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.

Nyckelord

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

Publikations- och innehållstyp

ref (ämneskategori)

art (ämneskategori)