Crystal structure of phage P22 tailspike protein complexed with Salmonella sp. O-antigen receptors.

Abstract

The O-antigenic repeating units of lipopolysaccharides from Salmonella serogroups A, B, and DI serve as receptors for the phage P22 tailspike protein, which also has receptor destroying endoglycosidase (endorhamnosidase) activity, integrating the functions of both hemagglutinin and neuraminidase in influenza virus. Crystal structures of the tailspike protein in complex with oligosaccharides, comprising two 0-antigenic repeating units from Sabnonella typhiumurium, SalmoneUa enteritidis, and Salmonella typhi 253Ty were determined at 1.8 A resolution. The active-site topology with Asp-392, Asp-395, and Glu-359 as catalytic residues was identified. Kinetics of binding and cleavage suggest a role of the receptor destroying endorhamnosidase activity primarily for detachment of newly assembled phages.The infection of Salmonella by phage P22 starts with the recognition of the 0-antigenic repeating units of the cell surface lipopolysaccharide by the homotrimeric tailspike protein (TSP), which is present in six copies on the attachment apparatus (1, 2). Phages that use lipopolysaccharide as receptors are faced with the chemical diversity of the 0-antigenic repeats, which differ in carbohydrate composition and stereochemistry of the 0-glycosidic linkage. In addition, microheterogeneity conceming the number of repeating units and additional modifications as acetylation or glucosylation known as form variation is an important feature of lipopolysaccharide (3-5). P22 has adapted to Salmonella serotypes A, B, and Dl ( Fig. 1), sharing a common trisaccharide repeating unit a-Dmannose-(1,4)-a-L-rhamnose-(1,3)-a-D-galactose for the 0-antigen but differing in their branching carbohydrate moieties, a 3,6-dideoxyhexose a-(1,3)-linked to D-mannose. Dideoxyhexoses are paratose (serogroup A), abequose (serotype B), or tyvelose (serotype D1) and reflect the correlation of serotype classification and chemical structure of the 0-antigenic repeats of Salmonella (6, 7). In addition, P22 tolerates the 0-antigen 122, which shows an a-(1,4)-linked D-glucose at D-galactose as in Salmonella typhi 253Ty (8). In the phage-host interaction, the dideoxyhexose can be viewed as a wobble position that allows for some flexibility in a specific interaction.Receptor destroying enzymatic activities are well known for viruses that use carbohydrates as receptors. Influenza A and B virus and paramyxovirus have neuraminidases releasing terminal N-acetylmuraminic acid from glycoproteins and glycolipids (9), whereas influenza virus C and some coronaviruses that recognize an 0-acetylated sialic acid epitope have a sialate 9-0-acetylesterase, removing an acetyl group (10, 11). Endoglycosidase or acetylesterase activities have also been demonstrated for a large number of phages acting on encapsulated Gram-negative bacteria like Escherichia coli, Salmonella, or Klebsiella (12). TSP possesses receptor destroying endorhamnosidase activity cleaving the a(1,3)-O-glycosidic bond between rhamnose and galactose of the 0-antigenic repeats (13,14). To elucidate the s...

Structural and Functional Insights into Intramolecular Fructosyl Transfer by Inulin Fructotransferase

“…␤-Helical folds have been identified in several classes of enzymes frequently associated with polysaccharides, including pectin lyase-related proteins (29), pectin methylesterase (30), and phage P22 tailspike protein (31). Recently, they have also been found in other polysaccharide-degrading enzymes in O-glycoside hydrolase families 28 (32), 49 (33), and 82 (34), as well as in virulence factors (35).…”

“…Indeed, most of the ␤-helix enzymes characterized to date function as monomers. Tailspike protein, which forms a trimer in a manner similar to that of BsIFTase, is the exception, although its proposed catalytic mechanism occurs within a monomeric structural context (31). Therefore, trimerization of the tailspike protein ␤-helix may not be required for catalysis and might instead be important for thermostability and protease resistance (38,39).…”

Enthalpic Barriers to the Hydrophobic Binding of Oligosaccharides to Phage P22 Tailspike Protein

“…Ionization does not appear to play a major role, as the dissociation constant of octasaccharide is only slightly influenced by different buffers (Tris or phosphate) and by different ionic strengths measured from 0 to 200 mM (35), and isothermal titration calorimetric experiments in Tris buffer showed no difference to phosphate buffer (data not shown). Comparison of the crystal structures shows that the conformation of the protein does not change from the free to the saccharide-bound form (27). The only difference is a lower temperature factor of some residues in the binding site, which is often seen in protein-ligand complexes.…”

“…The carbohydrate binding site runs parallel to the helix axis flanked by a 63-residue domain and three smaller loops inserted in the -helix ( Figure 1). Crystal structures of complexes with oligosaccharide fragments from different O-antigen serogroups disclosed the largest interaction surfaces of carbohydrates liganded to proteins known to date (25,27).…”

Bacteriophage Tailspikes and Bacterial O-Antigens as a Model System to Study Weak-Affinity Protein–Polysaccharide Interactions

“…12 TSP attain specificity via long and shallow binding grooves, and several complexes of TSP with oligosaccharide fragments up to nonamer length have been described. 13,14,15 The…”