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

Lactococcus lactis, a gram-positive bacterium widely used bythe dairy industry to manufacture cheeses, is subject to infectionby a diverse population of virulent phages. We have previouslydetermined the structures of three receptor binding proteins(RBPs) from lactococcal phages TP901-1, p2, and bIL170, eachof them having a distinct host range. Virulent phages p2 andbIL170 are classified within the 936 group, while the temperatephage TP901-1 is a member of the genetically distinct P335 polytheticgroup. These RBPs comprise three domains: the N-terminal domain,binding to the virion particle; a β-helical linker domain;and the C-terminal domain, bearing the receptor binding siteused for host recognition. Here, we have designed, expressed,and determined the structure of an RBP chimera in which theN-terminal and linker RBP domains of phage TP901-1 (P335) arefused to the C-terminal RBP domain of phage p2 (936). This chimeraexhibits a stable structure that closely resembles the parentalstructures, while a slight displacement of the linker made RBPdomain adaptation efficient. The receptor binding site is structurallyindistinguishable from that of native p2 RBP and binds glycerolwith excellent affinity.

INTRODUCTION

Top

INTRODUCTION

A broad number of products are manufactured by large-scale bacterialfermentation, including the value-added fermented dairy products.Most bacterial fermentation industries have experienced problemswith phage contamination. Phage outbreaks are costly and time-consumingbecause they can slow or arrest the fermentation process andadversely affect product quality (15). For decades, the dairyindustry has relied on an array of strategies to control thisnatural phenomenon, including rotation of their bacterial cultures(11, 24, 25). However, in spite of these efforts, new virulentlactococcal phages keep emerging. A better understanding ofthe various mechanisms affecting the genetic diversity of thephage population is necessary for optimal phage control strategies(18).

Lactococcal phages are among the most studied bacterial virusesbecause of the economic importance of their hosts. Hundredsof lactococcal phages have been isolated, and the vast majorityof them have a long, contractile tail, thereby belonging tothe Siphoviridae family (1). Lactococcus lactis phages are currentlyclassified into 10 genetically distinct groups (10), but onlymembers of 3 of them are highly adapted to multiply in milk,namely, the 936, c2, and P335 groups (11, 24, 25). The firststep for such an effective viral infection is host recognition,which necessitates the interaction between the adsorption devicelocated at the distal tail end of the phage and the cell surfacereceptor (32). Members of the 936 and P335 groups recognizetheir host through an interaction between their receptor bindingprotein (RBP) (13) and receptors, probably lipoteichoic acids,at the host cell surface (27, 29-31).

We have previously determined the crystal structures of threeRBPs, from the virulent lactococcal phages p2 (30, 31) and bIL170(936 group) (27) and from the temperate phage TP901-1 (P335group) (29). The RBPs of these phages have a similar architectureof three protomers related by a threefold axis. Each protomercomprises three domains: the N terminus (named shoulders inp2), the interlaced β-prism linker (the "neck" domain),and the jelly-roll domain (2) at the C terminus (the "head"domain). This last domain harbors a saccharide binding sitelikely involved in host recognition, as it binds with high affinityto phosphoglycerol, a component of teichoic acid (8, 19, 27,29-31). We have previously shown that the shoulder and neckdomains are highly conserved in the RBPs of 936-like phages(8, 19, 27, 29-31). The individuality of the RBP C-terminaldomain sequence likely dictates phage specificity for the receptor,which may specifically recognize different substitutions (H,GlcNAc, or D-Ala) of the phosphoglycerol moieties of the L.lactis teichoic acid polymers. Recently, the complete genomicsequence of the reference virulent phage P335 was determined,and comparative analysis revealed that the C terminus of itsRBP showed homology to the RBP of the virulent lactococcal phageP475 of the 936 group (17). Such homology between RBP head domainswas surprising because the two lactococcal phage groups rarelyshared common genes or domains. This observation suggested thatmodular shuffling of domains can occur between these otherwisegenetically distinct phage groups.

The overall fold of the N-terminal RBP domain is different in936- and P335-like phages. In the P335 group, the N-terminaldomain comprises a unique helix that fits into the rest of thephage baseplate (28, 29) (Fig. 1A), while in the 936 group,this 140-residue domain is a large β-sandwich with an external ${alpha}$ -helix (30) (Fig. 1B). Nonetheless, the N-terminal domains ofthe two RBPs may still be, related because both appear to bebuilt using a coiled coil, although the 936-like phages havean additional β-sandwich. The β-prism linkers (neckdomain) of the two phage groups also differ in sequence andin radius, but they have a similar fold, the latter being alsoclose to that of T4 phage short fiber (33). The linker domainof phage TP901-1 is wider than that of p2 and exhibits a repeatedmotif (G-X-Y-X-Y, where X is polar and Y nonpolar). Finally,the C-terminal domains of both species share the same fold,a jelly-roll motif (2) also found in adenovirus (5) and reovirus(3, 4, 6).

View larger version (54K):
[in this window]
[in a new window]

FIG. 1. Structures and sequences of RBPs from lactococcal phages. (A) Three-dimensional structure of the RBP from phage TP901-1 (P335 group; blue). (B) Three-dimensional structure of the RBP from phage p2 (936 group; magenta). (C) View of a model associating domains of TP901-1 (N terminus and linker domain, below red line, blue) and p2 (head, above red line, magenta) RBPs. (D) Three-dimensional crystal structure of chimera form 1 (yellow) assembled according to the model in panel C. (E) Sequence alignment of the RBPs of p2 (part) and TP901-1. The secondary structure is described above the alignment. The binding residues are shown with blue dots. The hinge proline (Pro 162/63) is identified by a red arrow. The chimera is composed of the N-terminal domain (residues 17 to 33) and the linker domain residues (residues 34 to 63) from phage TP901-1 RBP and the C-terminal domain (residues 163 to 264) from phage p2 RBP.

The question addressed here was whether exchange between theC-terminal domains of two phage groups would lead to a stableprotein with conserved binding capacity. To answer this question,we have generated an RBP chimera comprising the N-terminal andlinker domains of phage TP901-1 fused to the C-terminal domainof phage p2. We have produced this chimera and determined itscrystal structure and its sugar binding capacity. These resultsindicate that straightforward domain exchange produced a stablechimera with a conserved binding capacity and a structure closeto that of each of the parental parts.

MATERIALS AND METHODS

Top

MATERIALS AND METHODS

Lactococcal phages and host strains. Phages p2 and TP901-1 and their L. lactis host strains wereobtained from the Felix d'Hérelle Reference Center forBacterial Viruses (www.phage.ulaval.ca). L. lactis strains weregrown in M17 (Oxoid) supplemented with 0.5% glucose (GM17) withoutshaking at 30°C. When propagating phages, 10 mM CaCl₂ wasadded 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/shoulderand linker/neck domains of the lactococcal temperate phage TP901-1(amino acids 1 to 63) and the C-terminal/head domain of thevirulent lactococcal phage p2 RBP (amino acids 162 to 264) wasconstructed and cloned into the Gateway destination vector pDest17(Invitrogen). A common proline residue situated at the beginningof the C-terminal domain (proline 63 in TP901-1 and proline162 in p2) was used to define the boundary between the linkerdomain and C-terminal domain for constructing the chimera. PCRamplification of DNA sequences was performed using previouslydescribed plasmids (29, 30) containing the full-length RBP ofphage p2 or TP901-1. Amplification of the individual domainswas performed in two steps, with each domain first being amplified,followed by a joining step of amplification. The resulting vectorwas transformed into the Escherichia coli C41(DE3)/pLysS (Lucigen)strain for expression. Transformed cells were grown in 2x YTmedium at 37°C until an optical density at 600 nm of 0.5was reached. Expression was induced with 0.5 mM isopropyl-β-thiogalactoside(IPTG), and cells were left for 20 h at 17°C. Bacterialcells were recovered by centrifugation at 5,000 x 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/mllysozyme, EDTA-free antiproteases [Roche]) per liter of cellculture and frozen at –80°C.

Protein purification. Cells were thawed with shaking in the presence of 20 mM MgSO₄and 10 µg/ml of DNase. The lysate was then sonicated andcleared by a 30-min centrifugation at 21,400 x g. After filtrationon a 0.45-µm filter, supernatant was loaded on a 5 mlHiTrap nickel affinity column (GE Healthcare) equilibrated inimidazole buffer (50 mM Tris [pH 8.0], 300 mM NaCl, 10 mM imidazole).The column was washed and eluted with the same buffer containingincreased imidazole concentrations of 50 mM and 250 mM, respectively.The eluted protein was further purified on a HiLoad 26/60 Superdex200 (GE Healthcare) gel filtration column in a buffer containing1.8 mM KH₂PO₄, 10.1 mM Na₂HPO₄, 2.7 mM KCl, and 137 mM NaCl,pH 7.2. Purified material was concentrated to appropriate crystallizationconcentrations on an Amicon Ultra-15 centrifugal filter unitwith a 30-kDa cutoff.

Protein crystallization and crystal structure determination. Crystallization trials were performed using a sitting-drop techniqueimplemented on a nanodrop-dispensing robot (PixSys; Cartesian)in Greiner 96-well plates. At 291 K, initial crystals were obtainedunder Stura footprint screen (Molecular Dimensions Limited)conditions E3 (0.1 M sodium cacodylate [pH 5.5], 18% polyethyleneglycol [PEG] 2000 monomethyl ether) and F4 (0.2 M imidazolemalate [pH 6.0], 15% PEG 4000). Optimization of these conditionsyielded two different crystal morphologies with best-diffractingcrystals under modified condition E3 (0.1 M sodium cacodylate[pH 5.5], 12% PEG 2000 monomethyl ether) with a protein concentrationof 7.5 g/liter (form 1) and under modified condition F4 (0.2M imidazole malate [pH 6.1], 17% PEG 4000) with a protein concentrationof 6.3 g/liter (form 2). Data were collected at the Europeansynchrotron radiation facility (ESRF, Grenoble, France) on lineID14-EH1. Crystals were directly flash-frozen from their motherliquor in the nitrogen gas flow. Both data sets were integratedand reduced using MOSFLM and SCALA (7). Molecular replacementwas performed using the automated molecular replacement serverBALBES (20) for form 1 and using PHASER (22) with the p2 RBPC terminus trimer as a search model (Protein Data Bank number1BSD; residues 162 to 264) for form 2. Refinement was performedusing REFMAC (26), while rebuilding was done using COOT (16).Structure validation was performed using MolProbity (21). Thedata collection and refinement results are summarized in Table1. Structure superimpositions were performed with turbo-Frodo,and figures were generated with Pymol (9).

View this table:
[in this window]
[in a new window]

TABLE 1. Data collection and refinement statistics for both forms of the chimerical RBP

Fluorescence-quenching experiments. Fluorescence experiments were carried out on a Varian Eclipsespectrofluorimeter 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 recordingthe quenching of the intrinsic protein fluorescence upon additionof ligand aliquots. The excitation wavelength was 290 nm, andemission spectra were recorded in the range from 320 to 380nm. The excitation slit was 5 nm, while the emission slit was10 nm. Titrations were carried out at 20°C in a phosphatebuffer [1 µM protein in 10 mM (Na/Na₂) phosphate, 50 mMNaCl, pH 7.5]. The fluorescence intensities at the maximum ofemission (354 nm) were corrected for the buffer contributionbefore plotting and further analysis. The affinity was estimatedby plotting the decrease of fluorescence intensity at the emissionmaximum as [100 – (I_i – I_min)/(I₀ – I_min)·100]against the quencher concentration, where I₀ is the maximumfluorescence intensity of the protein alone, I_i is the fluorescenceintensity after the ith addition of quencher, and I_min is thefluorescence intensity at the saturating concentration of quencher.The K_diss value was estimated using Prism 3.02 (GraphPad SoftwareInc.) by nonlinear regression for a single binding site withthe equation Y = B_max·X/(K_diss + X), where B_max is themaximal binding, Y is the fluorescence intensity, X is the concentrationof ligand added, and K_diss is the concentration of ligand requiredto reach half-maximal binding.

RESULTS AND DISCUSSION

Top

RESULTS AND DISCUSSION

Structure of the RBP chimera. The chimera construct used in this study encoded 165 residuesfrom lactococcal phages p2 and TP901-1, an N terminus of 21residues 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 theRBP of the virulent phage p2 (936 species) (Fig. 1C and E),resulting in a RBP chimera (Fig. 1D) that could be producedand purified. The junction point, Pro 162 (p2 numbering) or63 (TP901-1 numbering), is located at the linker/head domainjunctions. It interrupts the last β-strand of the linkerand 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 (3.35Å), and the electron density starts at the same residuenumber 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 usingthe crystallographic threefold axis of the P2₁3 space species(Fig. 2A). On the other hand, form 2 diffracts with a betterresolution (1.65 Å) but is cleaved at the beginning ofthe β-helical linker domain and starts at residue 34. Form2 contains the expected trimer in the asymmetric unit, witheach protomer comprising residues 34 to 164 (Fig. 1 and 2A).

View larger version (49K):
[in this window]
[in a new window]

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 stretchcontaining Pro 162 (p2) or 63 (TP901-1) at the linker/head junctionof the RBP chimera is perfectly defined in the electron mapdensity (Fig. 3). Superimposition of the form 1 reconstitutedtrimer onto the trimer of form 2 yields a good root mean squaredeviation (RMSD) value of 0.49 Å for the 393 C ${alpha}$ carbonsin common (Fig. 2A).

View larger version (58K):
[in this window]
[in a new window]

FIG. 3. 2fo-fc 1.65-Å resolution electron density (stereo view) contoured at 1 ${sigma}$ 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 ofthe wild type from TP901-1, using the common domains, N terminus,and β-prism. The superimposition is excellent with bothforms, yielding RMSD values of 0.69 and 0.30 Å betweentheir C ${alpha}$ atoms for forms 1 and 2, respectively (Fig. 2B; Table2). Similarly, when superimposing the RBP C-terminal domainsof 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 thestructures of the individual domains are rigid enough to keepclose structures, independently of the domains upon which theyare grafted or the different space groups to which they belong.In the latter case, the superimposition of the wild-type linkerdomains indicates that the linker domain of phage TP901-1 isslightly 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 domainorientations (Table 2). The RMSD values still remain smallerthan that calculated between the C ${alpha}$ atoms of the RBPs of p2 andTP901-1, which is 1.53 Å. To adapt to the linker differencebetween phages p2 and TP901-1, a stretch of $~$ 10 residues (startingat the hinge proline of the linker/C terminus junction of thechimera) 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 p2and the binding domain of the chimera are very similar.

View this table:
[in this window]
[in a new window]

TABLE 2. Summary of superimposition results for both forms of the chimerical protein

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

View larger version (47K):
[in this window]
[in a new window]

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 occursin a large accessible loop (ELTSG ${wedge}$ GN) between two glycine residues(Fig. 4B). We never observed this cleavage in solution, butcrystal packing clearly indicates that the absent density cannotbe attributed to flexibility due to packing constraints. Anothercleavage site was previously observed in the RBP structure ofphage p2 while in complex with llama VHH domains (30) as wellas in the RBP structure of the lactococcal phage bIL170 (936group) (27). This cleavage occurs in a loop (T ${wedge}$ VSGSIDVP₁₆₂) atthe end of the last β-strand of the linker domain, justbefore the hinge proline 162. Interestingly, the cleavages inform 2 as well as in the wild-type RBPs of phages p2 and bIL170correspond to structural domain boundaries, with high solventaccessibility. It is noteworthy that small additions of proteasehave been used to induce cleavage at domain boundaries, thusfacilitating 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 phosphoglycerolwith high affinity. Glycerol was found in the putative receptorbinding site, close to a Trp or Phe residue, and establishingstrong hydrogen bonds with Arg, Glu, and His. These data ledus to postulate that the phage receptor at the bacterial cellsurface might be teichoic or lipoteichoic acids, which are polymersof substituted phosphoglycerol. In the RBP of the virulent lactococcalphage p2, a tryptophan is part of the binding site in the headdomain, which makes it possible to measure the affinity of theRBP chimera for glycerol using fluorescence quenching (27, 29-31).When glycerol is added in increasing concentrations to an RBPchimera solution of 1 µM, the maximum fluorescence isquenched 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 RBPof phage p2 and also for the RBP head domain of p2 expressedalone (27, 29-31), which indicates that the chimeric RBP isable to bind to glycerol.

In conclusion, we have shown here that an artificial RBP chimeracan be designed in a straightforward manner and is capable ofbinding activities. The RBP domain grafting resulted in stablechimerical structures in which the domains closely resemblethe wild-type structures. Such conserved structure was possibledue to adaptation through small displacements of the linkers.From a phage evolution standpoint, it suggests that lactococcalRBPs are built to efficiently exchange a head domain, whichmay lead to host range variation. Module shuffling also likelyhelps them to persist in a man-made environment containing distinctbacterial cells.

ACKNOWLEDGMENTS

This work was supported in part by the Marseille-Nice Genopoleand by a grant from the Agence Nationale de la Recherche (BLAN07-1_191968).S.M. acknowledges support from the Natural Sciences and EngineeringResearch Council of Canada (NSERC) Strategic Program.

FOOTNOTES

* Corresponding author. Mailing address: Architecture et Fonction des Macromolécules Biologiques, UMR 6098 CNRS, and Universités Aix-Marseille I & II, Campus de Luminy, Case 932, 13288 Marseille Cedex 09, France. Phone: 33 491 825 591. Fax: 33 491 266 720. E-mail: valerie.campanacci{at}afmb.univ-mrs.fr

Published ahead of print on 13 March 2009.

Supplemental material for this article is available at http://jb.asm.org/.

REFERENCES

Top