@article{BaxaWeintraubSeckler2020, author = {Baxa, Ulrich and Weintraub, Andrej and Seckler, Robert}, title = {Self-competitive inhibition of the bacteriophage P22 Tailspike endorhamnosidase by O-antigen oligosaccharides}, series = {Biochemistry}, volume = {59}, journal = {Biochemistry}, number = {51}, publisher = {American Chemical Society}, address = {Washington}, issn = {0006-2960}, doi = {10.1021/acs.biochem.0c00872}, pages = {4845 -- 4855}, year = {2020}, abstract = {The P22 tailspike endorhamnosidase confers the high specificity of bacteriophage P22 for some serogroups of Salmonella differing only slightly in their O-antigen polysaccharide. We used several biophysical methods to study the binding and hydrolysis of O-antigen fragments of different lengths by P22 tailspike protein. O-Antigen saccharides of defined length labeled with fluorophors could be purified with higher resolution than previously possible. Small amounts of naturally occurring variations of 0antigen fragments missing the nonreducing terminal galactose could be used to determine the contribution of this part to the free energy of binding to be similar to 7 kJ/mol. We were able to show via several independent lines of evidence that an unproductive binding mode is highly favored in binding over all other possible binding modes leading to hydrolysis. This is true even under circumstances under which the O-antigen fragment is long enough to be cleaved efficiently by the enzyme. The high-affinity unproductive binding mode results in a strong self-competitive inhibition in addition to product inhibition observed for this system. Self-competitive inhibition is observed for all substrates that have a free reducing end rhamnose. Naturally occurring O-antigen, while still attached to the bacterial outer membrane, does not have a free reducing end and therefore does not perform self-competitive inhibition.}, language = {en} } @article{KaufmannBaxaChipmanetal.2005, author = {Kaufmann, B. and Baxa, Ulrich and Chipman, P. R. and Rossmann, M. G. and Modrow, Susanne and Seckler, Robert}, title = {Parvovirus B19 does not bind to membrane-associated globoside in vitro}, issn = {0042-6822}, year = {2005}, abstract = {The glycosphingolipid globoside (globotetraosylceramide, Gb4Cer) has been proposed to be the cellular receptor of human parvovirus B19. Quantitative measurements of the binding of parvovirus B19 to Gb4Cer were performed to explore the molecular basis of the virus tropism. Solid-phase assays with fluorescence-labeled liposomes or (125)iodine-labeled empty capsids were used to characterize the specificity of binding. In addition, surface plasmon resonance on lipid layers, as well as isothermal titration microcalorimetry, was utilized for real-time analysis of the virus-receptor interaction. These studies did not confirm binding of Gb4Cer to recombinant B19 VP2 capsids, suggesting that Gb4Cer does not function on its own as the cellular receptor of human parvovirus B19, but might be involved in a more complex recognition event. The biochemical results were further confirmed by cryo-electron microscopy image reconstructions at 10 A resolution, in which the structures of empty capsids were compared with empty capsids incubated with Gb4Cer. (C) 2004 Elsevier Inc. All rights reserved}, language = {en} } @article{BaxaCooperWeintraubetal.2001, author = {Baxa, Ulrich and Cooper, Alan and Weintraub, N. and Pfeil, Wolfgang and Seckler, Robert}, title = {Enthalpic barriers to the hydrophobic binding of oligosaccharides to phage P22 tailspike protein}, year = {2001}, language = {en} } @article{FreibergMoronaVandenBoschetal.2003, author = {Freiberg, Alexander and Morona, Renato and Van den Bosch, Luisa and Jung, Christiane and Behlke, Joachim and Carlin, Nung and Seckler, Robert and Baxa, Ulrich}, title = {The tailspike protein of Shigella phage Sf6 : a structural homolog of Salmonella phage P22 tailspike protein without sequence similarity in the beta-helix domain}, issn = {0021-9258}, year = {2003}, abstract = {Bacteriophage Sf6 tailspike protein is functionally equivalent to the well characterized tailspike ofSalmonella phage P22, mediating attachment of the viral particle to host cell-surface polysaccharide. However, there is significant sequence similarity between the two 70-kDa polypeptides only in the N-terminal putative capsid-binding domains. The major, central part of P22 tailspike protein, which forms a parallel ;-helix and is responsible for saccharide binding and hydrolysis, lacks detectable sequence homology to the Sf6 protein. After recombinant expression in Escherichia coli as a soluble protein, the Sf6 protein was purified to homogeneity. As shown by circular dichroism and Fourier transform infrared spectroscopy, the secondary structure contents of Sf6 and P22 tailspike proteins are very similar. Both tailspikes are thermostable homotrimers and resist denaturation by SDS at room temperature. The specific endorhamnosidase activities of Sf6 tailspike protein toward fluorescence-labeled dodeca-, deca-, and octasaccharide fragments of Shigella O-antigen suggest a similar active site topology of both proteins. Upon deletion of the N-terminal putative capsid-binding domain, the protein still forms a thermostable, SDS-resistant trimer that has been crystallized. The observations strongly suggest that the tailspike of phage Sf6 is a trimeric parallel ;-helix protein with high structural similarity to its functional homolog from phage P22.}, language = {en} } @article{AndresHankeBaxaetal.2010, author = {Andres, Dorothee and Hanke, Christin and Baxa, Ulrich and Seul, Anait and Barbirz, Stefanie and Seckler, Robert}, title = {Tailspike interactions with lipopolysaccharide effect DNA ejection from phage P22 particles in vitro}, issn = {0021-9258}, doi = {10.1074/jbc.M110.169003}, year = {2010}, abstract = {Initial attachment of bacteriophage P22 to the Salmonella host cell is known to be mediated by interactions between lipopolysaccharide (LPS) and the phage tailspike proteins (TSP), but the events that subsequently lead to DNA injection into the bacterium are unknown. We used the binding of a fluorescent dye and DNA accessibility to DNase and restriction enzymes to analyze DNA ejection from phage particles in vitro. Ejection was specifically triggered by aggregates of purified Salmonella LPS but not by LPS with different O-antigen structure, by lipid A, phospholipids, or soluble O-antigen polysaccharide. This suggests that P22 does not use a secondary receptor at the bacterial outer membrane surface. Using phage particles reconstituted with purified mutant TSP in vitro, we found that the endorhamnosidase activity of TSP degrading the O-antigen polysaccharide was required prior to DNA ejection in vitro and DNA replication in vivo. If, however, LPS was pre-digested with soluble TSP, it was no longer able to trigger DNA ejection, even though it still contained five O-antigen oligosaccharide repeats. Together with known data on the structure of LPS and phage P22, our results suggest a molecular model. In this model, tail-spikes position the phage particles on the outer membrane surface for DNA ejection. They force gp26, the central needle and plug protein of the phage tail machine, through the core oligosaccharide layer and into the hydrophobic portion of the outer membrane, leading to refolding of the gp26 lazo-domain, release of the plug, and ejection of DNA and pilot proteins.}, language = {en} } @article{AndresBaxaHankeetal.2010, author = {Andres, Dorothee and Baxa, Ulrich and Hanke, Christin and Seckler, Robert and Barbirz, Stefanie}, title = {Carbohydrate binding of Salmonella phage P22 tailspike protein and its role during host cell infection}, issn = {0300-5127}, doi = {10.1042/Bst0381386}, year = {2010}, abstract = {TSPs (tailspike proteins) are essential infection organelles of bacteriophage P22. Upon infection, P22TSP binds to and cleaves the O-antigen moiety of the LPS (lipopolysaccharide) of its Salmonella host To elucidate the role of TSP during infection, we have studied binding to oligosaccharides and polysaccharides of Salmonella enteric Typhimurium and Enteritidis in vitro. P22TSP is a trimeric beta-helical protein with a carbohydrate-binding site on each subunit. Octasaccharide O-antigen fragments bind to P22TSP with micromolar dissociation constants. Moreover, P22TSP is an endorhamnosidase and cleaves the host O-antigen. Catalytic residues lie at the periphery of the high-affinity binding site, which enables unproductive binding modes, resulting in slow hydrolysis. However, the role of this hydrolysis function during infection remains unclear. Binding of polysaccharide to P22TSP is of high avidity with slow dissociation rates when compared with oligosaccharides. In vivo, the infection of Salmonella with phage P22 can be completely inhibited by the addition of LPS, indicating that binding of phage to its host via TSP is an essential step for infection.}, language = {en} } @article{BaxaSteinbacherWeintraubetal.1999, author = {Baxa, Ulrich and Steinbacher, Stefan and Weintraub, Andrej and Huber, Robert and Seckler, Robert}, title = {Mutations improving the folding of phage P22 tailspike protein affect its receptor binfing activity}, year = {1999}, language = {en} }