of S-layer properties, especially the details of surface-attachment. S-layers of Gram-positive bacteria often are attached through the interaction of S-layer homology (SLH) domain trimers with peptidoglycan-linked secondary cell wall polymers (SCWPs). Cocrystal structures of the SLH domain trimer from the Paenibacillus alvei S-layer protein SpaA (SpaA_SLH) with synthetic, terminal SCWP disaccharide and trisaccharide analogs, together with isothermal titration calorimetry binding analyses, reveal that while SpaA_SLH accommodates longer biologically relevant SCWP ligands within both its primary (G2) and secondary (G1) binding sites, the terminal pyruvylated ManNAc moiety serves as the nearly exclusive SCWP anchoring point. Binding is accompanied by displacement of a flexible loop adjacent to the receptor site that enhances the complementarity between protein and ligand, including electrostatic complementarity with the terminal pyruvate moiety. Remarkably, binding of the pyruvylated monosaccharide SCWP fragment alone is sufficient to cause rearrangement of the receptor-binding sites in a manner necessary to accommodate longer SCWP fragments. The observation of multiple conformations in longer oligosaccharides bound to the protein, together with the demonstrated functionality of two of the three SCWP receptor-binding sites, reveals how the SpaA_SLH-SCWP interaction has evolved to accommodate longer SCWP ligands and alleviate the strain inherent to bacterial S-layer adhesion during growth and division.

Keywords: S-layer; SLH domain; X-ray crystallography; cell wall anchoring; isothermal titration calorimetry; secondary cell wall polymer.

Figure 1

The cell surface of many prokaryotes is coated in a surface layer (S-layer) consisting of one or more (glyco)proteins arranged in a repeating two-dimensional array.A, schematic diagram of S-layer protein binding on Gram-positive bacteria mediated via specialized S-layer homology (SLH) domains that recognize and bind peptidoglycan-linked secondary cell wall polymers (SCWPs), with the inset showing the structure of the SCWP from P. alvei CCM 2051^T (11, 27, 30); and (B) the synthetic ligand analogs used in this study corresponding to the terminal disaccharide and trisaccharide units of the P. alvei CCM 2051^T SCWP ligand.

Figure 2

Ribbon models for each SpaA_SLH co-crystal structure inTable 2. 2F_o-F_c electron density maps contoured to 1σ shown for the corresponding model of bound ligand for (A) disaccharide-bound SpaA_SLH; (B) trisaccharide-bound SpaA_SLH; (C) disaccharide-bound SpaA_SLH/G109A with ligands shown from both molecules of the asymmetric unit of the co-crystal structure; and (D) disaccharide-bound SpaA_SLH/G46A/G109A double mutant. Grooves 1, 2, and 3 are indicated on each structure as G1, G2, and G3, respectively. SLH1 orange, SLH2 blue, and SLH3 aquamarine. Ligand atoms are colored by element with oxygen red, nitrogen blue, and carbon white. 2F_o-F_c electron density maps are depicted as gray mesh.

Figure 3

Only the terminal monosaccharide of the di- and trisaccharide ligands forms significant direct contacts with the SpaA_SLH,protein, with the internal saccharide residues forming mainly bridging water contacts.A, the synthetic terminal disaccharide analog binds to G2 of SpaA_SLH, with the GlcNAc residue forming no direct contact to the protein. B, the synthetic terminal trisaccharide analog binds to G2 of SpaA_SLH, with the GlcNAc residue forming a single hydrogen bond interaction with the protein. C, the synthetic terminal disaccharide analog binds to G1 of the single mutant with the terminal ManNAc again forming the only direct interactions with the protein. D, the GlcNAc residue bound to G2 of the wild-type SpaA_SLH is disordered over two conformations, with the second conformation corresponding to the GlcNAc orientation observed in trisaccharide-bound SpaA_SLH structure. Water molecules are depicted as light green spheres, while atoms are colored by element with oxygen red, nitrogen blue, protein carbon white, and ligand carbon green (or tan for disaccharide conformation 2).

Figure 4

SpaA_SLHresidues SLH 30-31-32 that border the monosaccharide receptor site shift in response to terminal ManNAc binding to accommodate longer, more biologically relevant SCWP ligands.A, G2 of unliganded SpaA_SLH (grey; PDB 6CWC) overlapped with G2 of monosaccharide-bound SpaA_SLH (purple; PDB 6CWH) showing the relative displacement of these residues in G2 caused by binding of monosaccharide. B, the overlap of G2 of disaccharide-bound SpaA_SLH (green) with G2 of monosaccharide-bound SpaA_SLH (purple; PDB 6CWH) shows similar shifts, indicating that it is the binding of the monosaccharide, which is largely responsible for the shifts. C, G1 of unliganded SpaA_SLH (grey) is overlapped with G1 of monosaccharide-bound SpaA_SLH/G109A single mutant (purple; PDB 6CWN) showing the relative displacement of these residues in G1 caused by binding of the monosaccharide. D, the overlap of G1 of disaccharide-bound SpaA_SLH/G109A (green) with G1 of monosaccharide-bound SpaA_SLH (purple, PDB: 6CWN) shows similar shifts, indicating again that it is the binding of the monosaccharide, which is largely responsible for the shifts. All carbon atoms are colored according to the model descriptions above, with oxygen and nitrogen atoms colored in red and blue, respectively.