title: Single amino acid exchange in bacteriophage HK620 tailspike protein results in thousand-fold increase of its oligosaccharide affinity.
abstract: 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.
authors.affiliation: Physikalische Biochemie, Universität Potsdam, Germany.
authors.forename: Nina K
authors.initials: NK
authors.lastname: Broeker
authors.forename: Ulrich
authors.initials: U
authors.lastname: Gohlke
authors.forename: Jürgen J
authors.initials: JJ
authors.lastname: Müller
authors.forename: Charlotte
authors.initials: C
authors.lastname: Uetrecht
authors.forename: Udo
authors.initials: U
authors.lastname: Heinemann
authors.forename: Robert
authors.initials: R
authors.lastname: Seckler
authors.forename: Stefanie
authors.initials: S
authors.lastname: Barbirz
mesh_terms: D000596:Amino Acids; D001216:Asparagine / Q000235:genetics / Q000378:metabolism; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D020134:Catalytic Domain; D003090:Coliphages / Q000378:metabolism*; D018360:Crystallography, X-Ray; D006026:Glycoside Hydrolases; D006860:Hydrogen Bonding; D008958:Models, Molecular; D019081:O Antigens / Q000737:chemistry / Q000378:metabolism; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D013499:Surface Properties; D013816:Thermodynamics; D017376:Viral Tail Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D000596:Amino Acids; D019081:O Antigens; D009844:Oligosaccharides; D017376:Viral Tail Proteins; D001224:Aspartic Acid; D001216:Asparagine; D006026:Glycoside Hydrolases; C083268:tailspike protein, bacteriophage