TY - JOUR A1 - Bröker, Nina Kristin A1 - Gohlke, Ulrich A1 - Müller, Jürgen J. A1 - Uetrecht, Charlotte A1 - Heinemann, Udo A1 - Seckler, Robert A1 - Barbirz, Stefanie T1 - Single amino acid exchange in bacteriophage HK620 tailspike protein results in thousand-fold increase of its oligosaccharide affinity JF - Glycobiology N2 - Bacteriophage HK620 recognizes and cleaves the O-antigen polysaccharide of Escherichia coli serogroup O18A1 with its tailspike protein (TSP). HK620TSP binds hexasaccharide fragments with low affinity, but single amino acid exchanges generated a set of high-affinity mutants with submicromolar dissociation constants. Isothermal titration calorimetry showed that only small amounts of heat were released upon complex formation via a large number of direct and solvent-mediated hydrogen bonds between carbohydrate and protein. At room temperature, association was both enthalpy- and entropy-driven emphasizing major solvent rearrangements upon complex formation. Crystal structure analysis showed identical protein and sugar conformers in the TSP complexes regardless of their hexasaccharide affinity. Only in one case, a TSP mutant bound a different hexasaccharide conformer. The extended sugar binding site could be dissected in two regions: first, a hydrophobic pocket at the reducing end with minor affinity contributions. Access to this site could be blocked by a single aspartate to asparagine exchange without major loss in hexasaccharide affinity. Second, a region where the specific exchange of glutamate for glutamine created a site for an additional water molecule. Side-chain rearrangements upon sugar binding led to desolvation and additional hydrogen bonding which define this region of the binding site as the high-affinity scaffold. KW - bacterial O-antigen KW - carbohydrate interaction KW - site-directed mutagenesis KW - structural thermodynamics KW - tailspike protein Y1 - 2013 U6 - https://doi.org/10.1093/glycob/cws126 SN - 0959-6658 VL - 23 IS - 1 SP - 59 EP - 68 PB - Oxford Univ. Press CY - Cary ER - TY - JOUR A1 - Andres, Dorothee A1 - Gohlke, Ulrich A1 - Bröker, Nina Kristin A1 - Schulze, Stefan A1 - Rabsch, Wolfgang A1 - Heinemann, Udo A1 - Barbirz, Stefanie A1 - Seckler, Robert T1 - An essential serotype recognition pocket on phage P22 tailspike protein forces Salmonella enterica serovar Paratyphi A O-antigen fragments to bind as nonsolution conformers JF - Glycobiology N2 - Bacteriophage P22 recognizes O-antigen polysaccharides of Salmonella enterica subsp. enterica (S.) with its tailspike protein (TSP). In the serovars S. Typhimurium, S. Enteritidis, and S. Paratyphi A, the tetrasaccharide repeat units of the respective O-antigens consist of an identical main chain trisaccharide but different 3,6-dideoxyhexose substituents. Here, the epimers abequose, tyvelose and paratose determine the specific serotype. P22 TSP recognizes O-antigen octasaccharides in an extended binding site with a single 3,6-dideoxyhexose binding pocket. We have isolated S. Paratyphi A octasaccharides which were not available previously and determined the crystal structure of their complex with P22 TSP. We discuss our data together with crystal structures of complexes with S. Typhimurium and S. Enteritidis octasaccharides determined earlier. Isothermal titration calorimetry showed that S. Paratyphi A octasaccharide binds P22 TSP less tightly, with a difference in binding free energy of similar to 7 kJ mol(-1) at 20 degrees C compared with S. Typhimurium and S. Enteritidis octasaccharides. Individual protein-carbohydrate contacts were probed by amino acid replacements showing that the dideoxyhexose pocket contributes to binding of all three serotypes. However, S. Paratyphi A octasaccharides bind in a conformation with an energetically unfavorable phi/epsilon glycosidic bond angle combination. In contrast, octasaccharides from the other serotypes bind as solution-like conformers. Two water molecules are conserved in all P22 TSP complexes with octasaccharides of different serotypes. They line the dideoxyhexose binding pocket and force the S. Paratyphi A octasaccharides to bind as nonsolution conformers. This emphasizes the role of solvent as part of carbohydrate binding sites. KW - bacterial O-antigen KW - carbohydrate interaction KW - paratose KW - structural thermodynamics KW - tailspike protein Y1 - 2013 U6 - https://doi.org/10.1093/glycob/cws224 SN - 0959-6658 VL - 23 IS - 4 SP - 486 EP - 494 PB - Oxford Univ. Press CY - Cary ER -