Phage P22 tailspike protein: crystal structure of the head-binding domain at 2.3 A, fully refined structure of the endorhamnosidase at 1.56 A resolution, and the molecular basis of O-antigen recognition and cleavage - PubMed (original) (raw)

Phage P22 tailspike protein: crystal structure of the head-binding domain at 2.3 A, fully refined structure of the endorhamnosidase at 1.56 A resolution, and the molecular basis of O-antigen recognition and cleavage

S Steinbacher et al. J Mol Biol. 1997.

Abstract

The tailspike protein of Salmonella phage P22 is a viral adhesion protein with both receptor binding and destroying activities. It recognises the O-antigenic repeating units of cell surface lipopolysaccharide of serogroup A, B and D1 as receptor, but also inactivates its receptor by endoglycosidase (endorhamnosidase) activity. In the final step of bacteriophage P22 assembly six homotrimeric tailspike molecules are non-covalently attached to the DNA injection apparatus, mediated by their N-terminal, head-binding domains. We report the crystal structure of the head-binding domain of P22 tailspike protein at 2.3 A resolution, solved with a recombinant telluromethionine derivative and non-crystallographic symmetry averaging. The trimeric dome-like structure is formed by two perpendicular beta-sheets of five and three strands, respectively in each subunit and caps a three-helix bundle observed in the structure of the C-terminal receptor binding and cleaving fragment, reported here after full refinement at 1.56 A resolution. In the central part of the receptor binding fragment, three parallel beta-helices of 13 complete turns are associated side-by-side, while the three polypeptide strands merge into a single domain towards their C termini, with close interdigitation at the junction to the beta-helix part. Complex structures with receptor fragments from S. typhimurium, S. enteritidis and S. typhi253Ty determined at 1.8 A resolution are described in detail. Insertions into the beta-helix form the O-antigen binding groove, which also harbours the active site residues Asp392, Asp395 and Glu359. In the intact structure of the tailspike protein, head-binding and receptor-binding parts are probably linked by a flexible hinge whose function may be either to deal with shearing forces on the exposed, 150 A long tailspikes or to allow them to bend during the infection process.

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Figures

Figure 1

Figure 1

Diagram of Salmonella phage P22. The icosahedral head is formed by approximately 420 molecules gp5. The portal protein (gp1) 12-mer is inserted into one icosahedral facet. Up to six tailspike molecules (gp9) are attached to a slender neck structure formed by gp4, gp10 and gp26. This contact is mediated by the N-terminal, head-binding domain. Minor components (gp7, gp16 and gp20) are associated with the portal protein or the DNA.

Figure 2

Figure 2

Schematic representation of the topology of one subunit of the head-binding domain. β-Sheet A comprises five strands (A1 from 100 to 109, A2 from 89 to 94; A3 from 29 to 32, A4 from 65 to 67, A5 from 72 to 74), β-sheet B comprises three strands (B1 from 81 to 83, B2 from 49 to 54, B3 from 56 to 62). In the trimer, β-sheet B is extended by the N-terminal strand of a neighbouring subunit (residues 7 to 9). The secondary structure was analysed with DSSP (Kabsch & Sander, 1983).

Figure 3

Figure 3

Stereoview of the head-binding domain. The β-sheets A and B form the walls of a dome-like structure. (a) Side view perpendicular to the trimer axis. The side contacts between the subunits are not very extensive. (b) Top view. A cluster of the three Phe22 residues at the molecular triad stabilises the head-binding domain. (c) Bottom view. The N-terminal polypetide strands extend to the neighbouring subunits and shield the single Met89 from the solvent, which was replaced by Met(Te) for heavy atom derivative preparation. The Figure was produced with MOLSCRIPT (Kraulis, 1991).

Figure 3

Figure 3

Stereoview of the head-binding domain. The β-sheets A and B form the walls of a dome-like structure. (a) Side view perpendicular to the trimer axis. The side contacts between the subunits are not very extensive. (b) Top view. A cluster of the three Phe22 residues at the molecular triad stabilises the head-binding domain. (c) Bottom view. The N-terminal polypetide strands extend to the neighbouring subunits and shield the single Met89 from the solvent, which was replaced by Met(Te) for heavy atom derivative preparation. The Figure was produced with MOLSCRIPT (Kraulis, 1991).

Figure 3

Figure 3

Stereoview of the head-binding domain. The β-sheets A and B form the walls of a dome-like structure. (a) Side view perpendicular to the trimer axis. The side contacts between the subunits are not very extensive. (b) Top view. A cluster of the three Phe22 residues at the molecular triad stabilises the head-binding domain. (c) Bottom view. The N-terminal polypetide strands extend to the neighbouring subunits and shield the single Met89 from the solvent, which was replaced by Met(Te) for heavy atom derivative preparation. The Figure was produced with MOLSCRIPT (Kraulis, 1991).

Figure 4

Figure 4

Stereoview of the electrostatic potential of the head-binding domain from −7kT/e− (red) to +7kT/e− (blue). The three monomers are labelled I to III. The view is perpendicular to the molecular triad (green triangle). Charged residues mainly cluster at the domain interface, whereas the centre of β-sheet A is dominated by polar and hydrophobic residues corresponding to a neutral potential. The saltbridge Arg19/Asp100# is located at the subunit interface (B). The Figure was produced with GRASP (Nicholls et al., 1993).

Figure 5

Figure 5

Stereoview of the tailspike protein with bound O-antigen receptor fragment from S. typhi253Ty. The head-binding domain caps a three helix-bundle present at the C terminus of the receptor binding and catalytic fragment. The active site is located 80 Å above the C terminus.

Figure 6

Figure 6

Structurally based amino acid alignment within the β-helix main body of the tailspike protein. Each row corresponds to a β-helix turn (1 to 13) and each column to a ladder of aligned residues, pointing either into the interior of the β-helix (i) or to the outside (a). Dots indicate insertion or deletions into the β-helix. The sheets termed A (blue), B (yellow) and C (green) (Steinbacher et al., 1994) correspond to the sheets PB2, PB3 and PB1 in pectate lyase C from Ewinia chrysanthemi(Yoder et al., 1993). Only short repetitive elements of identical amino acids are present.

Figure 7

Figure 7

(a) Schematic structure of Salmonella LPS, consisting of a lipid A membrane anchor, a common core of carbohydrates and a varying number of 4 to 30 O-antigenic repeats. (b) Chemical structure of Salmonella O-antigenic repeats of serotypes A (Par, paratose), B (Abe, abequose) and D1 (Tyv, tyvelose) α-(1,3)-linked to Man of the trisaccharide repeat α-Man-(1–4)-α-Rha-(1–3)-α-Gal. Randomly α-(1–4)-linked Glc represents O-antigen 122. The arrow marks the cleavage site.

Figure 7

Figure 7

(a) Schematic structure of Salmonella LPS, consisting of a lipid A membrane anchor, a common core of carbohydrates and a varying number of 4 to 30 O-antigenic repeats. (b) Chemical structure of Salmonella O-antigenic repeats of serotypes A (Par, paratose), B (Abe, abequose) and D1 (Tyv, tyvelose) α-(1,3)-linked to Man of the trisaccharide repeat α-Man-(1–4)-α-Rha-(1–3)-α-Gal. Randomly α-(1–4)-linked Glc represents O-antigen 122. The arrow marks the cleavage site.

Figure 8

Figure 8

Stereoview of the 2_F_o − _F_c electron density of (a) S. typhimurium and (b) S. enteritidis O-antigen octasaccharides at 1.8 Å resolution contoured at 1σ. Repeating unit I is proximal, repeating unit II distal from the active site.

Figure 9

Figure 9

(a) Solvent accessible surface of O-antigen binding site (blue) with bound O-antigen octasaccharide from S. enteritidis (red). (b) Stereoview of the binding site. The O-antigen receptor is recognised in a 20 Å cleft formed between the dorsal and the ventral fin domain.

Figure 10

Figure 10

Stereoview of the 2_F_o − _F_c electron density of the terminal rhamnose I (Rha I) of the O-antigen fragment from S. typhi253Ty bound to the active site at 1.8 Å resolution contoured at 1σ. The discontinuity of the electron density between C2 and C3 is also observed for S. typhimurium and S. enteritidis receptor fragments. The main conformation adopted by Rha I corresponds to a twisted boat conformation with C1 in α-configuration as in the substrate.

Figure 11

Figure 11

Stereoview of the active site with Rha I of the bound product. The steric situation suggests an inverting mechanism. A water molecule tightly bound also in the absence of the carbohydrate is activated by Glu359 and Asp395 as general bases. Asp392 serves as general acid and is H-bonded to O1 of Rha I.

Figure 12

Figure 12

Stereoview of the 2_F_o − _F_c electron density of the 3,6-dideoxyhexoses in repeating unit II pointing towards the protein surface. (a) Abequose (serotype B) and (b) tyvelose (serotype D1) at 1.8 Å resolution contoured at 1σ.

Figure 13

Figure 13

Stereoview of the binding site for 3,6-dideoxyhexoses in repeating unit II. Multiple host specificity is achieved by alternative contacts between the 3,6-dideoxyhexose and the protein, which are partly mediated by two water molecules. The distances between Abe O2 and Asp303 and Tyv O4 and Glu309 are 2.7 and 3.2 Å, respectively.

Figure 14

Figure 14

Stereoview of the 2_F_o − _F_c electron density of glucose in repeating unit I of S. typhi253Ty O-antigen introduced by form variation at 1.8 Å resolution contoured at 1σ.

Figure 15

Figure 15

Stereoview of the binding site for glucose I of O-antigen 122 introduced by form variation. Lys302 has to reorient its side-chain to open the binding site. This is the only rearrangement of side-chains observed upon receptor binding.

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