Abstract
Self-assembling protein surface (S-) layers are common cell envelope structures of prokaryotes and have critical roles from structural maintenance to virulence. S-layers of Gram-positive bacteria are often attached through the interaction of S-layer homology (SLH) domain trimers with peptidoglycan-linked secondary cell wall polymers (SCWPs). Here we present an in-depth characterization of this interaction, with co-crystal structures of the three consecutive SLH domains from the Paenibacillus alvei S-layer protein SpaA with defined SCWP ligands. The most highly conserved SLH domain residue SLH-Gly29 is shown to enable a peptide backbone flip essential for SCWP binding in both biophysical and cellular experiments. Furthermore, we find that a significant domain movement mediates binding by two different sites in the SLH domain trimer, which may allow anchoring readjustment to relieve S-layer strain caused by cell growth and division.
Highlights
Self-assembling protein surface (S-) layers are common cell envelope structures of prokaryotes and have critical roles from structural maintenance to virulence
The crystal structures and binding analyses presented here of P. alvei SpaASLH, SpaASLH/G109A, and SpaASLH/G46A/G109A with synthetic secondary cell wall polymers (SCWPs) fragments provide novel insights into the functional contributions of many conserved S-layer homology (SLH) domain residues, including SLH-Trp[13], SLH-Gly[29], and SLH-Arg[43], which precisely interlock from three different SLH domains to generate two active binding sites
These SLH domains appear to be specific for the non-reducing-end Pyr-β-D-ManNAc moiety of P. alvei SCWP, as opposed to internal occurrences of Pyr-β-D-ManNAc with β-D-GlcNAc-(1 → 3) linkages. This specificity is consistent with that of B. anthracis SapSLH for its SCWP that is pyruvylated only on the terminal ManNAc40,41, which may indicate a general trend in the recognition of terminal anionic moieties of SCWPs by SLH domains in other organisms
Summary
Despite the lack of binding measured by ITC, a co-crystal structure with the internal disaccharide was obtained, again indicating that binding is enhanced significantly in the crystalline environment compared to in solution In both molecules in the AU of this structure, the 4,6-Pyr-β-D-ManNAcOMe moiety of the disaccharide is bound in the G2 pocket in a manner similar to the monosaccharide ligand (Supplementary Fig. 8). The first G of each motif is within a turn exiting α1 and transitioning into this loop, and the hydrophobic residues VF, LM, and LF are oriented toward the hydrophobic core of each SLH domain lobe In both G1 and G2, the non-conserved SLH-28 residue is observed to make a hydrogen bond from its backbone carbonyl to the side chain of the conserved SLH-Arg[43] of the TRAE motif. The hydrogen bond between the SLH-28 backbone carbonyl and SLH-Arg[43] in the unliganded state may support the proper folding of the SLH domain trimer, as these residues precisely interlock
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