Single amino acid exchange in bacteriophage HK620 tailspike protein results in thousand-fold increase of its oligosaccharide affinity
2013 (English)In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 23, no 1, 59-68 p.Article in journal (Refereed) Published
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.
Place, publisher, year, edition, pages
2013. Vol. 23, no 1, 59-68 p.
bacterial O-antigen, carbohydrate interaction, site-directed mutagenesis, structural thermodynamics, tailspike protein, bacteriophage hk620 tailspike protein, carbohydrate, glutamic acid, glutamine, mutant protein, O antigen, oligosaccharide, solvent, unclassified drug, virus protein, amino acid substitution, article, bacteriophage, bacteriophage hk620, binding site, complex formation, crystal structure, dissociation constant, enthalpy, Escherichia coli, heat, hydrogen bond, hydrophobicity, isothermal titration calorimetry, nonhuman, priority journal, protein binding, reaction analysis, room temperature
IdentifiersURN: urn:nbn:se:uu:diva-192025DOI: 10.1093/glycob/cws126OAI: oai:DiVA.org:uu-192025DiVA: diva2:600400