Crystal Structure of a Chimeric Receptor Binding Protein Constructed from Two Lactococcal Phages

Lactococcal phages and host strains.Phages p2 and TP901-1 and their L. lactis host strains were obtained from the Felix d'Hérelle Reference Center for Bacterial Viruses (www.phage.ulaval.ca). L. lactis strains were grown in M17 (Oxoid) supplemented with 0.5% glucose (GM17) without shaking at 30°C. When propagating phages, 10 mM CaCl₂ was added to the medium. Phage DNA was isolated as previously reported (14).

Cloning and bacterial expression.A gene coding for a chimeric RBP protein containing the N-terminal/shoulder and linker/neck domains of the lactococcal temperate phage TP901-1 (amino acids 1 to 63) and the C-terminal/head domain of the virulent lactococcal phage p2 RBP (amino acids 162 to 264) was constructed and cloned into the Gateway destination vector pDest17 (Invitrogen). A common proline residue situated at the beginning of the C-terminal domain (proline 63 in TP901-1 and proline 162 in p2) was used to define the boundary between the linker domain and C-terminal domain for constructing the chimera. PCR amplification of DNA sequences was performed using previously described plasmids (29, 30) containing the full-length RBP of phage p2 or TP901-1. Amplification of the individual domains was performed in two steps, with each domain first being amplified, followed by a joining step of amplification. The resulting vector was transformed into the Escherichia coli C41(DE3)/pLysS (Lucigen) strain for expression. Transformed cells were grown in 2× YT medium at 37°C until an optical density at 600 nm of 0.5 was reached. Expression was induced with 0.5 mM isopropyl-β-thiogalactoside (IPTG), and cells were left for 20 h at 17°C. Bacterial cells were recovered by centrifugation at 5,000 × g for 10 min. Bacterial pellets were then resuspended in 40 ml of lysis buffer (50 mM Tris [pH 8.0], 300 mM NaCl, 10 mM imidazole, 0.25 mg/ml lysozyme, EDTA-free antiproteases [Roche]) per liter of cell culture and frozen at −80°C.

Protein purification.Cells were thawed with shaking in the presence of 20 mM MgSO₄ and 10 μg/ml of DNase. The lysate was then sonicated and cleared by a 30-min centrifugation at 21,400 × g. After filtration on a 0.45-μm filter, supernatant was loaded on a 5 ml HiTrap nickel affinity column (GE Healthcare) equilibrated in imidazole buffer (50 mM Tris [pH 8.0], 300 mM NaCl, 10 mM imidazole). The column was washed and eluted with the same buffer containing increased imidazole concentrations of 50 mM and 250 mM, respectively. The eluted protein was further purified on a HiLoad 26/60 Superdex 200 (GE Healthcare) gel filtration column in a buffer containing 1.8 mM KH₂PO₄, 10.1 mM Na₂HPO₄, 2.7 mM KCl, and 137 mM NaCl, pH 7.2. Purified material was concentrated to appropriate crystallization concentrations on an Amicon Ultra-15 centrifugal filter unit with a 30-kDa cutoff.

Protein crystallization and crystal structure determination.Crystallization trials were performed using a sitting-drop technique implemented on a nanodrop-dispensing robot (PixSys; Cartesian) in Greiner 96-well plates. At 291 K, initial crystals were obtained under Stura footprint screen (Molecular Dimensions Limited) conditions E3 (0.1 M sodium cacodylate [pH 5.5], 18% polyethylene glycol [PEG] 2000 monomethyl ether) and F4 (0.2 M imidazole malate [pH 6.0], 15% PEG 4000). Optimization of these conditions yielded two different crystal morphologies with best-diffracting crystals under modified condition E3 (0.1 M sodium cacodylate [pH 5.5], 12% PEG 2000 monomethyl ether) with a protein concentration of 7.5 g/liter (form 1) and under modified condition F4 (0.2 M imidazole malate [pH 6.1], 17% PEG 4000) with a protein concentration of 6.3 g/liter (form 2). Data were collected at the European synchrotron radiation facility (ESRF, Grenoble, France) on line ID14-EH1. Crystals were directly flash-frozen from their mother liquor in the nitrogen gas flow. Both data sets were integrated and reduced using MOSFLM and SCALA (7). Molecular replacement was performed using the automated molecular replacement server BALBES (20) for form 1 and using PHASER (22) with the p2 RBP C terminus trimer as a search model (Protein Data Bank number 1BSD; residues 162 to 264) for form 2. Refinement was performed using REFMAC (26), while rebuilding was done using COOT (16). Structure validation was performed using MolProbity (21). The data collection and refinement results are summarized in Table 1. Structure superimpositions were performed with turbo-Frodo, and figures were generated with Pymol (9).

Fluorescence-quenching experiments.Fluorescence experiments were carried out on a Varian Eclipse spectrofluorimeter using a quartz cuvette in a right-angle configuration; the light paths were 0.4 and 1 cm for the excitation and emission, respectively. The interaction with RBP was monitored by recording the quenching of the intrinsic protein fluorescence upon addition of ligand aliquots. The excitation wavelength was 290 nm, and emission spectra were recorded in the range from 320 to 380 nm. The excitation slit was 5 nm, while the emission slit was 10 nm. Titrations were carried out at 20°C in a phosphate buffer [1 μM protein in 10 mM (Na/Na₂) phosphate, 50 mM NaCl, pH 7.5]. The fluorescence intensities at the maximum of emission (354 nm) were corrected for the buffer contribution before plotting and further analysis. The affinity was estimated by plotting the decrease of fluorescence intensity at the emission maximum as [100 − (I_i − I_min)/(I₀ − I_min)·100] against the quencher concentration, where I₀ is the maximum fluorescence intensity of the protein alone, I_i is the fluorescence intensity after the ith addition of quencher, and I_min is the fluorescence intensity at the saturating concentration of quencher. The K_diss value was estimated using Prism 3.02 (GraphPad Software Inc.) by nonlinear regression for a single binding site with the equation Y = B_max·X/(K_diss + X), where B_max is the maximal binding, Y is the fluorescence intensity, X is the concentration of ligand added, and K_diss is the concentration of ligand required to reach half-maximal binding.

Structure of the RBP chimera.The chimera construct used in this study encoded 165 residues from lactococcal phages p2 and TP901-1, an N terminus of 21 residues from the cloning vector, and the His₆ tag. Specifically, residues 1 to 63 were from the RBP of the temperate phage TP901-1 (P335 species) and were fused to residues 163 to 264 from the RBP of the virulent phage p2 (936 species) (Fig. 1C and E), resulting in a RBP chimera (Fig. 1D) that could be produced and purified. The junction point, Pro 162 (p2 numbering) or 63 (TP901-1 numbering), is located at the linker/head domain junctions. It interrupts the last β-strand of the linker and redirects the amino acid chain to the top (Fig. 1C and E).

Crystallization of this RBP chimera led to two crystal forms (Table 1). Form 1 diffracts to a relatively low resolution (3.35 Å), and the electron density starts at the same residue number as the native RBP from the temperate phage TP901-1 (27, 29-31). It contains a cleaved protomer in the asymmetric unit, comprising residues 17 to 165 (chimera numbering). The trimer, found in native wild-type lactococcal RBPs, was generated using the crystallographic threefold axis of the P2₁3 space species (Fig. 2A). On the other hand, form 2 diffracts with a better resolution (1.65 Å) but is cleaved at the beginning of the β-helical linker domain and starts at residue 34. Form 2 contains the expected trimer in the asymmetric unit, with each protomer comprising residues 34 to 164 (Fig. 1 and 2A).

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FIG. 2.

Superimposition and comparison of the RBPs from lactococcal phages p2 and TP901-1 as well as their chimera. (A) Superimposition of chimera form 1 (yellow) and form 2 (blue). (B) Superimposition, using the N-terminal and linker domains, of form 2 (yellow) on the wild-type RBP of phage TP901-1 (pink). (C) Superimposition, using the C-terminal domains, of form 2 (yellow) on the wild-type RBP of phage p2 (green). Inset, 90° view of the β-prism linker domains of p2 (green) and the chimera (yellow), illustrating the larger size of the latter.

In both crystal forms, the electron density map is well defined, considering the respective resolutions. In particular, the stretch containing Pro 162 (p2) or 63 (TP901-1) at the linker/head junction of the RBP chimera is perfectly defined in the electron map density (Fig. 3). Superimposition of the form 1 reconstituted trimer onto the trimer of form 2 yields a good root mean square deviation (RMSD) value of 0.49 Å for the 393 Cα carbons in common (Fig. 2A).

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FIG. 3.

2fo-fc 1.65-Å resolution electron density (stereo view) contoured at 1σ of the stretch of residues linking the swapped domains of the chimera, including the hinge proline.

Comparison of the chimera structures with the wild-type structures.We have superimposed the RBP structures of the chimera and of the wild type from TP901-1, using the common domains, N terminus, and β-prism. The superimposition is excellent with both forms, yielding RMSD values of 0.69 and 0.30 Å between their Cα atoms for forms 1 and 2, respectively (Fig. 2B; Table 2). Similarly, when superimposing the RBP C-terminal domains of the chimera and phage p2, the RMSD values are even better, at 0.47 Å and 0.28 Å for forms 1 and 2, respectively (Fig. 2C; Table 2). These two comparisons indicate that the structures of the individual domains are rigid enough to keep close structures, independently of the domains upon which they are grafted or the different space groups to which they belong. In the latter case, the superimposition of the wild-type linker domains indicates that the linker domain of phage TP901-1 is slightly larger than that of phage p2 (Fig. 2C, inset). Indeed, when superimpositions are applied to the whole RBP structures, the RMSD values become worse, due to the differences in domain orientations (Table 2). The RMSD values still remain smaller than that calculated between the Cα atoms of the RBPs of p2 and TP901-1, which is 1.53 Å. To adapt to the linker difference between phages p2 and TP901-1, a stretch of ∼10 residues (starting at the hinge proline of the linker/C terminus junction of the chimera) adopts a different track from that in the TP901-1 structure, leading to a C terminus domain rotation of ∼8 degrees. Finally, the receptor binding sites of the wild-type RBP of phage p2 and the binding domain of the chimera are very similar.

Interpreting the cleavage sites within a structural context.As indicated above, the analyses of the two crystal forms of the RBP chimera revealed protease cleavages during the course of crystallization (Fig. 4). The reasons for the cleavages are unknown, although traces of contaminating proteases from the expression organisms cannot be ruled out. Interestingly, the cleavage site in form 1 was also previously reported in the structure of the native RBP from phage TP901-1 (29). The freshly prepared protein is intact, but it is cleaved in a few days. We can see the amino acid chain from threonine 17 in the electron map density (Fig. 4A). Since the structure of the intact RBP protein of TP901-1 is still unknown, we cannot interpret this cleavage in terms of residue accessibility.

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FIG. 4.

Identification of the protease cleavage sites on RBPs and their correlation with domain junctions. (A) Chimera form 1 and its cleavage after Glu16. (B) Chimera form 2 and its cleavage after Thr30, between the N terminus and linker domains. (C) RBP head domain of phage p2 from the complex with llama VHHs.

In contrast, the cleavage observed in chimera form 2 occurs in a large accessible loop (ELTSG∧GN) between two glycine residues (Fig. 4B). We never observed this cleavage in solution, but crystal packing clearly indicates that the absent density cannot be attributed to flexibility due to packing constraints. Another cleavage site was previously observed in the RBP structure of phage p2 while in complex with llama VHH domains (30) as well as in the RBP structure of the lactococcal phage bIL170 (936 group) (27). This cleavage occurs in a loop (T∧VSGSIDVP₁₆₂) at the end of the last β-strand of the linker domain, just before the hinge proline 162. Interestingly, the cleavages in form 2 as well as in the wild-type RBPs of phages p2 and bIL170 correspond to structural domain boundaries, with high solvent accessibility. It is noteworthy that small additions of protease have been used to induce cleavage at domain boundaries, thus facilitating crystallization of individual domains (12).

Interaction with glycerol.We have shown previously that the RBPs of phages p2 (27, 29-31), TP901-1 (29), and bIL170 (27) bind glycerol and phosphoglycerol with high affinity. Glycerol was found in the putative receptor binding site, close to a Trp or Phe residue, and establishing strong hydrogen bonds with Arg, Glu, and His. These data led us to postulate that the phage receptor at the bacterial cell surface might be teichoic or lipoteichoic acids, which are polymers of substituted phosphoglycerol. In the RBP of the virulent lactococcal phage p2, a tryptophan is part of the binding site in the head domain, which makes it possible to measure the affinity of the RBP chimera for glycerol using fluorescence quenching (27, 29-31). When glycerol is added in increasing concentrations to an RBP chimera solution of 1 μM, the maximum fluorescence is quenched by ∼30% (see Fig. S1 in the supplemental material). Analysis of the decrease curve yields a K_diss value of 80 nM, a value slightly better than that measured for wild-type RBP of phage p2 and also for the RBP head domain of p2 expressed alone (27, 29-31), which indicates that the chimeric RBP is able to bind to glycerol.

In conclusion, we have shown here that an artificial RBP chimera can be designed in a straightforward manner and is capable of binding activities. The RBP domain grafting resulted in stable chimerical structures in which the domains closely resemble the wild-type structures. Such conserved structure was possible due to adaptation through small displacements of the linkers. From a phage evolution standpoint, it suggests that lactococcal RBPs are built to efficiently exchange a head domain, which may lead to host range variation. Module shuffling also likely helps them to persist in a man-made environment containing distinct bacterial cells.