Bacteriophage Tailspikes and Bacterial O-Antigens as a Model System to Study Weak-Affinity Protein–Polysaccharide Interactions

Abstract

Understanding interactions of bacterial surface polysaccharides with receptor protein scaffolds is important for the development of antibiotic therapies. The corresponding protein recognition domains frequently form low-affinity complexes with polysaccharides that are difficult to address with experimental techniques because of the conformational flexibility of the polysaccharide. In this work, we studied the tailspike protein (TSP) of the bacteriophage Sf6. Sf6TSP binds and hydrolyzes the high-rhamnose, serotype Y O-antigen polysaccharide of the Gram-negative bacterium Shigella flexneri (S. flexneri) as a first step of bacteriophage infection. Spectroscopic analyses and enzymatic cleavage assays confirmed that Sf6TSP binds long stretches of this polysaccharide. Crystal structure analysis and saturation transfer difference (STD) NMR spectroscopy using an enhanced method to interpret the data permitted the detailed description of affinity contributions and flexibility in an Sf6TSP-octasaccharide complex. Dodecasaccharide fragments corresponding to three repeating units of the O-antigen in complex with Sf6TSP were studied computationally by molecular dynamics simulations. They showed that distortion away from the low-energy solution conformation found in the octasaccharide complex is necessary for ligand binding. This is in agreement with a weak-affinity functional polysaccharide-protein contact that facilitates correct placement and thus hydrolysis of the polysaccharide close to the catalytic residues. Our simulations stress that the flexibility of glycan epitopes together with a small number of specific protein contacts provide the driving force for Sf6TSP-polysaccharide complex formation in an overall weak-affinity interaction system.

Increasing the Affinity of an O-Antigen Polysaccharide Binding Site in Shigella Flexneri Bacteriophage Sf6 Tailspike Protein

“…This illustrates that the amphiphilic bindingp artners interacti na na queous environment,o ften involvingr ather shallowp rotein surface grooves. [7] This is ac lear difference to glycanb inding sites found in antibodies that featured eep hydrophobic pockets and nm dissociation constants. [8] Glycan binding proteins thus provide ar ich pool of highly specific sites suitable for variousa pplications,b ut their high-affinity engineering is difficult due to the various thermodynamic effects governing the complex formation.…”

“…Sf6TSP is aw ell-studied,w eak-affinity carbohydrate binding protein asa nalysed with NMR spectroscopy,X -ray crystallography andc omputational analyses. [7] An Sf6TSP hydrolysis deficient variant with alaninee xchanges for active site carboxylate residues (Sf6TSP E366A D399A, Sf6TSP EADA )w as used to construct af luorescent probe sensitivef or the O-antigen polysaccharideo nS. flexneri bacteria.…”

“…[29][30][31] TSPs have been also used as models ystems to computationally link solvent network structures to experimental thermodynamic signatures of oligosaccharide ligand binding. [9] Bacteriophage Sf6 TSP specifically binds to the O-antigen of its host, Shigella flexneri, with the serogroup Yr epeat unit [7,32] Sf6TSP is ah ighly stable trimeric protein with endorhamnosidase activity,p roducing oligosaccharides of mainly 2R U, that is, octasaccharides, from the S. flexneri O-antigen polysaccharide. [7,[32][33][34] Sf6TSP has three independent, elongated glycan binding sites for oligosaccharide O-antigen fragments produced by hydrolysis.…”

“…[9] Bacteriophage Sf6 TSP specifically binds to the O-antigen of its host, Shigella flexneri, with the serogroup Yr epeat unit [7,32] Sf6TSP is ah ighly stable trimeric protein with endorhamnosidase activity,p roducing oligosaccharides of mainly 2R U, that is, octasaccharides, from the S. flexneri O-antigen polysaccharide. [7,[32][33][34] Sf6TSP has three independent, elongated glycan binding sites for oligosaccharide O-antigen fragments produced by hydrolysis. Thorough glycan bindings ite description in Sf6TSP was achieved by a combination of MD simulations, X-ray crystallography and NMR spectroscopy ( Figure 1).…”

“…Thorough glycan bindings ite description in Sf6TSP was achieved by a combination of MD simulations, X-ray crystallography and NMR spectroscopy ( Figure 1). [7] Its N-terminal end anchors Sf6TSP to the phage tail and capsid,a ccordingly it can sample the O-antigen protrudingp erpendicularly from the bacterial cell surface.…”