Crystal Structure of ORF210 from E. coli O157:H1 Phage CBA120 (TSP1), a Putative Tailspike Protein

Abstract

Bacteriophage tailspike proteins act as primary receptors, often possessing endoglycosidase activity toward bacterial lipopolysaccharides or other exopolysaccharides, which enable phage absorption and subsequent DNA injection into the host. Phage CBA120, a contractile long-tailed Viunalikevirus phage infects the virulent Escherichia coli O157:H7. This phage encodes four putative tailspike proteins exhibiting little amino acid sequence identity, whose biological roles and substrate specificities are unknown. Here we focus on the first tailspike, TSP1, encoded by the orf210 gene. We have discovered that TSP1 is resistant to protease degradation, exhibits high thermal stability, but does not cleave the O157 antigen. An immune-dot blot has shown that TSP1 binds strongly to non-O157:H7 E. coli cells and more weakly to K. pneumoniae cells, but exhibits little binding to E. coli O157:H7 strains. To facilitate structure-function studies, we have determined the crystal structure of TSP1 to a resolution limit of 1.8 Å. Similar to other tailspikes proteins, TSP1 assembles into elongated homotrimers. The receptor binding region of each subunit adopts a right-handed parallel β helix, reminiscent yet not identical to several known tailspike structures. The structure of the N-terminal domain that binds to the virion particle has not been seen previously. Potential endoglycosidase catalytic sites at the three subunit interfaces contain two adjacent glutamic acids, unlike any catalytic machinery observed in other tailspikes. To identify potential sugar binding sites, the crystal structures of TSP1 in complexes with glucose, α-maltose, or α-lactose were determined. These structures revealed that each sugar binds in a different location and none of the environments appears consistent with an endoglycosidase catalytic site. Such sites may serve to bind sugar units of a yet to be identified bacterial exopolysaccharide.

Structure and function of bacteriophage CBA120 ORF211 (TSP2), the determinant of phage specificity towards E. coli O157:H7

“…When heated from 20 to 95 °C, the protein unfolded cooperatively with a melting temperature (T m ) of 82.8 °C (data not shown). Similar high thermal stability of tailspikes, thought to be required to withstand harsh environmental conditions, has been reported in the literature for TSP1 of phage CBA120 (T m = 80.7 °C), the TSP of phage P22 (T m = 88.4 °C) and the TSP of phage HK620 (T m = 80 °C) 9 , 16 , 17 . However, the thermal stability of TSP3 from phage CBA120 is considerably lower (T m = 61.8 °C) 10 .…”

“…1 b). TSP2 head domain is strikingly smaller than the head domains of TSP1, TSP3, and TSP4; whereas each of the latter TSP head domains contains two adjoining fold modules, termed previously D1 and D2 9 , 10 , TSP2 head domain contains only the D1 unit and lacks the D2 unit (Figs. 1 b and 4 a).…”

“…The C-terminal domains of TSP glycosidases contain two adjoining fold modules, which we termed previously D3 and D4 9 , 10 . The enzymatic machinery resides on D3.…”

“…After binding to the bacterial lipopolysaccharide (LPS), TSPs exhibit lyase, glycosidase or esterase activity directed toward the O-antigen part of the LPS that eventually results in irreversible binding of the phage particle to its bacterial target 7 , 8 . For consistency, the terminology used to describe the architecture of TSP1 and TSP3 structures will be used here 9 , 10 . TSPs form homotrimers, which in the case of TSP1 and TSP3 are broadly divided into two domains.…”

Function of bacteriophage G7C esterase tailspike in host cell adsorption

“…The two N-terminal domains show 70% sequence identity to the N-terminal region of the CBA120 phage tailspike 1 (orf210, PDB code 4OJ5) (Chen et al, 2014). The structures of the two proteins are very similar and can be superimposed (as rigid two-domain units) on each other with a RMSD of 0.5 Å between the 142 aligned C-alpha atoms.…”