@article{ZaccheusBroekerLundborgetal.2012,
  author    = {Zaccheus, Mona V. and Br{\"o}ker, Nina Kristin and Lundborg, Magnus and Uetrecht, Charlotte and Barbirz, Stefanie and Widmalm, Goran},
  title     = {Structural studies of the O-antigen polysaccharide from Escherichia coli TD2158 having O18 serogroup specificity and aspects of its interaction with the tailspike endoglycosidase of the infecting bacteriophage HK620},
  series = {Carbohydrate research},
  volume    = {357},
  journal   = {Carbohydrate research},
  number    = {8},
  publisher = {Elsevier},
  address   = {Oxford},
  issn      = {0008-6215},
  doi       = {10.1016/j.carres.2012.05.022},
  pages     = {118 -- 125},
  year      = {2012},
  abstract  = {We have analyzed the O-antigen polysaccharide of the previously uncharacterized Escherichia coli strain TD2158 which is a host of bacteriophage HK620. This bacteriophage recognizes and cleaves the polysaccharide with its tailspike protein (TSP). The polysaccharide preparation as well as oligosaccharides obtained from HK620TSP endoglycosidase digests were analyzed with NMR spectroscopy. Additionally, sugar analysis was performed on the O-antigen polysaccharide and MALDI-TOF MS was used in oligosaccharide analysis. The present study revealed a heterogeneous polysaccharide with a hexasaccharide repeating unit of the following structure: alpha-D-Glcp-(1 -> 6) vertical bar vertical bar 2)-alpha-L-Rhap-(1 -> 6)-alpha-D-Glcp-(1 -> 4)-alpha-D-Galp-(1 -> 3)-alpha-D-GlcpNAc- (1 ->vertical bar beta-D-Glcp/beta-D-GlcpNAc-(1 -> 3) A repeating unit with a D-GlcNAc substitution of D-Gal has been described earlier as characteristic for serogroup O18A1. Accordingly, we termed repeating units with D-Glc substitution at D-Gal as O18A2. NMR analyses of the polysaccharide confirmed that O18A1- and O18A2-type repeats were present in a 1:1 ratio. However, HK620TSP preferentially bound the D-GlcNAc- substituted O18A1-type repeating units in its high affinity binding pocket with a dissociation constant of 140 mu M and disfavored the O18A2-type having a beta-D-Glcp-(1 -> 3)-linked group. As a result, in hexasaccharide preparations, O18A1 and O18A2 repeats were present in a 9: 1 ratio stressing the clear preference of O18A1- type repeats to be cleaved by HK620TSP.},
  language  = {en}
}
@article{KunstmannGohlkeBroekeretal.2018,
  author    = {Kunstmann, Ruth Sonja and Gohlke, Ulrich and Br{\"o}ker, Nina Kristin and Roske, Yvette and Heinemann, Udo and Santer, Mark and Barbirz, Stefanie},
  title     = {Solvent networks tune thermodynamics of oligosaccharide complex formation in an extended protein binding site},
  series = {Journal of the American Chemical Society},
  volume    = {140},
  journal   = {Journal of the American Chemical Society},
  number    = {33},
  publisher = {American Chemical Society},
  address   = {Washington},
  issn      = {0002-7863},
  doi       = {10.1021/jacs.8b03719},
  pages     = {10447 -- 10455},
  year      = {2018},
  abstract  = {The principles of protein-glycan binding are still not well understood on a molecular level. Attempts to link affinity and specificity of glycan recognition to structure suffer from the general lack of model systems for experimental studies and the difficulty to describe the influence of solvent. We have experimentally and computationally addressed energetic contributions of solvent in protein-glycan complex formation in the tailspike protein (TSP) of E. coli bacteriophage HK620. HK620TSP is a 230 kDa native trimer of right-handed, parallel beta-helices that provide extended, rigid binding sites for bacterial cell surface O-antigen polysaccharides. A set of high affinity mutants bound hexa- or pentasaccharide O-antigen fragments with very similar affinities even though hexasaccharides introduce an additional glucose branch into an occluded protein surface cavity. Remarkably different thermodynamic binding signatures were found for different mutants; however, crystal structure analyses indicated that no major oligosaccharide or protein topology changes had occurred upon complex formation. This pointed to a solvent effect. Molecular dynamics simulations using a mobility-based approach revealed an extended network of solvent positions distributed over the entire oligosaccharide binding site. However, free energy calculations showed that a small water network inside the glucose-binding cavity had the most notable influence on the thermodynamic signature. The energy needed to displace water from the glucose binding pocket depended on the amino acid at the entrance, in agreement with the different amounts of enthalpy-entropy compensation found for introducing glucose into the pocket in the different mutants. Studies with small molecule drugs have shown before that a few active water molecules can control protein complex formation. HK620TSP oligosaccharide binding shows that similar fundamental principles also apply for glycans, where a small number of water molecules can dominate the thermodynamic signature in an extended binding site.},
  language  = {en}
}
@article{BroekerGohlkeMuelleretal.2013,
  author    = {Br{\"o}ker, Nina Kristin and Gohlke, Ulrich and M{\"u}ller, J{\"u}rgen J. and Uetrecht, Charlotte and Heinemann, Udo and Seckler, Robert and Barbirz, Stefanie},
  title     = {Single amino acid exchange in bacteriophage HK620 tailspike protein results in thousand-fold increase of its oligosaccharide affinity},
  series = {Glycobiology},
  volume    = {23},
  journal   = {Glycobiology},
  number    = {1},
  publisher = {Oxford Univ. Press},
  address   = {Cary},
  issn      = {0959-6658},
  doi       = {10.1093/glycob/cws126},
  pages     = {59 -- 68},
  year      = {2013},
  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.},
  language  = {en}
}
@inproceedings{DonohueSmithBroekeretal.2015,
  author    = {Donohue, Patrick and Smith, Mychal Daijon and Br{\"o}ker, Nina Kristin and Doering, Carolin and Mattos, Carla and Barbirz, Stefanie},
  title     = {Multiple Solvent Crystal Structures of phage P22 tailspike protein: An analysis of binding site hot spots and surface hydration},
  series = {The FASEB journal : the official journal of the Federation of American Societies for Experimental Biology},
  volume    = {29},
  booktitle = {The FASEB journal : the official journal of the Federation of American Societies for Experimental Biology},
  publisher = {Federation of American Societies for Experimental Biology},
  address   = {Bethesda},
  issn      = {0892-6638},
  pages     = {1},
  year      = {2015},
  language  = {en}
}
@phdthesis{Broeker2012,
  author    = {Br{\"o}ker, Nina Kristin},
  title     = {Die Erkennung komplexer Kohlenhydrate durch das Tailspike Protein aus dem Bakteriophagen HK620},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-60366},
  school      = {Universit{\"a}t Potsdam},
  year      = {2012},
  abstract  = {Kohlenhydrate stellen aufgrund der strukturellen Vielfalt und ihrer oft exponierten Lage auf Zelloberfl{\"a}chen wichtige Erkennungsstrukturen dar. Die Wechselwirkungen von Proteinen mit diesen Kohlenhydraten vermitteln einen spezifischen Informationsaustausch. Protein-Kohlenhydrat-Interaktionen und ihre Triebkr{\"a}fte sind bislang nur teilweise verstanden, da nur wenig strukturelle Daten von Proteinen im Komplex mit vorwiegend kleinen Kohlenhydraten erh{\"a}ltlich sind. Mit der vorliegenden Promotionsarbeit soll ein Beitrag zum Verst{\"a}ndnis von Protein-Kohlenhydrat-Wechselwirkungen durch Analysen struktureller Thermodynamik geleistet werden, um zuk{\"u}nftig Vorhersagen mit zuverl{\"a}ssigen Algorithmen zu erlauben. Als Modellsystem zur Erkennung komplexer Kohlenhydrate diente dabei das Tailspike Protein (TSP) aus dem Bakteriophagen HK620. Dieser Phage erkennt spezifisch seinen E. coli-Wirt anhand der Oberfl{\"a}chenzucker, der sogenannten O-Antigene. Dabei binden die TSP des Phagen das O-Antigen des Lipopolysaccharids (LPS) und weisen zudem eine hydrolytische Aktivit{\"a}t gegen{\"u}ber dem Polysaccharid (PS) auf. Anhand von isolierten Oligosacchariden des Antigens (Typ O18A1) wurde die Bindung an HK620TSP und verschiedener Varianten davon systematisch analysiert. Die Bindung der komplexen Kohlenhydrate durch HK620TSP zeichnet sich durch große Interaktionsfl{\"a}chen aus. Durch einzelne Aminos{\"a}ureaustausche im aktiven Zentrum wurden Varianten generiert, die eine tausendfach erh{\"o}hte Affinit{\"a}t (KD ~ 100 nM) im Vergleich zum Wildtyp-Protein (KD ~ 130 μM) aufweisen. Dabei zeichnet sich das System dadurch aus, dass die Bindung bei Raumtemperatur nicht nur enthalpisch, sondern auch entropisch getrieben wird. Ursache f{\"u}r den g{\"u}nstigen Entropiebeitrag ist die große Anzahl an Wassermolek{\"u}len, die bei der Bindung des Hexasaccharids verdr{\"a}ngt werden. R{\"o}ntgenstrukturanalysen zeigten f{\"u}r alle TSP-Komplexe außer f{\"u}r Variante D339N unabh{\"a}ngig von der Hexasaccharid-Affinit{\"a}t analoge Protein- und Kohlenhydrat-Konformationen. Dabei kann die Bindestelle in zwei Regionen unterteilt werden: Zum einen befindet sich am reduzierenden Ende eine hydrophobe Tasche mit geringen Beitr{\"a}gen zur Affinit{\"a}tsgenerierung. Der Zugang zu dieser Tasche kann ohne große Affinit{\"a}tseinbuße durch einen einzelnen Aminos{\"a}ureaustausch (D339N) blockiert werden. In der zweiten Region kann durch den Austausch eines Glutamats durch ein Glutamin (E372Q) eine Bindestelle f{\"u}r ein zus{\"a}tzliches Wassermolek{\"u}l generiert werden. Die Rotation einiger Aminos{\"a}uren bei Kohlenhydratbindung f{\"u}hrt zur Desolvatisierung und zur Ausbildung von zus{\"a}tzlichen Wasserstoffbr{\"u}cken, wodurch ein starker Affinit{\"a}tsgewinn erzielt wird. HK620TSP ist nicht nur spezifisch f{\"u}r das O18A1-Antigen, sondern erkennt zudem das um eine Glucose verk{\"u}rzte Oligosaccharid des Typs O18A und hydrolysiert polymere Strukturen davon. Studien zur Bindung von O18A-Pentasaccharid zeigten, dass sich die Triebkr{\"a}fte der Bindung im Vergleich zu dem zuvor beschriebenen O18A1-Hexasaccharid verschoben haben. Durch Fehlen der Seitenkettenglucose ist die Bindung im Vergleich zu dem O18A1-Hexasaccharid weniger stark entropisch getrieben (Δ(-TΔS) ~ 10 kJ/mol), w{\"a}hrend der Enthalpiebeitrag zu der Bindung g{\"u}nstiger ist (ΔΔH ~ -10 kJ/mol). Insgesamt gleichen sich diese Effekte aus, wodurch sehr {\"a}hnliche Affinit{\"a}ten der TSP-Varianten zu O18A1-Hexasaccharid und O18A-Pentasaccharid gemessen wurden. Durch die Bindung der Glucose werden aus einer hydrophoben Tasche vier Wassermolek{\"u}le verdr{\"a}ngt, was entropisch stark beg{\"u}nstigt ist. Unter enthalpischen Aspekten ist dies ebenso wie einige Kontakte zwischen der Glucose und einigen Resten in der Tasche eher ung{\"u}nstig. Die Bindung der Glucose in die hydrophobe Tasche an HK620TSP tr{\"a}gt somit nicht zur Affinit{\"a}tsgenerierung bei und es bleibt zu vermuten, dass sich das O18A1-Antigen-bindende HK620TSP aus einem O18A-Antigen-bindenden TSP evolution{\"a}r herleitet. In dem dritten Teilprojekt der Dissertation wurde der Infektionsmechanismus des Phagen HK620 untersucht. Es konnte gezeigt werden, dass analog zu dem verwandten Phagen P22 die Ejektion der DNA aus HK620 allein durch das Lipopolysaccharid (LPS) des Wirts in vitro induziert werden kann. Die Morphologie und Kettenl{\"a}nge des LPS sowie die Aktivit{\"a}t von HK620TSP gegen{\"u}ber dem LPS erwiesen sich dabei als essentiell. So konnte die DNA-Ejektion in vitro auch durch LPS aus Bakterien der Serogruppe O18A induziert werden, welches ebenfalls von dem TSP des Phagen gebunden und hydrolysiert wird. Diese Ergebnisse betonen die Rolle von TSP f{\"u}r die Erkennung der LPS-Rezeptoren als wichtigen Schritt f{\"u}r die Infektion durch die Podoviren HK620 und P22.},
  language  = {de}
}
@article{SchmidtRabschBroekeretal.2016,
  author    = {Schmidt, Andreas and Rabsch, Wolfgang and Br{\"o}ker, Nina Kristin and Barbirz, Stefanie},
  title     = {Bacteriophage tailspike protein based assay to monitor phase variable glucosylations in Salmonella O-antigens},
  series = {BMC microbiology},
  volume    = {16},
  journal   = {BMC microbiology},
  publisher = {BioMed Central},
  address   = {London},
  issn      = {1471-2180},
  doi       = {10.1186/s12866-016-0826-0},
  pages     = {2214 -- 2226},
  year      = {2016},
  abstract  = {Background: Non-typhoid Salmonella Typhimurium (S. Typhimurium) accounts for a high number of registered salmonellosis cases, and O-serotyping is one important tool for monitoring epidemiology and spread of the disease. Moreover, variations in glucosylated O-antigens are related to immunogenicity and spread in the host. However, classical autoagglutination tests combined with the analysis of specific genetic markers cannot always reliably register phase variable glucose modifications expressed on Salmonella O-antigens and additional tools to monitor O-antigen glucosylation phenotypes of S. Typhimurium would be desirable. Results: We developed a test for the phase variable O-antigen glucosylation state of S. Typhimurium using the tailspike proteins (TSP) of Salmonella phages 9NA and P22. We used this ELISA like tailspike adsorption (ELITA) assay to analyze a library of 44 Salmonella strains. ELITA was successful in discriminating strains that carried glucose 1-6 linked to the galactose of O-polysaccharide backbone (serotype O1) from non-glucosylated strains. This was shown by O-antigen compositional analyses of the respective strains with mass spectrometry and capillary electrophoresis. The ELITA test worked rapidly in a microtiter plate format and was highly O-antigen specific. Moreover, TSP as probes could also detect glucosylated strains in flow cytometry and distinguish multiphasic cultures differing in their glucosylation state. Conclusions: Tailspike proteins contain large binding sites with precisely defined specificities and are therefore promising tools to be included in serotyping procedures as rapid serotyping agents in addition to antibodies. In this study, 9NA and P22TSP as probes could specifically distinguish glucosylation phenotypes of Salmonella on microtiter plate assays and in flow cytometry. This opens the possibility for flow sorting of cell populations for subsequent genetic analyses or for monitoring phase variations during large scale O-antigen preparations necessary for vaccine production.},
  language  = {en}
}
@article{AndresGohlkeBroekeretal.2013,
  author    = {Andres, Dorothee and Gohlke, Ulrich and Br{\"o}ker, Nina Kristin and Schulze, Stefan and Rabsch, Wolfgang and Heinemann, Udo and Barbirz, Stefanie and Seckler, Robert},
  title     = {An essential serotype recognition pocket on phage P22 tailspike protein forces Salmonella enterica serovar Paratyphi A O-antigen fragments to bind as nonsolution conformers},
  series = {Glycobiology},
  volume    = {23},
  journal   = {Glycobiology},
  number    = {4},
  publisher = {Oxford Univ. Press},
  address   = {Cary},
  issn      = {0959-6658},
  doi       = {10.1093/glycob/cws224},
  pages     = {486 -- 494},
  year      = {2013},
  abstract  = {Bacteriophage P22 recognizes O-antigen polysaccharides of Salmonella enterica subsp. enterica (S.) with its tailspike protein (TSP). In the serovars S. Typhimurium, S. Enteritidis, and S. Paratyphi A, the tetrasaccharide repeat units of the respective O-antigens consist of an identical main chain trisaccharide but different 3,6-dideoxyhexose substituents. Here, the epimers abequose, tyvelose and paratose determine the specific serotype. P22 TSP recognizes O-antigen octasaccharides in an extended binding site with a single 3,6-dideoxyhexose binding pocket. We have isolated S. Paratyphi A octasaccharides which were not available previously and determined the crystal structure of their complex with P22 TSP. We discuss our data together with crystal structures of complexes with S. Typhimurium and S. Enteritidis octasaccharides determined earlier. Isothermal titration calorimetry showed that S. Paratyphi A octasaccharide binds P22 TSP less tightly, with a difference in binding free energy of similar to 7 kJ mol(-1) at 20 degrees C compared with S. Typhimurium and S. Enteritidis octasaccharides. Individual protein-carbohydrate contacts were probed by amino acid replacements showing that the dideoxyhexose pocket contributes to binding of all three serotypes. However, S. Paratyphi A octasaccharides bind in a conformation with an energetically unfavorable phi/epsilon glycosidic bond angle combination. In contrast, octasaccharides from the other serotypes bind as solution-like conformers. Two water molecules are conserved in all P22 TSP complexes with octasaccharides of different serotypes. They line the dideoxyhexose binding pocket and force the S. Paratyphi A octasaccharides to bind as nonsolution conformers. This emphasizes the role of solvent as part of carbohydrate binding sites.},
  language  = {en}
}