Crystal structure of ORF210 from E. coli O157:H1 phage CBA120 (TSP1), a putative tailspike protein.

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

Characterization of AlgMsp, an alginate lyase from Microbulbifer sp. 6532A.

Alginate is a polysaccharide produced by certain seaweeds and bacteria that consists of mannuronic acid and guluronic acid residues. Seaweed alginate is used in food and industrial chemical processes, while the biosynthesis of bacterial alginate is associated with pathogenic Pseudomonas aeruginosa. Alginate lyases cleave this polysaccharide into short oligo-uronates and thus have the potential to be utilized for both industrial and medicinal applications. An alginate lyase gene, algMsp, from Microbulbifer sp. 6532A, was synthesized as an E.coli codon-optimized clone. The resulting 37 kDa recombinant protein, AlgMsp, was expressed, purified and characterized. The alginate lyase displayed highest activity at pH 8 and 0.2 M NaCl. Activity of the alginate lyase was greatest at 50°C; however the enzyme was not stable over time when incubated at 50°C. The alginate lyase was still highly active at 25°C and displayed little or no loss of activity after 24 hours at 25°C. The activity of AlgMsp was not dependent on the presence of divalent cations. Comparing activity of the lyase against polymannuronic acid and polyguluronic acid substrates showed a higher turnover rate for polymannuronic acid. However, AlgMSP exhibited greater catalytic efficiency with the polyguluronic acid substrate. Prolonged AlgMsp-mediated degradation of alginate produced dimer, trimer, tetramer, and pentamer oligo-uronates

Statistics on data collection and refinements of sugar-bound TSP1 CBA120 structures.

a<p>The values in parentheses are for the highest resolution shell.</p><p><i>R</i>_merge = Σ<i>_hkl</i> [(Σ<i>_j</i>|<i>I_j</i>−<<i>I</i>>|)/Σ<i>_j</i>|<i>I_j</i>|].</p><p><i>R</i>_work = Σ<i>_hkl</i> | |<i>F_o</i>|−|<i>F_c</i>| |/Σ<i>_hkl</i> |<i>F_o</i>|, where <i>F_o</i> and <i>F_c</i> are the observed and calculated structure factors, respectively.</p><p><i>R</i>_free is computed from 5% (TSP1/Glucose) or 2,000 (TSP1/Lactose and TSP1/Maltose) randomly selected reflections that were omitted from the refinement.</p

Effect of temperature on alginate lyase activity and stability of activity.

<p>(A) Effect of temperature on activity with (filled circles) and without NaCl (open circles). Activity was determined, after 10 minutes at the target temperature, by the increase in absorbance at 235 nm over the absorbance of the control reaction without enzyme. (B) Thermostability of alginate lyase activity. AlgMsp was incubated at 50°C (circles), 37°C (squares), and 25°C (triangles) for 24 hours in 20 mM Tris, 200 mM NaCl, pH 8. Samples were tested at 0, 0.5, 1, 2, 4, 8, and 24 hours for residual activity as above. Activity was normalized to 100% for the most active sample.</p

ESI-mass spectrometry analyses of peak fractions from FPLC separation of the oligo-uronates from AlgMsp digestion of alginate.

<p>DPx is degree of polymerization. DPx and ΔDPx represent saturated and unsaturated oligo-uronates, respectively. (A) Mass spectrum of FPLC fraction 1, the 13.4 ml elution peak, showing predominance of ΔDP5 eluent. Inset: MS/MS product ion spectrum of <i>m/z</i> 439 peak, showing the methodology used to elucidate the structure of all observed ESI-MS anion peaks. The <i>m/z</i> 703 and <i>m/z</i> 721 peaks are indicative of the double charge of the precursor anion, <i>m/z</i> 439. The annotated peak series, {[ΔDP<i>x</i>-1H]⁻, <i>x</i> = 1–4}, and the peaks denoted with asterisks ([DP4-2H]²⁻ (<i>m/z</i> 360), {[DP3-H]⁻ (<i>m/z</i> 545), and {[DP4-H]⁻ (<i>m/z</i> 721), confirm that the composition of the <i>m/z</i> 439 precursor anion, is consistent with [ΔDP5-2H]²⁻. (B) Mass spectrum of FPLC fraction 2, the 14.0 ml elution peak, showing predominance of ΔDP4 eluent. (C) Mass spectrum of FPLC fraction 3, the 14.7 ml elution peak, showing predominance of ΔDP3 eluent. (D) Mass spectrum of FPLC fraction 4, the 15.6 ml elution peak, showing predominance of ΔDP2 eluent.</p

Effects of pH and NaCl on alginate lyase activity of AlgMsp.

<p>(A) Effect of pH in the absence of NaCl on activity in 20 mM Tris (circles) or BP buffer (triangles). (B) Effect of NaCl on activity in 20 mM Tris buffer pH 8. (C) Effect of pH on activity in the presence of 200 mM NaCl in 20 mM Tris (circles) or BP buffer (triangles). Activity determined by the initial velocity of increase in absorbance at 235 nm over a 10 minute incubation at 25°C. Activity was normalized to 100% for the most active sample.</p

Structure of TSP1 from bacteriophage CBA120.

<p>(A) “Side view” of the homotrimer. The three monomers are colored in green, cyan, and magenta. The Zn²⁺ is shown as an orange sphere and indicated by an arrow. The “hole” in the catalytic domain is indicated. (B) The structure of TSP1 monomer. The N-terminal head binding domain and a C-terminal receptor-binding domain are further divided into four subdomains, D1, D2, D3, and D4 colored in blue, red, green, and cyan, respectively. The D3-D4 intervening region that bends the β-helical axis is colored in grey. (C) and (D) “Top view” (down from the N-terminus) and “bottom view” (down from the C-terminus), respectively. (E) Anomalous difference map calculated with diffraction data collected at the zinc absorption edge peak (1.28283 Å). The calculated phases included only the protein atoms. The Zn²⁺ coordinates His25 of each subunit and a water molecule. The anomalous difference map (magenta cage) is contoured at 15σ.</p

Kinetics of AlgMsp.

<p>Kinetics of AlgMsp.</p

Stereoscopic representation of the environment of sugars bound to TSP1.

<p>Ligands and interacting protein residues are shown as stick models and water molecules as spheres. The atom color scheme is as follows: Nitrogen – blue, oxygen – red, ligand carbon – yellow, protein carbon – green. Hydrogen bonds are shown as dashed lines. (A) Glucose. (B) Lactose. (C) Maltose. The cartoon models are colored gray in (A) and (C). The cartoon models of two TSP1 molecules that contribute to the lactose binding are colored green and cyan in (B).</p

Effects of divalent cations on AlgMsp activity.

<p>Effects of divalent cations on AlgMsp activity.</p