TY - JOUR A1 - Landström, Jens A1 - Nordmark, Eva-Lisa A1 - Eklund, Robert A1 - Weintraub, Andrej A1 - Seckler, Robert A1 - Widmalm, Göran T1 - Interaction of a Salmonella enteritidis O-antigen octasaccharide with the phage P22 tailspike protein by NMR spectroscopy and docking studies N2 - The tailspike protein P22 recognizes an octasaccharide derived from the O-antigen polysaccharide of Salmonella enteritidis in a shallow groove and molecular docking successfully identifies this binding region on the protein surface. Analysis by 2D 1H,1H-T-ROESY and transferred NOESY NMR experiments indicate that the bound octasaccharide ligand has a conformation similar to that observed in solution. The results from a saturation transfer difference NMR experiment show that a large number of protons in the octasaccharide are in close contact with the protein as a result of binding. A comparison of the crystal structure of the complex and a molecular dynamics simulation of the octasaccharide with explicit water molecules suggest that only minor conformational changes are needed upon binding to the tailspike protein. Y1 - 2008 UR - http://www.springerlink.com/content/w3146138p25r2456/ U6 - https://doi.org/10.1007/s10719-007-9065-9 SN - 0282-0080 ER - TY - JOUR A1 - Zaccheus, Mona V. A1 - Bröker, Nina Kristin A1 - Lundborg, Magnus A1 - Uetrecht, Charlotte A1 - Barbirz, Stefanie A1 - Widmalm, Goran T1 - Structural studies of the O-antigen polysaccharide from Escherichia coli TD2158 having O18 serogroup specificity and aspects of its interaction with the tailspike endoglycosidase of the infecting bacteriophage HK620 JF - Carbohydrate research N2 - We have analyzed the O-antigen polysaccharide of the previously uncharacterized Escherichia coli strain TD2158 which is a host of bacteriophage HK620. This bacteriophage recognizes and cleaves the polysaccharide with its tailspike protein (TSP). The polysaccharide preparation as well as oligosaccharides obtained from HK620TSP endoglycosidase digests were analyzed with NMR spectroscopy. Additionally, sugar analysis was performed on the O-antigen polysaccharide and MALDI-TOF MS was used in oligosaccharide analysis. The present study revealed a heterogeneous polysaccharide with a hexasaccharide repeating unit of the following structure: alpha-D-Glcp-(1 -> 6) vertical bar vertical bar 2)-alpha-L-Rhap-(1 -> 6)-alpha-D-Glcp-(1 -> 4)-alpha-D-Galp-(1 -> 3)-alpha-D-GlcpNAc- (1 ->vertical bar beta-D-Glcp/beta-D-GlcpNAc-(1 -> 3) A repeating unit with a D-GlcNAc substitution of D-Gal has been described earlier as characteristic for serogroup O18A1. Accordingly, we termed repeating units with D-Glc substitution at D-Gal as O18A2. NMR analyses of the polysaccharide confirmed that O18A1- and O18A2-type repeats were present in a 1:1 ratio. However, HK620TSP preferentially bound the D-GlcNAc- substituted O18A1-type repeating units in its high affinity binding pocket with a dissociation constant of 140 mu M and disfavored the O18A2-type having a beta-D-Glcp-(1 -> 3)-linked group. As a result, in hexasaccharide preparations, O18A1 and O18A2 repeats were present in a 9: 1 ratio stressing the clear preference of O18A1- type repeats to be cleaved by HK620TSP. KW - Escherichia coli KW - Tailspike KW - Endoglycosidase KW - Lipopolysaccharide KW - NMR KW - Mass spectrometry Y1 - 2012 U6 - https://doi.org/10.1016/j.carres.2012.05.022 SN - 0008-6215 VL - 357 IS - 8 SP - 118 EP - 125 PB - Elsevier CY - Oxford ER - 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 -