TY  - JOUR
A1  - Barbirz, Stefanie
A1  - Müller, Jürgen J.
A1  - Uetrecht, Charlotte
A1  - Clark, Alvin J.
A1  - Heinemann, Udo
A1  - Seckler, Robert
T1  - Crystal structure of Escherichia coli phage HK620 tailspike : podoviral tailspike endoglycosidase modules are evolutionarily related
N2  - Bacteriophage HK620 infects Escherichia coli H and is closely related to Shigella phage Sf6 and Salmonella phage P22. All three Podoviridae recognize and cleave their respective host cell receptor polysaccharide by homotrimeric tailspike proteins. The three proteins exhibit high sequence identity in the 110 residues of their N-terminal particle- binding domains, but no apparent sequence similarity in their major, receptor-binding parts. We have biochemically characterized the receptor-binding part of HK620 tailspike and determined its crystal structure to 1.38 Å resolution. Its major domain is a right-handed parallel ;-helix, as in Sf6 and P22 tailspikes. HK620 tailspike has endo-N- acetylglucosaminidase activity and produces hexasaccharides of an O18A1-type O-antigen. As indicated by the structure of a hexasaccharide complex determined at 1.6 Å resolution, the endoglycosidase-active sites are located intramolecularly, as in P22, and not between subunits, as in Sf6 tailspike. In contrast, the extreme C-terminal domain of HK620 tailspike forms a ;-sandwich, as in Sf6 and unlike P22 tailspike. Despite the different folds, structure-based sequence alignments of the C-termini reveal motifs conserved between the three proteins. We propose that the tailspike genes of P22, Sf6 and HK620 have a common precursor and are not mosaics of unrelated gene fragments.
Y1  - 2008
UR  - http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2958.2008.06311.x/pdf
SN  - 0950-382X
ER  - 
TY  - JOUR
A1  - Bröker, Nina Kristin
A1  - Gohlke, Ulrich
A1  - Müller, Jürgen J.
A1  - Uetrecht, Charlotte
A1  - Heinemann, Udo
A1  - Seckler, Robert
A1  - Barbirz, Stefanie
T1  - Single amino acid exchange in bacteriophage HK620 tailspike protein results in thousand-fold increase of its oligosaccharide affinity
JF  - Glycobiology
N2  - 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.
KW  - bacterial O-antigen
KW  - carbohydrate interaction
KW  - site-directed mutagenesis
KW  - structural thermodynamics
KW  - tailspike protein
Y1  - 2013
U6  - https://doi.org/10.1093/glycob/cws126
SN  - 0959-6658
VL  - 23
IS  - 1
SP  - 59
EP  - 68
PB  - Oxford Univ. Press
CY  - Cary
ER  -