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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 couldBacteriophage 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.show moreshow less

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Metadaten
Author:Nina Kristin Bröker, Ulrich Gohlke, Jürgen J. Müller, Charlotte Uetrecht, Udo Heinemann, Robert SecklerORCiDGND, Stefanie BarbirzORCiDGND
DOI:https://doi.org/10.1093/glycob/cws126
ISSN:0959-6658 (print)
Parent Title (English):Glycobiology
Publisher:Oxford Univ. Press
Place of publication:Cary
Document Type:Article
Language:English
Year of first Publication:2013
Year of Completion:2013
Release Date:2017/03/26
Tag:bacterial O-antigen; carbohydrate interaction; site-directed mutagenesis; structural thermodynamics; tailspike protein
Volume:23
Issue:1
Pagenumber:10
First Page:59
Last Page:68
Funder:Deutsche Forschungsgemeinschaft [BA 4046/1-1]
Organizational units:Mathematisch-Naturwissenschaftliche Fakultät / Institut für Biochemie und Biologie
Peer Review:Referiert