TY - JOUR A1 - Kang, Yu A1 - Gohlke, Ulrich A1 - Engström, Olof A1 - Hamark, Christoffer A1 - Scheidt, Tom A1 - Kunstmann, Ruth Sonja A1 - Heinemann, Udo A1 - Widmalm, Göran A1 - Santer, Mark A1 - Barbirz, Stefanie T1 - Bacteriophage Tailspikes and Bacterial O-Antigens as a Model System to Study Weak-Affinity Protein-Polysaccharide Interactions JF - Journal of the American Chemical Society N2 - 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 due to 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. Y1 - 2016 U6 - https://doi.org/10.1021/jacs.6b00240 SN - 0002-7863 VL - 138 SP - 9109 EP - 9118 PB - American Chemical Society CY - Washington ER - TY - GEN A1 - Kunstmann, Ruth Sonja A1 - Engström, Olof A1 - Wehle, Marko A1 - Widmalm, Göran A1 - Santer, Mark A1 - Barbirz, Stefanie T1 - Increasing the affinity of an O-Antigen polysaccharide binding site in Shigella flexneri bacteriophage Sf6 tailspike protein T2 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - Broad and unspecific use of antibiotics accelerates spread of resistances. Sensitive and robust pathogen detection is thus important for a more targeted application. Bacteriophages contain a large repertoire of pathogen-binding proteins. These tailspike proteins (TSP) often bind surface glycans and represent a promising design platform for specific pathogen sensors. We analysed bacteriophage Sf6 TSP that recognizes the O-polysaccharide of dysentery-causing Shigella flexneri to develop variants with increased sensitivity for sensor applications. Ligand polyrhamnose backbone conformations were obtained from 2D H-1,H-1-trNOESY NMR utilizing methine-methine and methine-methyl correlations. They agreed well with conformations obtained from molecular dynamics (MD), validating the method for further predictions. In a set of mutants, MD predicted ligand flexibilities that were in good correlation with binding strength as confirmed on immobilized S. flexneri O-polysaccharide (PS) with surface plasmon resonance. In silico approaches combined with rapid screening on PS surfaces hence provide valuable strategies for TSP-based pathogen sensor design. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 1417 KW - carbohydrates KW - molecular dynamics simulations KW - NMR spectroscopy KW - protein-carbohydrate interactions KW - surface plasmon resonance Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-519418 SN - 1866-8372 IS - 32 ER - TY - JOUR A1 - Kunstmann, Ruth Sonja A1 - Engström, Olof A1 - Wehle, Marko A1 - Widmalm, Göran A1 - Santer, Mark A1 - Barbirz, Stefanie T1 - Increasing the affinity of an O-Antigen polysaccharide binding site in Shigella flexneri bacteriophage Sf6 tailspike protein JF - Chemistry – A European Journal N2 - Broad and unspecific use of antibiotics accelerates spread of resistances. Sensitive and robust pathogen detection is thus important for a more targeted application. Bacteriophages contain a large repertoire of pathogen-binding proteins. These tailspike proteins (TSP) often bind surface glycans and represent a promising design platform for specific pathogen sensors. We analysed bacteriophage Sf6 TSP that recognizes the O-polysaccharide of dysentery-causing Shigella flexneri to develop variants with increased sensitivity for sensor applications. Ligand polyrhamnose backbone conformations were obtained from 2D H-1,H-1-trNOESY NMR utilizing methine-methine and methine-methyl correlations. They agreed well with conformations obtained from molecular dynamics (MD), validating the method for further predictions. In a set of mutants, MD predicted ligand flexibilities that were in good correlation with binding strength as confirmed on immobilized S. flexneri O-polysaccharide (PS) with surface plasmon resonance. In silico approaches combined with rapid screening on PS surfaces hence provide valuable strategies for TSP-based pathogen sensor design. KW - carbohydrates KW - molecular dynamics simulations KW - NMR spectroscopy KW - protein-carbohydrate interactions KW - surface plasmon resonance Y1 - 2020 U6 - https://doi.org/10.1002/chem.202000495 SN - 0947-6539 SN - 1521-3765 VL - 26 IS - 32 SP - 7263 EP - 7273 PB - Wiley-VCH CY - Weinheim ER -