Structural thermodynamics of complex carbohydat recognition by proteins
Final Report Abstract
Aim of the project was to explore origins of affinity, specificity and energetics of protein-carbohydrate interactions on large protein interfaces. During the first funding period, we could establish a set of highly stable model proteins from bacteriophages that recognize oligosaccharides from the Gram-negative bacterial cell surface. We defined the origin of high-affinity hot spots on a glycan-binding protein interface and the impact of glycan dynamics on ligand affinity in the presence of structural water molecules in the binding site. This work has set up the basis for further studies in the second funding period that focused on the interdisciplinary analysis of our model systems with computational, NMR-, X-ray crystallography and calorimetry studies. Here, emphasis lay on the description of ligand dynamics in solution in the presence of low-affinity binding sites. With a high-affinity model system we were able to quantify the energetics of desolvation both with computational and experimental methods. This is an exceptional result for oligosaccharide-protein complexes where the field still lacks data on contributions of single water molecules to overall affinity of complex formation. Our model systems have focused on neutral, i.e. uncharged oligosaccharide ligands. In the second funding period we therefore aimed at defining protein interfaces with anionic oligosaccharides. Anionic glycans are widespread in protective extracellular matrices of microorganisms. They are an important research target because development of new antibiotic strategies implies a thorough understanding of all protein interaction sites with these ligands during synthesis, transport and interactions with the immune system or in biofilms. We have engineered fragments of polysialic acid binding proteins with different binding site architectures. However, preparation and handling of polysialic acid fragments was more time consuming than expected and so far, interaction data is not yet available. Instead we have isolated a biofilm modifying enzyme from the plant pathogen Pantoea stewartii. We tested interactions with anionic purified exopolysaccharide preparations and found strong impact on microviscosity, stressing that biofilm producing bacteria as well as bacteriophages possess proteins that shape the biophysical properties of polyanionic extracellular matrices in a defined manner.
Publications
- (2010) Carbohydrate binding of Salmonella phage P22 tailspike protein and its role during host cell infection, Biochem. Soc. Trans. 38:1386-1389
Dorothee Andres, Ulrich Baxa, Kristin Hanke, Robert Seckler and Stefanie Barbirz
(See online at https://doi.org/10.1042/BST0381386) - Tailspike interactions with lipopolysaccharide effect DNA ejection from phage P22 particles in vitro. J. Biol. Chem. 2010, 285 (47), 36768
Andres, D.; Hanke, C.; Baxa, U.; SeuI, A.; Barbirz, S.; Seckler, R.
(See online at https://doi.org/10.1074/jbc.M110.169003) - Tail morphology controls DNA release in two Salmonella phages with one lipopolysaccharide receptor recognition system. Mol. Microbiol. 2012, 83 (6), 1244
Andres, D.; Roske, Y.; Doering, C.; Heinemann, U.; Seckler, R.; Barbirz, S.
(See online at https://doi.org/10.1111/j.1365-2958.2012.08006.x) - An essential serotype recognition pocket on phage P22 tailspike protein forces Salmonella enterica serovar Paratyphi A O-antigen fragments to bind as nonsolution conformers. Glycobiology 2013, 23 (4), 486
Andres, D.; Gohlke, U.; Broeker, N. K.; Schulze, S.; Rabsch, W.; Heinemann, U.; Barbirz, S.; Seckler, R.
(See online at https://doi.org/10.1093/glycob/cws224) - Single amino acid exchange in bacteriophage HX620 tailspike protein results in thousand-fold increase of its oligosaccharide affinity. Glycobiology 2013, 23 (1), 59
Broeker, N. K.; Gohlke, U.; Müller, J. J.; Uetrecht, C.; Heinemann, U.; Seckler, R.; Barbirz, S.
(See online at https://doi.org/10.1093/glycob/cws126) - Conformational Diversity of O-antigen Polysaccharides of the Gram-negative Bactermm Shigella flexneri Serotype Y. J Phys Chem B 2014,118, 2523
Kang, Y.; Barbirz, S.; Lipowski, R.; Santer, M.
(See online at https://doi.org/10.1021/jp4111713) - Bacteriophage tailspike protein based assay to monitor phase variable glucosylations in Salmonella O-antigens. BMC Microbiology 2016,16, 207
Schmidt, A.; Rabsch, W.; Broeker, N. K.; Barbirz, S.
(See online at https://doi.org/10.1186/s12866-016-0826-0) - Bacteriophage Tailspikes and Bacterial O-Antigens as a Model System to Study Weak-Affinity Protein-Polysaccharide Interactions. J. Am. Chem. Soc. 2016,138 (29), 9109
Kang, Y.; Gohlke, U.; Engstrdm, O.; Hamark, C.; Scheidt, T.; Kunstmann, S.; Heinemann, U.; Widmalm, G.; Santer, M.; Barbirz, S.
(See online at https://doi.org/10.1021/jacs.6b00240) - (2017) Complex carbohydrate recognition by proteins: Fundamental insights from bacteriophage cell adhesion systems, Perspectives in Science 11: 45-52
Nina K. Broeker, Dorothee Andres, Yu Kang, Ulrich Gohlke, Andreas Schmidt, Sonja Kunstmann, Mark Santer, Stefanie Barbirz
(See online at https://doi.org/10.1016/j.pisc.2016.10.001) - (2017) Not a barrier but a key: Hozo bacteriophages exploit host's O-antigen as an essential receptor to initiate infection. Mol. Microbiol. 105: 353-357
Nina K. Broeker, Stefanie Barbirz
(See online at https://doi.org/10.1111/mmi.13729) - Bacteriophage Sf6 Tailspike Protein for Detection of Shigella flexneri Pathogens. Viruses-Basel 2018,10 (8)
Kunstmann, S.; Scheidt, T.; Buchwald, S.; Helm, A.; Mulard, L. A.; Fruth, A.; Barbirz, S.
(See online at https://doi.org/10.3390/v10080431) - In Vitro Studies of Lipopolysaccharidc-Mediated DNA Release of Podovirus HK620. Viruses-Basel 2018,10 (6)
Broeker, N. K.; Kiele, F.; Casjens, S. R.; Gilcrease, E. B.; Thalhammer, A.; Koetz, J.; Barbirz, S.
(See online at https://doi.org/10.3390/v10060289) - Solvent Networks Tune Thennodynamics of Oligosaccharide Complex Formation in an Extended Protein Binding Site. J. Am. Chem. Soc. 2018,140 (33), 10447
Kunstmann, S.; Gohlke, U.; Broeker, N. K.; Roske, Y.; Heinemann, U.; Santer, M.; Barbirz, S.
(See online at https://doi.org/10.1021/jacs.8b03719)
