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.

Gohlke, U. (författare)
Müller, J. J. (författare)
Uetrecht, CharlotteUppsala universitet,Molekylär biofysik(Swepub:uu)chaue348 (författare)
Heinemann, U. (författare)
Seckler, R. (författare)
Barbirz, S. (författare)
Uppsala universitetMolekylär biofysik (creator_code:org_t)

Ingår i:Glycobiology: Oxford University Press (OUP)23:1, s. 59-680959-66581460-2423

Av författaren/redakt...: Broeker, N. K.; Gohlke, U.; Müller, J. J.; Uetrecht, Charlo ...; Heinemann, U.; Seckler, R.; visa fler...; Barbirz, S.; visa färre...

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