A poly-proline II helix in YadA from Yersinia enterocolitica serotype O:9 facilitates heparin binding through electrostatic interactions.

Poly-proline II helices are secondary structure motifs frequently found in ligand-binding sites. They exhibit increased flexibility and solvent exposure compared to the strongly hydrogen-bonded α-helices or ß-strands and can therefore easily be misinterpreted as completely unstructured regions with an extremely high rotational freedom. Here, we show that the adhesin YadA of Yersinia enterocolitica serotype O:9 contains a poly-proline II helix interaction motif in the N-terminal region. The motif is involved in the interaction of YadAO:9 with heparin, a host glycosaminoglycan. We show that the basic residues within the N-terminal motif of YadA are required for electrostatic interactions with the sulfate groups of heparin. Biophysical methods including CD spectroscopy, solution-state NMR and SAXS all independently support the presence of a poly-proline helix allowing YadAO:9 binding to the rigid heparin. Lastly, we show that host cells deficient in sulfation of heparin and heparan sulfate are not targeted by YadAO:9 -mediated adhesion. We speculate that the YadAO:9 -heparin interaction plays an important and highly strain-specific role in the pathogenicity of Yersinia enterocolitica serotype O:9.

A trimeric coiled-coil motif binds bacterial lipopolysaccharides with picomolar affinity.

α-helical coiled-coils are ubiquitous protein structures in all living organisms. For decades, modified coiled-coils sequences have been used in biotechnology, vaccine development, and biochemical research to induce protein oligomerization, and form self-assembled protein scaffolds. A prominent model for the versatility of coiled-coil sequences is a peptide derived from the yeast transcription factor, GCN4. In this work, we show that its trimeric variant, GCN4-pII, binds bacterial lipopolysaccharides (LPS) from different bacterial species with picomolar affinity. LPS molecules are highly immunogenic, toxic glycolipids that comprise the outer leaflet of the outer membrane of Gram-negative bacteria. Using scattering techniques and electron microscopy, we show how GCN4-pII breaks down LPS micelles in solution. Our findings suggest that the GCN4-pII peptide and derivatives thereof could be used for novel LPS detection and removal solutions with high relevance to the production and quality control of biopharmaceuticals and other biomedical products, where even minuscule amounts of residual LPS can be lethal.

Catching a SPY: Using the SpyCatcher-SpyTag and Related Systems for Labeling and Localizing Bacterial Proteins.

The SpyCatcher-SpyTag system was developed seven years ago as a method for protein ligation. It is based on a modified domain from a Streptococcus pyogenes surface protein (SpyCatcher), which recognizes a cognate 13-amino-acid peptide (SpyTag). Upon recognition, the two form a covalent isopeptide bond between the side chains of a lysine in SpyCatcher and an aspartate in SpyTag. This technology has been used, among other applications, to create covalently stabilized multi-protein complexes, for modular vaccine production, and to label proteins (e.g., for microscopy). The SpyTag system is versatile as the tag is a short, unfolded peptide that can be genetically fused to exposed positions in target proteins; similarly, SpyCatcher can be fused to reporter proteins such as GFP, and to epitope or purification tags. Additionally, an orthogonal system called SnoopTag-SnoopCatcher has been developed from an S. pneumoniae pilin that can be combined with SpyCatcher-SpyTag to produce protein fusions with multiple components. Furthermore, tripartite applications have been produced from both systems allowing the fusion of two peptides by a separate, catalytically active protein unit, SpyLigase or SnoopLigase. Here, we review the current state of the SpyCatcher-SpyTag and related technologies, with a particular emphasis on their use in vaccine development and in determining outer membrane protein localization and topology of surface proteins in bacteria.

Insights into the autotransport process of a trimeric autotransporter, Yersinia Adhesin A (YadA).

Trimeric autotransporter adhesins (TAAs) are a subset of a larger protein family called the type V secretion systems. They are localized on the cell surface of Gram-negative bacteria, function as mediators of attachment to inorganic surfaces and host cells, and thus include important virulence factors. Yersinia adhesin A (YadA) from Yersinia enterocolitica is a prototypical TAA that is used extensively to study the structure and function of the type Vc secretion system. A solid-state NMR study of the membrane anchor domain of YadA previously revealed a flexible stretch of small residues, termed the ASSA region, that links the membrane anchor to the stalk domain. In this study, we present evidence that single amino acid proline substitutions produce two different conformers of the membrane anchor domain of YadA; one with the N-termini facing the extracellular surface, and a second with the N-termini located in the periplasm. We propose that TAAs adopt a hairpin intermediate during secretion, as has been shown before for other subtypes of the type V secretion system. As the YadA transition state intermediate can be isolated from the outer membrane, future structural studies should be possible to further unravel details of the autotransport process.

1H, 13C, 15N backbone assignment of the human heat-labile enterotoxin B-pentamer and chemical shift mapping of neolactotetraose binding.

The major virulence factor of enterotoxigenic Escherichia coli is the heat-labile enterotoxin (LT), an AB5 toxin closely related to the cholera toxin. LT consists of six subunits, the catalytically active A-subunit and five B-subunits arranged as a pentameric ring (LTB), which enable the toxin to bind to the epithelial cells in the intestinal lumen. LTB has two recognized binding sites; the primary binding site is responsible for anchoring the toxin to its main receptor, the GM1-ganglioside, while the secondary binding site recognizes blood group antigens. Herein, we report the 1H, 13C, 15N main chain assignment of LTB from human isolates (hLTB; 103 a.a. per subunit, with a total molecular mass of 58.5 kDa). The secondary structure was predicted based on 13C', 13Cα, 13Cß, 1HN and 15N chemical shifts and compared to a published crystal structure of LTB. Neolactotetraose (NEO) was titrated to hLTB and chemical shift perturbations were measured. The chemical shift perturbations were mapped onto the crystal structure, confirming that NEO binds to the primary binding site of hLTB and competes with GM1-binding. Our new data further lend support to the hypothesis that binding at the primary binding site is transmitted to the secondary binding site of the toxin, where it may influence the binding to blood group antigens.