35130
2013
2013
eng
486
494
9
4
23
article
Oxford Univ. Press
Cary
1
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An essential serotype recognition pocket on phage P22 tailspike protein forces Salmonella enterica serovar Paratyphi A O-antigen fragments to bind as nonsolution conformers
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.
Glycobiology
10.1093/glycob/cws224
0959-6658
wos:2011-2013
WOS:000315631900007
Barbirz, S (reprint author), Univ Potsdam, Karl Liebknecht Str 24-25, D-14476 Golm, Germany., barbirz@uni-potsdam.de; seckler@uni-potsdam.de
Leibniz Graduate School of Molecular Biophysics; [BA4046/1-1]
Dorothee Andres
Ulrich Gohlke
Nina Kristin Bröker
Stefan Schulze
Wolfgang Rabsch
Udo Heinemann
Stefanie Barbirz
Robert Seckler
eng
uncontrolled
bacterial O-antigen
eng
uncontrolled
carbohydrate interaction
eng
uncontrolled
paratose
eng
uncontrolled
structural thermodynamics
eng
uncontrolled
tailspike protein
Institut für Biochemie und Biologie
Referiert
35431
2013
2013
eng
59
68
10
1
23
article
Oxford Univ. Press
Cary
1
--
--
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Single amino acid exchange in bacteriophage HK620 tailspike protein results in thousand-fold increase of its oligosaccharide affinity
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.
Glycobiology
10.1093/glycob/cws126
0959-6658
wos:2011-2013
WOS:000311642500006
Barbirz, S (reprint author), Univ Potsdam, Karl Liebknecht Str 24-25, D-14476 Potsdam, Germany., barbirz@uni-potsdam.de
Deutsche Forschungsgemeinschaft [BA 4046/1-1]
Nina Kristin Bröker
Ulrich Gohlke
Jürgen J. Müller
Charlotte Uetrecht
Udo Heinemann
Robert Seckler
Stefanie Barbirz
eng
uncontrolled
bacterial O-antigen
eng
uncontrolled
carbohydrate interaction
eng
uncontrolled
site-directed mutagenesis
eng
uncontrolled
structural thermodynamics
eng
uncontrolled
tailspike protein
Institut für Biochemie und Biologie
Referiert