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

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 (SpaASLH) with synthetic, terminal SCWP disaccharide and trisaccharide analogs, together with isothermal titration calorimetry binding analyses, reveal that while SpaASLH 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 SpaASLH-SCWP interaction has evolved to accommodate longer SCWP ligands and alleviate the strain inherent to bacterial S-layer adhesion during growth and division.

Lipoteichoic acid mediates binding of a Lactobacillus S-layer protein.

The Gram-positive lactic acid bacterium Lactobacillus buchneri CD034 is covered by a two-dimensional crystalline, glycoproteinaceous cell surface (S-) layer lattice. While lactobacilli are extensively exploited as cell surface display systems for applied purposes, questions about how they stick their cell wall together are remaining open. This also includes the identification of the S-layer cell wall ligand. In this study, lipoteichoic acid was isolated from the L. buchneri CD034 cell wall as a significant fraction of the bacterium's cell wall glycopolymers, structurally characterized and analyzed for its potential to mediate binding of the S-layer to the cell wall. Combined component analyses and 1D- and 2D-nuclear magnetic resonance spectroscopy (NMR) revealed the lipoteichoic acid to be composed of on average 31 glycerol-phosphate repeating units partially substituted with α-d-glucose, and with an α-d-Galp(1→2)-α-d-Glcp(1→3)-1,2-diacyl-sn-Gro glycolipid anchor. The specificity of binding between the L. buchneri CD034 S-layer protein and purified lipoteichoic acid as well as their interaction force of about 45 pN were obtained by single-molecule force spectroscopy; this value is in the range of typical ligand-receptor interactions. This study sheds light on a functional implication of Lactobacillus cell wall architecture by showing direct binding between lipoteichoic acid and the S-layer of L. buchneri CD034.

Influence of quercetin on the interaction of gliclazide with human serum albumin - spectroscopic and docking approaches.

Protein-binding interactions are displacement reactions which have been implicated as the causative mechanisms in many drug-drug interactions. Thus, the aim of presented study was to analyse human serum albumin-binding displacement interaction between two ligands, hypoglycaemic drug gliclazide and widely distributed plant flavonoid quercetin. Fluorescence analysis was used in order to investigate the effect of substances on intrinsic fluorescence of human serum albumin (HSA) and to define binding and quenching properties of ligand-albumin complexes in binary and ternary systems, respectively. Both ligands showed the ability to bind to HSA, although to a different extent. The displacement effect of one ligand from HSA by the other one has been described on the basis of the quenching curves and binding constants comparison for the binary and ternary systems. According to the fluorescence data analysis, gliclazide presents a substance with a lower binding capacity towards HSA compared with quercetin. Results also showed that the presence of quercetin hindered the interaction between HSA and gliclazide, as the binding constant for gliclazide in the ternary system was remarkably lower compared with the binary system. This finding indicates a possibility for an increase in the non-bound fraction of gliclazide which can lead to its more significant hypoglycaemic effect. Additionally, secondary and tertiary structure conformational alterations of HSA upon binding of both ligands were investigated using synchronous fluorescence, circular dichroism and FT-IR. Experimental data were complemented with molecular docking studies. Obtained results provide beneficial information about possible interference upon simultaneous co-administration of the food/dietary supplement and drug.

Molecular modelling and site-directed mutagenesis provide insight into saccharide pyruvylation by the Paenibacillus alvei CsaB enzyme.

Pyruvylation is a biologically versatile but mechanistically unexplored saccharide modification. 4,6-Ketal pyruvylated N-acetylmannosamine within bacterial secondary cell wall polymers serves as a cell wall anchoring epitope for proteins possessing a terminal S-layer homology domain trimer. The pyruvyltransferase CsaB from Paenibacillus alvei served as a model to investigate the structural basis of the pyruvyltransfer reaction by a combination of molecular modelling and site-directed mutagenesis together with an enzyme assay using phosphoenolpyruvate (PEP; donor) and synthetic ß-D-ManNAc-(1 → 4)-α-D-GlcNAc-diphosphoryl-11-phenoxyundecyl (acceptor). CsaB protein structure modelling was done using Phyre2 and I-Tasser based on the partial crystal structure of the Schizosaccharomyces pombe pyruvyltransferase Pvg1p and by AlphaFold. The models informed the construction of twelve CsaB mutants targeted at plausible PEP and acceptor binding sites and KM and kcat values were determined to evaluate the mutants, indicating the importance of a loop region for catalysis. R148, H308 and K328 were found to be critical to PEP binding and insight into acceptor binding was obtained from an analysis of Y14 and F16 mutants, confirming the modelled binding sites and interactions predicted using Molecular Operating Environment. These data lay the basis for future mechanistic studies of saccharide pyruvylation as a novel target for interference with bacterial cell wall assembly.

Functional Characterization of Enzymatic Steps Involved in Pyruvylation of Bacterial Secondary Cell Wall Polymer Fragments.

Various mechanisms of protein cell surface display have evolved during bacterial evolution. Several Gram-positive bacteria employ S-layer homology (SLH) domain-mediated sorting of cell-surface proteins and concomitantly engage a pyruvylated secondary cell-wall polymer as a cell-wall ligand. Specifically, pyruvate ketal linked to ß-D-ManNAc is regarded as an indispensable epitope in this cell-surface display mechanism. That secondary cell wall polymer (SCWP) pyruvylation and SLH domain-containing proteins are functionally coupled is supported by the presence of an ortholog of the predicted pyruvyltransferase CsaB in bacterial genomes, such as those of Bacillus anthracis and Paenibacillus alvei. The P. alvei SCWP, consisting of pyruvylated disaccharide repeats [→4)-ß-D-GlcNAc-(1→3)-4,6-Pyr-ß-D-ManNAc-(1→] serves as a model to investigate the widely unexplored pyruvylation reaction. Here, we reconstituted the underlying enzymatic pathway in vitro in combination with synthesized compounds, used mass spectrometry, and nuclear magnetic resonance spectroscopy for product characterization, and found that CsaB-catalyzed pyruvylation of ß-D-ManNAc occurs at the stage of the lipid-linked repeat. We produced the P. alvei TagA (PAV_RS07420) and CsaB (PAV_RS07425) enzymes as recombinant, tagged proteins, and using a synthetic 11-phenoxyundecyl-diphosphoryl-α-GlcNAc acceptor, we uncovered that TagA is an inverting UDP-α-D-ManNAc:GlcNAc-lipid carrier transferase, and that CsaB is a pyruvyltransferase, with synthetic UDP-α-D-ManNAc and phosphoenolpyruvate serving as donor substrates. Next, to substitute for the UDP-α-D-ManNAc substrate, the recombinant UDP-GlcNAc-2-epimerase MnaA (PAV_RS07610) of P. alvei was included in this in vitro reconstitution system. When all three enzymes, their substrates and the lipid-linked GlcNAc primer were combined in a one-pot reaction, a lipid-linked SCWP repeat precursor analog was obtained. This work highlights the biochemical basis of SCWP biosynthesis and bacterial pyruvyl transfer.

Structural basis of cell wall anchoring by SLH domains in Paenibacillus alvei.

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.