Single amino acid exchange in bacteriophage HK620 tailspike protein results in thousand-fold increase of its oligosaccharide affinity

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Item Type:	Article
Title:	Single amino acid exchange in bacteriophage HK620 tailspike protein results in thousand-fold increase of its oligosaccharide affinity
Creators Name:	Broeker, N.K., Gohlke, U., Müller, J.J., Uetrecht, C., Heinemann, U., Seckler, R. and Barbirz, S.
Abstract:	Bacteriophage HK620 recognizes and cleaves the Oantigen polysaccharide of E. 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: Firstly, 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. Secondly, a region where 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.
Keywords:	Tailspike Protein, Structural Thermodynamics, Carbohydrate Interaction, Site-Directed Mutagenesis, Bacterial Oantigen
Source:	Glycobiology
ISSN:	0959-6658
Publisher:	Oxford University Press
Volume:	23
Number:	1
Page Range:	59-68
Date:	January 2013
Official Publication:	https://doi.org/10.1093/glycob/cws126
PubMed:	View item in PubMed

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