JOURNAL OF BACTERIOLOGY, Feb. 2009, p. 728–734 Vol. 191, No. 30021-9193/09/$08.00�0 doi:10.1128/JB.01363-08Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Crystal Structure of ORF12 from Lactococcus lactis Phage p2Identifies a Tape Measure Protein Chaperone�†

Marina Siponen,1 Giuliano Sciara,1 Manuela Villion,2,4 Silvia Spinelli,1 Julie Lichiere,1Christian Cambillau,1 Sylvain Moineau,2,3,4* and Valerie Campanacci1*

Architecture et Fonction des Macromolecules Biologiques, UMR 6098 CNRS and Universites d’Aix-Marseille I & II, Campus deLuminy, case 932, 13288 Marseille cedex 09, France,1 and Groupe de Recherche en Ecologie Buccale (GREB), Faculte de

Medecine Dentaire,2 Felix d’Herelle Reference Center for Bacterial Viruses,3 and Departement de Biochimie et deMicrobiologie, Faculte des Sciences et de Genie,4 Universite Laval, Quebec City, Quebec, Canada G1K 7P4

Received 30 September 2008/Accepted 20 November 2008

We report here the characterization of the nonstructural protein ORF12 of the virulent lactococcal phage p2,which belongs to the Siphoviridae family. ORF12 was produced as a soluble protein, which forms largeoligomers (6- to 15-mers) in solution. Using anti-ORF12 antibodies, we have confirmed that ORF12 is notfound in the virion structure but is detected in the second half of the lytic cycle, indicating that it is alate-expressed protein. The structure of ORF12, solved by single anomalous diffraction and refined at 2.9-Åresolution, revealed a previously unknown fold as well as the presence of a hydrophobic patch at its surface.Furthermore, crystal packing of ORF12 formed long spirals in which a hydrophobic, continuous crevice wasidentified. This crevice exhibited a repeated motif of aromatic residues, which coincided with the same repeatedmotif usually found in tape measure protein (TMP), predicted to form helices. A model of a complex betweenORF12 and a repeated motif of the TMP of phage p2 (ORF14) was generated, in which the TMP helix fittedexquisitely in the crevice and the aromatic patches of ORF12. We suggest, therefore, that ORF12 might act asa chaperone for TMP hydrophobic repeats, maintaining TMP in solution during the tail assembly of thelactococcal siphophage p2.

During industrial milk fermentation, Lactococcus lactis cellsare added to transform milk into an array of fermented prod-ucts such as cheese. However, this manufacturing process maybe impaired by lytic phages present in the factory environmentas well as in the milk itself (30). Due to the destructive effectsof phage infections on bacterial fermentation, much effort hasbeen undertaken to isolate and study the biodiversity of thesebacteriophages. Lactococcal bacteriophages belong to at least10 different genetically distinct species of double-strandedDNA viruses (9). Of them, three lactococcal phage species, allbelonging to the Siphoviridae family, are the major source ofproblems in milk fermentation, namely, the 936, P335, and c2species (7, 28, 29). Furthermore, members of the 936 speciesare by far responsible for the majority of infections (50 to 80%)(1, 24, 41). Numerous phages of the 936 species have beenisolated, and several have been characterized at the genomelevel (25). However, little is known concerning their molecularmechanisms of infection, although we recently solved the struc-ture of the receptor-binding protein (RBP) of our model 936-

like phage, namely, the virulent phage p2 (38, 43), and ofphages belonging to the P335 species (27, 34, 37, 38).

As with all viruses, bacteriophage genomes are quite com-pact, leaving little room for noncoding sequences (4). In fact,phage genes are disposed in an operon-type organization (4),and the order of genes corresponds to the different phases ofthe infection cycle. Moreover, genes are often in clusters (re-ferred to as modules), with gene products from adjacent genesgenerally found to interact with each other. Interestingly,phage genome organization, including individual gene order, isoften conserved within a given species, particularly within theSiphoviridae family. In the case of L. lactis virulent phagesbelonging to the 936 or P335 species, this principle appliesparticularly to the morphogenesis gene module, which includesall the genes coding for the phage structural protein genes. Forthe tail assembly, a module comprises a set of genes betweenthe portal protein, which is connecting the tail to the capsid,and the RBP, which is located at the tip of the tail and isinvolved in host recognition (39, 43).

The characterization of tail assembly genes of lactococcalphages has been more extensive for temperate siphophagesbelonging to the P335 species (27, 34, 37, 38). Because of thesimilarities in genome organization, the findings in this phagespecies can, in some cases, be used as clues toward understand-ing the morphology of 936-like phages. For the temperatephage Tuc2009 (P335 species), all structural proteins requiredfor tail and baseplate assembly have been identified (27, 34, 37,38). Genes located between those coding for the tape measureprotein (TMP) and BppL (RBP) were identified as corre-sponding to components of the baseplate structure, located atthe tail distal end. Furthermore, a gene coding for the major

* Corresponding author. Mailing address for Valerie Campanacci:Architecture et Fonction des Macromolecules Biologiques, UMR 6098CNRS and Universites d’Aix-Marseille I & II, Campus de Luminy,case 932, 13288 Marseille cedex 09, France. Phone: 33 491 82 55 91.Fax: 33 491-266-720. E-mail: valerie.campanacci@afmb.univ-mrs.fr.Mailing address for Sylvain Moineau: Groupe de Recherche enEcologie Buccale (GREB), Faculte de Medecine Dentaire, UniversiteLaval, Quebec City, Quebec, Canada G1K 7P4. Phone: (418) 656-3712.Fax: (418) 656-2861. E-mail: Sylvain.Moineau@bcm.ulaval.ca.

† Supplemental material for this article may be found at http://jb.asm.org/.

� Published ahead of print on 1 December 2008.

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tail protein (MTP) was also identified at a position upstreamfrom tmp. Between the genes coding for the MTP and thosecoding for the TMP in Tuc2009 are two gene products identi-fied as gpG and gpGT, which are not present in the phageparticle. These two proteins were named based on their likelyrole analogous to the tail assembly proteins present in coli-phage lambda, a model virus belonging to the Siphoviridaefamily (21, 27, 47). gpGT has an essential role in lambda tailassembly, acting prior to tail shaft assembly, while the role ofgpG in tail assembly is not known (21). Both gpG and gpGTare also absent from mature lambda virions (21). It has beenargued that they may act as assembly chaperones (47).

A close examination of 936 genomes indicates the presenceof two genes coding for gpG and gpGT-like proteins. Analysisof the phage p2 genome, closely related to that of lactococcalphage sk1 (6), revealed that the putative tail assembly proteinscould correspond to gene products ORF12 and ORF13. Thesetwo genes are followed by the TMP gene corresponding toorf14, other genes coding for other structural proteins, and theRBP gene orf18. During our ongoing investigation of the struc-ture of phage p2, we report here the cloning, expression, andcrystal structure of ORF12 in order to decipher its role in thetail assembly process.

MATERIALS AND METHODS

Bacterial strains and phage. Lactococcus lactis subsp. cremoris MG1363 (14)was grown at 30°C in M17 supplemented with 0.5% glucose (GM17). In phage p2(31) infection experiments, 10 mM CaCl2 was added to plates or medium.Propagation of phages and determination of the titers of the lysates were per-formed as described previously (12).

Intracellular detection of ORF12 during phage infection. L. lactis MG1363was grown in GM17 until the optical density at 600 nm reached 0.5, and then itwas infected with virulent phage p2 at a multiplicity of infection of 5. Sampleswere taken at 5-min intervals and flash-frozen (�80°C). Cell pellets were resus-pended in 10 mM Tris-Cl, pH 8.0, 1 mM EDTA, 0.3% sodium dodecyl sulfate(SDS) and lysed with a bead beater. The cytoplasmic extracts were then dosed bya standard Bradford assay, and 5 �g of each sample was migrated on a 15%SDS-polyacrylamide gel. The gel was electrotransferred (30 V) overnight at 4°Cwith a transblot apparatus (Bio-Rad) onto a polyvinylidene difluoride membrane(Hybond P; GE Healthcare) using Tris-glycine-methanol buffer (25 mM Tris, pH8.3, 192 mM glycine, 10% methanol). The intracellular production of ORF12during the infection was subsequently detected with a protein A purified anti-ORF12 antibody (Davids Biotechnologie GmbH, Germany). Briefly, the mem-brane was first blocked with 5% skim milk in PBST (phosphate buffer supple-mented with 0.1% Tween 20) for at least 1 h on a rotational shaker. Themembrane was then treated with a primary antibody, anti-ORF12, diluted1:100,000 (in blocking buffer) for 1 h at room temperature. Following washeswith PBST, the anti-ORF12 antibody was detected after a 1-h incubation with asecondary antibody, horseradish peroxidase-labeled anti-rabbit immunoglobulinG, diluted 1:100,000 in blocking buffer (Rockland Immunochemicals). Afterother washes in PBST, the membrane was rinsed with phosphate-buffered salinebefore the final detection with the ECL Plus detection kit (GE Healthcare)following the manufacturer’s instructions.

ORF12 cloning, expression, and purification. The orf12 gene of phage p2 wascloned into the Gateway destination vector pETG-20A (Arie Geerlof, EMBL,Hamburg, Germany) for protein production according to the standard Gatewayprotocols and using the following primers for the initial PCR: forward primer,5�-GGGGACAAGTTTGTACAAAAAAGCAGGCTTAGAAAACCTGTACTTCCAGGGTGCAAAACAATTGAGTACAGCACG-3�; reverse primer, 5�-GGGGACCACTTTGTACAAGAAAGCTGGGTTTATTAAATTTCTTTCTGCCACAATTCG-3�. The att sequences are in italic, the tobacco etch virus (TEV) recog-nition site coding sequence is in bold, and stop codons are underlined. The finalconstruct encoded a thioredoxin fusion protein containing an N-terminal hexa-histidine tag followed by a TEV protease recognition site. Protein expression wasdone in the Escherichia coli Rosetta(DE3)pLysS strain (Novagen). Production ofthe selenomethionine (SeMet)-labeled protein was performed by blocking themethionine biosynthesis pathway (11). Briefly, cells were grown at 37°C in M9

broth supplemented with 0.1 mM CaCl2, 4 mM MgSO4, 1.2% glycerol, 100mg/liter lysine, 100 mg/liter phenylalanine, 100 mg/liter threonine, 50 mg/literisoleucine, 50 mg/liter leucine, 50 mg/liter valine, 50 mg/liter SeMet, and 1ml/liter oligonucleotide elements. When the optical density reached 0.5, proteinexpression was induced with 0.5 mM isopropyl-�-thiogalactoside (IPTG) andcells were left overnight at 25°C. Bacterial cells were then harvested by centri-fugation at 3,300 � g for 10 min, resuspended in lysis buffer (50 mM Tris-HCl,pH 8.0, 300 mM NaCl, 0.25 mg/ml lysozyme, and EDTA-free antiproteasecocktail) (Roche), and frozen at �80°C.

Pellets were quickly thawed at 37°C followed by an incubation with shaking at4°C in the presence of 20 mM MgSO4 and 10 �g/ml of DNase. Cells were thensonicated and cleared by centrifugation at 21,400 � g. After filtration (0.45-�m-pore-size filter), the supernatant was loaded on a 5-ml HiTrap nickel affinitycolumn (GE Healthcare) preequilibrated with buffer (10 mM Tris, pH 8.0, 300mM NaCl, 10 mM imidazole). Proteins were eluted using the same buffer con-taining 50 mM and 250 mM imidazole. Prior to TEV protease cleavage, bufferwas changed to 10 mM Tris, pH 8.0, 300 mM NaCl, 10 mM imidazole on aHiPrep 26/10 desalting (GE Healthcare) column. The cleavage was performed at4°C overnight using a 1:10 (wt/wt) ratio of TEV protease to target protein. Aftercleavage, protein was recovered in the flowthrough fraction of a 5-ml HiTrapnickel affinity column. The eluted protein was further purified on a HiLoad 26/60Superdex 200 (GE Healthcare) gel filtration column in a buffer containing 10mM Tris, pH 8.0, and 300 mM NaCl. Purified material was visualized on a 15%SDS-polyacrylamide gel and concentrated to appropriate crystallization concen-trations on an Amicon Ultra-15 centrifugal filter unit with a cutoff size of 5 kDa.

ORF12 biochemical and biophysical characterization. Purified protein wasfirst analyzed for size and incorporation of SeMet by matrix-assisted laser de-sorption ionization–time of flight mass spectrometry and trypsin peptide massfingerprinting (Bruker Autoflex2; Daltonics Bremen, Germany). Then, analyticalsize-exclusion chromatography was carried out with online multiangle static lightscattering on an Alliance 2695 high-pressure liquid chromatography system (Wa-ters) on a silica gel KW804 column (Shodex) in 10 mM Tris, pH 8.0, 300 mMNaCl at a flow rate of 0.5 ml/min. The protein was loaded at a concentration of6 mg/ml or 8 mg/ml. Detection was performed using a UV/VIS absorbancephotodiode array detector (2996; Waters), a triple-angle static light-scatteringdetector (MiniDAWN Treos; Wyatt Technology), a quasielastic light-scatteringinstrument (Dynapro; Wyatt Technology), and a differential refractometer (Op-tilab rEX; Wyatt Technology) (37). Molecular weight and hydrodynamic radiusdeterminations were performed with the ASTRA V software (Wyatt Technol-ogy) using a dn/dc value of 0.175 ml/g.

Crystallization, data collection, structure determination, analysis, and topo-logical modeling. Crystallization trials were performed using a sitting drop vapordiffusion technique implemented on a nanodrop-dispensing robot (PixSys orHoneybee-X8; Cartesian Inc.) in Greiner 288-well plates (10, 19, 40). The pro-tein was initially screened using commercially available crystallization screens:crystal screens I and II (Molecular Dimensions Ltd.), Stura footprint screens(Molecular Dimensions Ltd.), and the NeXtal SM1 suite (Qiagen). The initialcrystal hit was obtained in the Stura II footprinting screen condition number 2(0.1 M HEPES, pH 7.5, 18% polyethylene glycol 600). Optimization screens wereprepared by varying buffer pH values and precipitant concentrations based onthis initial crystallization buffer hit (10, 19, 40). All crystallization plates werestored in a thermoregulated room at 291 K. After 48 h, diffracting crystals grewto a dimension of 50 � 50 � 150 �m in 0.1 M HEPES, pH 6.8, 15.9% polyeth-ylene glycol 600 with the use of 0.1 �l of protein mixed with 0.2 �l of reservoirsolution. Crystals were quick-frozen in a liquid nitrogen flux with 10% glycerol asa cryoprotectant. A complete data set was collected at the European synchrotronradiation facility (Grenoble, France) on beamline ID14-EH4. A total of 360images were collected with an oscillation range of 1° at a wavelength of 0.9785 Å.

The data set was integrated and reduced using MOSFLM and SCALA fromthe CCP4 suite (8). Phases were calculated with SHELX (36). Phase extension to2.9 Å was performed, and a partial model was built using RESOLVE (42). Cyclesof manual model rebuilding were carried out using Coot (13). Refinement wasperformed with REFMAC (32) using TLS (Translation/Libration/Screw) seg-ments defined by the TLS Motion Determination server (http://skuld.bmsc.washington.edu/�tlsmd/). Threefold noncrystallographic symmetry restraintswere applied throughout refinement to the homotrimer found in the asymmetricunit. The final structure was analyzed using PROCHECK (20) and MolProbity(22). A summary of the structure determination and refinement statistics ispresented in Table 1. Interaction models were generated using Coot (13) andrefined using Turbo-Frodo (35). An all-canonical �-helix was built with Turbo-Frodo. The helix was subsequently slightly bent at the display to adapt to thegroove shape and geometrically refined with the Turbo-Frodo “refine” option.The helix was further docked in ORF12 groove manually at the display with the

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Turbo-Frodo FBRT option. Figures were generated with Pymol (http://pymol.sourceforge.net/) and Turbo-Frodo (35).

Protein structure accession number. Final coordinates and structure factorswere deposited in the Protein Data Bank (PDB) (http://www.rcsb.org/PDB) withcode 3D8L.

RESULTS AND DISCUSSION

Production and biochemical characterization of ORF12from the virulent lactococcal phage p2 of the 936 species.Using well-established laboratory screening procedures (44–46), the cloning and subsequent overproduction of a SeMet-labeled ORF12 full-length protein were successfully carriedout. Optimal conditions gave a yield of approximately 8 mg ofsoluble and purified protein per liter of E. coli culture inminimal medium. Furthermore, the protein was shown to bequite soluble, up to 10 mg/ml. The combination of goodprotein yield and the high level of protein solubility allowed usboth to carry out biophysical characterization and to performcrystallization trials for ORF12.

Basic characterization on a 15% SDS-polyacrylamide gelidentified a single 10-kDa band (expected size, 10,552 Da). TheSeMet protein was also subjected to matrix-assisted laser de-sorption ionization–time of flight mass spectroscopy, whichidentified a 10.7-kDa protein. Analysis of trypsin digests bymass spectroscopy also confirmed the full incorporation ofthree SeMets in the protein. A first indication of the oligomer-ization state of ORF12 arose from protein elution off the

HiLoad 26/60 Superdex 200 gel filtration column. Based onthe calibration curve established for this particular column,ORF12 was identified by SDS-polyacrylamide gel electro-phoresis in fractions eluting between 55 and 65 kDa (data notshown). This suggested that the protein is likely present as ahexamer in solution. ORF12 was then subjected to weight andsize analysis using MALS/UV/RI (multiangle light scatter-ing-UV absorbance-refractive index) spectroscopy (37), whichconfirmed a higher oligomerization state in solution (Fig. 1).The native protein (Fig. 1, blue line) was injected at a concen-tration of 6 mg/ml. The curve exhibited a slow decrease fromthe main peak, which corresponded to a mass of 58 3 kDa,comparable to a theoretical mass of 64.1 kDa for a hexamer.The SeMet-labeled ORF12 was injected at 8 mg/ml and exhib-ited a comparable behavior but with the main peak at a massof 158 8 kDa (Fig. 1, red line), which corresponded to a15-mer polymerization (theoretical size, 160.3 kDa). In bothcases, however, all forms between the oligomer and the mono-mer were observed. The hydrophobic effect of the SeMet la-beling explains the higher oligomerization state of ORF12 (2).

Structure of ORF12. Crystal structure determination yieldeda solution with a well-defined electron density map relative tothe 2.9-Å resolution. The resulting refined structure had anR/Rfree of 20.3/25.3 (Table 1 shows a complete list of datacollection and refinement statistics). The asymmetric unit ofthese R32 (a � b � 158.3, c � 99.5; � � � � 90.0, � � 120.0)crystals contained three monomers of ORF12 (Fig. 2A). Theelectron density allowed the reconstruction of all 91 aminoacids (aa) in chains A and B (Fig. 2B), while the first 3 aa ofchain C could not be modeled. These three molecules could besuperimposed with main-chain root mean square deviation(RMSD) values of 0.32 to 0.45 Å, as determined by the Su-perPose server (26), within the experimental error at this res-olution. The coordinates of ORF12 were submitted to different

FIG. 1. Online multiangle laser light-scattering, absorbance, andrefractive index analysis of ORF12 in solution. The abscissa indicatesthe time of elution from the high-pressure liquid chromatographycolumn; the left ordinate indicates the molar mass in g/mol (Da). Theabsorption peaks are in blue (native) and red (SeMet), and the dashedlines indicate the molar masses. The experimental masses are given inblack (kDa). The native ORF12 has been injected at a concentration of6 mg/ml, and the SeMet derivative has been injected at 8 mg/ml.

TABLE 1. Data collection and refinement statistics for p2 ORF12

Parameter Valuea

Data collectionPDB access code ...................................................3D8LSpace group ...........................................................R 3 2Unit cell (A) ..........................................................158.3 158.3 99.5

90.0 90.0 120.0Beamline ................................................................ID14-EH4Detector..................................................................ADSC Q315Wavelength (A) .....................................................0.9785Rotation range (°).................................................360Resolution range (A)............................................35.0–2.90 (3.06–

2.90)No. of observations ...............................................230,325 (31,122)No. of unique reflections .....................................10,739 (1,556)Completeness.........................................................99.9 (100)Redundancy ...........................................................21.4 (20.0)I/sI ...........................................................................29.1 (6.4)Rsym (%) .................................................................9.2 (44.1)

RefinementResolution range (A)............................................30.0–2.9 (2.98–2.90)No. of unique reflections .....................................10,218No. of atoms of protein per water/buffer ..........22,114R/Rfree all (last shell) ............................................25.3/20.3 (33.5/29.4)RMSD

Bonds (A) ..........................................................0.015Angles (°) ...........................................................1.591

Mean B value (A2)................................................Protein ................................................................64.43Water ..................................................................51.33

Ramachandran (%)Favored region ..................................................92.8Allowed regions.................................................5.7Generously allowed regions.............................1.5

a All values in parentheses belong to the last shell.

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structure analysis servers (DALI and ProFunc) (16) with aview to the identification of closely related structures andhence a putative function. No structural neighbors were iden-tified, meaning that ORF12 has an original, previously unob-served fold.

ORF12 is an entirely �-helical protein, composed of five�-helices in total (H1 to H5) (Fig. 1). All helices, with the

exception of helix H2, are perfectly amphiphilic helices ori-ented in such a way as to form a hydrophobic cleft on the innersurface of the protein. H2, on the other hand, has few aminoacids involved in the cleft and a majority of outside surfaceresidues. The fifth helix (H5) both serves to form the nonpolarcleft and serves in the interaction between two monomers. Twotypes of interfaces can be observed between the three mono-mers of the asymmetric unit in the crystals. The first twomonomers (chains A and B) interact side by side through asingle helix (helix H5) with the third monomer (chain C) sittingin a head-to-tail position with the second (chain B). The side-by-side helices are also oriented in a head-to-tail manner withGlu 82 of one chain H bonding with Lys 89 of the other andvice versa (Fig. 3C). The interface formed by the head-to-tailassociation of two monomers creates an extended hydrophobiccleft whose surface is formed in large part by the nonpolar faceof helix H3. The interface is mainly formed by nonpolar inter-actions of residues present in the loops and strands betweenthe defined helices. Notably, one monomer has a 10-aa loopbetween H4 and H5 and a 6-aa loop between H2 and H3 andthe first two residues of H3 at the surface. The other presentsboth the N and the C termini, including the last residues of H5,plus the first two residues of H2. This later helix includes Phe20, which forms with the C-terminal residue Trp 87 an aro-matic residue pocket situated in the hydrophobic cleft (see Fig.S1 in the supplemental material).

The three monomers of the asymmetric unit have very fewburied residues in their core (probe radius, 1.6 Å; no cutoff).Chains A, B, and C have 87, 86, and 84 surface-exposed aminoacid residues, respectively (with a special note concerningchain C, which has three residues missing), indicating thatvirtually all residues are either accessible to solvent or availablefor interactions among themselves in the crystal packing.

As mentioned above, the monomers in the asymmetric unitreveal two types of interactions, which were analyzed for theindividual monomer positions in the asymmetric unit trimerusing the EMBL-EBI Protein Interfaces, Surfaces and Assem-blies (PISA) server (http://www.ebi.ac.uk/msd-srv/prot_int/cgi-bin/piserver). The largest surface interaction area was foundto involve monomers B and C in the trimer, with an interfaceof 642 Å2. A smaller surface was observed between the inter-actions described above for H5 of chains A and B (149 Å2),while A and C had virtually no interactions (Fig. 2A). Thelarger surface area is involved in the B-C-type interaction,which forms the aromatic pocket at the interface of the twomonomers.

Generating symmetry mates of the ORF12 trimer leads tothe formation of two large spirals of compactly packed mono-mers (Fig. 3). A cross section of the spirals revealed an innersolvent channel of 36 Å with a complete diameter of 80 Å. Thetwo spirals display weak mutual contacts, namely, those de-scribed above between monomers A and B. The contactswithin the spiral are like those described for monomers B andC, with interaction surface areas of 709 Å2 for monomer B andits interacting partner, monomer A, in a symmetry mate. TheB-C interface corresponds to 642 Å2, while C and its symmetrymate A have an interface of 754 Å2. Therefore, the averagevalue for this type of interaction is 702 Å2, corresponding to11% of the total surface area of the ORF12 monomer. Thiskind of surface area is comparable to that observed for Fab/

FIG. 2. Structure of p2 ORF12. (A) Crystallographic trimer ribbonrepresentation with monomers identified by their letter in the PDB(3D8L). The ribbon is colored blue to red from the N to the Cterminus. The surface interaction areas between each monomer aregiven. (B) Stereo view of ORF12 monomer, with the same coloring asin panel A. The helices are numbered 1 to 5.

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protein interactions (23) and is large enough to be significantfor true biological interactions (17). We therefore propose thata crystallographic spiral corresponds to the elongation of theoligomers (15-mers in the case of SeMet ORF12) observed insolution, triggered by the decrease in solubility resulting fromthe increase of the concentration of precipitant occurring dur-ing the crystallization process.

ORF12 is a nonstructural protein but is expressed duringphage infection. Phage p2 was purified through a CsCl gradientto obtain highly concentrated preparations (1011 to 1012 PFU/ml), which were used to infect L. lactis MG1363. Then, a timecourse infection was performed and samples were taken atintervals. Intracellular extracts were tested for the productionof ORF12 (Fig. 4). The phage protein ORF12 was first de-tected at 15 min after the beginning of the infection and at theexpected size of 10 kDa, confirming that it is a late-expressedphage protein (5). The production of ORF12 then peaked at30 min, and its concentration started to decrease coincidingwith lysis of the host culture. This is in agreement with previousdata that estimated the latency period of phage p2 (the timefrom infection to release of new phage progeny) as being up to30 min (15). As expected, we could not find ORF12 in thestructure of phage p2; thus, its role in the phage infectionprocess was still unknown.

Putative function of ORF12: model of the TMP repeat seg-ment and its binding to ORF12. Each spiral formed by thecrystal packing of the nonstructural phage p2 protein ORF12

displays several noteworthy features. Firstly, nonpolar crevicesare located regularly at the inner face of the spiral, within acontinuous cleft. In contrast, the external face of the spiralexhibits polar residues facing the solvent. Secondly, within thenonpolar cleft, a highly repetitive motif of aromatic residuescan be identified (Fig. 5A).

Based on its position in the genome, and by comparison withother Siphoviridae, we hypothesized that ORF12 may play arole in tail assembly. Due to the peculiar characteristics of theTMP (TMP/ORF14) of phage p2, with two hydrophilic do-mains at each sequence extremity, but with a long hydrophobichelix, likely insoluble by itself (Fig. 5D), we examined thepossibility that ORF12 might be a chaperone complexing tothe hydrophobic central part of TMP and maintaining it insolution before its assembly with the MTP to form the phagetail. The TMP acts as a ruler or template that measures lengthduring tail assembly (18). Besides the N (1 to 522 aa) and C(898 to 999 aa) termini of the TMP, which are likely globularand interact with the portal protein on the N-terminal side andwith the baseplate on the C-terminal side, the middle partexhibits a 40-aa repeat of evenly spaced aromatic residues (Fig.5D). Furthermore, this type of repeat (although varying inlength from phage to phage) appears to be a common featureof TMPs in Siphoviridae (3, 6, 27).

Since this repeat region of the TMP molecule is predicted tobe an �-helix, we generated a small 42-mer helical model ofthis repeat to verify if it could bind within the twisted hydro-phobic crevice identified in the large twisted spiral structure ofORF12 (Fig. 3). This structure-structure docking revealed thatthe amphiphilic helix has aromatic residues correctly spaced tofit into the aromatic residue binding pockets of the ORF12spiral (Fig. 3B and C; see also Fig. S2 in the supplementalmaterial). These residues are observed on the nonpolar surfaceof the hydrophobic crevice (Fig. 3A), while the other side ofthe spiral displays a solvent-exposed polar side bearing manycharged residues.

A possible function of ORF12 might therefore be to coverthe central segment of p2 TMP composed of repeated hydro-phobic motifs, to keep them in solution. The sequences of theN terminus of TMP (1 to 521 aa) and its C terminus (898 to 999

FIG. 3. View of the spirals formed by ORF12 crystal packing. The crystal is formed from such spirals packed side by side. (A) Side view of thetwo spirals side by side. Each monomer is colored brown, violet, and yellow repeatedly. (B) View of the spirals rotated by 90° around the verticalaxis. The gross dimensions of the spiral are displayed. (C) Stick representation of a stretch of the two side-by-side-positioned spirals illustratingthe dense network of charge interactions, but loose packing, between the two spirals. The color code is blue for positively charged residues, redfor negatively charged residues, pale green for semipolar residues, green for methionines, yellow for aliphatics, and violet for aromatics.

FIG. 4. Intracellular detection of ORF12 during phage p2 infec-tion of L. lactis MG1363. Samples were taken at various timeintervals after phage infection, and ORF12 was detected by West-ern blot analysis. For ORF12, 5 ng of purified protein and, for p2,1 � 1010 PFU of CsCl-purified phages were loaded, which corre-sponded to 7.8 1.5 �g.

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aa) resemble more globular proteins and are probably solubleby themselves. We hypothesize that maintaining the TMP insolution would help the assembly of MTPs around the TMPhydrophobic helix, a scheme seen in the virulent Bacillussiphophage SPP1 tail structure (33).

Conclusion. Using structural genomic approaches, it is ex-pected that the knowledge of the three-dimensional structureof a protein of unknown function may reveal its function if itsfold resembles that of a protein of known function. The struc-ture of p2 ORF12 being novel, such an approach was notpossible. However, because the number of open readingframes in phages is limited and gene organization may beconserved, genomic comparisons may be the source of func-tional hypotheses, which can then be checked in silico or ex-perimentally. Here, we suggest, based on strong topologicalobservations, that the nonstructural ORF12 of phage p2 (andpossibly other siphophages) may serve as a chaperone of theTMP central domain to maintain it in solution and present it toMTPs to facilitate the tail assembly process. The perfect matchof ORF12 spiral (observed as an oligomer in solution) hydro-phobic patches with the aromatic residues of TMP repeatsleads us to postulate that a complex between the TMP residues522 to 898 and a spiral of the �70-mer might be stable longenough in solution to allow MTP to approach TMP and the tailto be formed.

ACKNOWLEDGMENTS

We thank Arie Geerlof, who kindly provided the Gateway plasmidpETG-20A for His-thioredoxin fusion. We are grateful to DeniseTremblay and Helene Deveau for helpful discussion.

This work was supported in part by the Marseille-Nice Genopole, bythe company BioXtal, and by a grant from the Agence Nationale de laRecherche (BLAN07-1_191968) to C.C. and V.C. and by a strategicgrant from the Natural Sciences and Engineering Research Council ofCanada (NSERC) to S.M.

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