Modular Structure of the Receptor Binding Proteins of Lactococcus lactis Phages

Lactococcus lactis is a Gram-positive bacterium widely used by the dairy industry. Several industrial L. lactis strains are sensitive to various distinct bacteriophages. Most of them belong to the Siphoviridae family and comprise several species, among which the 936 and P335 are prominent. Members of these two phage species recognize their hosts through the interaction of their receptor-binding protein (RBP) with external cell wall saccharidices of the host, the “receptors.” We report here the 1.65 Å resolution crystal structure of the RBP from phage TP901-1, a member of the P335 species. This RBP of 163 amino acids is a homotrimer comprising three domains: a helical N terminus, an interlaced β-prism, and a β-barrel, the head domain (residues 64-163), which binds a glycerol molecule. Fluorescence quenching experiments indicated that the RBP exhibits high affinity for glycerol, muramyl-dipeptide, and other saccharides in solution. The structural comparison of this RBP with that of lactococcal phage p2 RBP, a member of the 936 species (Spinelli, S., Desmyter, A., Verrips, C. T., de Haard, J. W., Moineau, S., and Cambillau, C. (2006) Nat. Struct. Mol. Biol. 13, 85-89) suggests a large extent of modularity in RBPs of lactococcal phages. Lactococcus lactis is a Gram-positive bacterium widely used by the dairy industry. Several industrial L. lactis strains are sensitive to various distinct bacteriophages. Most of them belong to the Siphoviridae family and comprise several species, among which the 936 and P335 are prominent. Members of these two phage species recognize their hosts through the interaction of their receptor-binding protein (RBP) with external cell wall saccharidices of the host, the “receptors.” We report here the 1.65 Å resolution crystal structure of the RBP from phage TP901-1, a member of the P335 species. This RBP of 163 amino acids is a homotrimer comprising three domains: a helical N terminus, an interlaced β-prism, and a β-barrel, the head domain (residues 64-163), which binds a glycerol molecule. Fluorescence quenching experiments indicated that the RBP exhibits high affinity for glycerol, muramyl-dipeptide, and other saccharides in solution. The structural comparison of this RBP with that of lactococcal phage p2 RBP, a member of the 936 species (Spinelli, S., Desmyter, A., Verrips, C. T., de Haard, J. W., Moineau, S., and Cambillau, C. (2006) Nat. Struct. Mol. Biol. 13, 85-89) suggests a large extent of modularity in RBPs of lactococcal phages. Phages of Lactococcus lactis are a major problem in industrial milk fermentation, because they are ubiquitous within their process environments as well as within pasteurized milk (1Moineau S. Tremblay D. Labrie S. ASM News. 2002; 68: 388-393Google Scholar). They belong to several different species of the Siphoviridae family (small isometric capsid and long noncontractile tail), among which the genetically distinct species 936, P335, and c2 are the three prominent (2Moineau S. Fortier J. Ackermann H.-W. Can. J. Microbiol. 1992; 38: 875-882Crossref Scopus (88) Google Scholar, 3Moineau S.M. Borkaev B.J. Holler S.A. Walker J.K. Kondo E.R. Vedamuthu Vandenbergh P.A. J. Dairy Sci. 1996; 79: 2104-2111Abstract Full Text PDF Scopus (95) Google Scholar, 4Bissonnette F. Labrie S. Deveau H. Lamoureux M. Moineau S. J. Dairy Sci. 2000; 83: 620-627Abstract Full Text PDF PubMed Scopus (70) Google Scholar, 5Josephsen J. Nielsen E.W. Milchwissenschaft. 1988; 43: 219-223Google Scholar, 6Josephsen J. Neve H. Salminen S. von Wright A. Lactic Acid Bacteria: Microbiology and Functional Aspects. Marcel Dekker, Inc., New York1998: 385-436Google Scholar, 7Moineau S. Pandian S. Klaenhammer T.R. Appl. Environ. Microbiol. 1993; 59: 197-202Crossref PubMed Google Scholar). The first steps of phage infection require interactions between the phage receptor-binding proteins (RBPs) 3The abbreviations used are: RBP, receptor binding protein; ORF, open reading frame; r.m.s., root mean square; EM, electron microscopy; BppL, baseplate protein lower. 3The abbreviations used are: RBP, receptor binding protein; ORF, open reading frame; r.m.s., root mean square; EM, electron microscopy; BppL, baseplate protein lower. (8Dupont K. Vogensen F.K. Neve H. Bresciani J. Josephsen J. Appl. Environ. Microbiol. 2004; 70: 5818-5824Crossref PubMed Scopus (69) Google Scholar, 9De Haard H.J. Bezemer S. Ledeboer A.M. Muller W.H. Boender P.J. Moineau S. Coppelmans M.C. Verkleij A.J. Frenken L.G. Verrips C.T. J. Bacteriol. 2005; 187: 4531-4541Crossref PubMed Scopus (64) Google Scholar) and the receptors at the host cell surface. These mediating RBPs are located at the distal structure of their long tail (150-200 nm). Lactococcal phages from species 936 or P335 bind to carbohydrate receptors at the surface of the cell wall, the exact nature of them being still unknown (10Deveau H. Van Calsteren M.R. Moineau S. Appl. Environ. Microbiol. 2002; 68: 4364-4369Crossref PubMed Scopus (88) Google Scholar, 11Dupont K. Janzen T. Vogensen F.K. Josephsen J. Stuer-Lauridsen B. Appl. Environ. Microbiol. 2004; 70: 5825-5832Crossref PubMed Scopus (63) Google Scholar, 12Geller B.L. Ngo H.T. Mooney D.T. Su P. Dunn N. J. Dairy Sci. 2005; 88: 900-907Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 13Valyasevi R. Sandine W.E. Geller B.L. Appl. Environ. Microbiol. 1990; 56: 1882-1889Crossref PubMed Google Scholar, 14Valyasevi R. Sandine W.E. Geller B.L. J. Dairy Sci. 1994; 77: 1-6Abstract Full Text PDF PubMed Scopus (38) Google Scholar, 15Tremblay D.M. Tegoni M. Spinelli S. Campanacci V. Blangy S. Huyghe C. Desmyter A. Labrie S. Moineau S. Cambillau C. J. Bacteriol. 2006; 188: 2400-2410Crossref PubMed Scopus (92) Google Scholar). A better understanding at a molecular level of this recognition mechanism would increase the possibility of designing novel tools to inactivate the RBPs, thereby preventing phage infection. In this context, solving the structure of lactococcal phage RBPs is a significant step in understanding the phage-host interactions. To this end, we have previously determined the first crystal structure of a RBP from a lactococcal phage, namely from the lytic phage p2 (936 species) (16Spinelli S. Desmyter A. Verrips C.T. de Haard H.J. Moineau S. Cambillau C. Nat. Struct. Mol. Biol. 2006; 13: 85-89Crossref PubMed Scopus (107) Google Scholar). This RBP is formed of three monomers related by a 3-fold noncrystallographic axis, each assembling three domains, from N to C terminus: the shoulders, the interlaced neck, and the heads. We have shown that this last domain harbors the putative saccharide-binding site, which can be blocked by a llama immunoglobulin VH domain of camelid antibody heavy chain (9De Haard H.J. Bezemer S. Ledeboer A.M. Muller W.H. Boender P.J. Moineau S. Coppelmans M.C. Verkleij A.J. Frenken L.G. Verrips C.T. J. Bacteriol. 2005; 187: 4531-4541Crossref PubMed Scopus (64) Google Scholar, 16Spinelli S. Desmyter A. Verrips C.T. de Haard H.J. Moineau S. Cambillau C. Nat. Struct. Mol. Biol. 2006; 13: 85-89Crossref PubMed Scopus (107) Google Scholar, 17Ledeboer A.M. Bezemer S. de Hiaard J.J. Schaffers I.M. Verrips C.T. van Vliet C. Dusterhoft E.M. Zoon P. Moineau S. Frenken L.G. J. Dairy Sci. 2002; 85: 1376-1382Abstract Full Text PDF PubMed Scopus (43) Google Scholar). We recently showed that the binding of a glycerol molecule to the head domain led to the identification of the residues of the saccharide-binding site (15Tremblay D.M. Tegoni M. Spinelli S. Campanacci V. Blangy S. Huyghe C. Desmyter A. Labrie S. Moineau S. Cambillau C. J. Bacteriol. 2006; 188: 2400-2410Crossref PubMed Scopus (92) Google Scholar). The lactococcal temperate phage TP901-1 belongs to the P335 species. It has a genome size of 37,667 bp with 56 open reading frames (ORFs) (18Brondsted L. Ostergaard S. Pedersen M. Hammer K. Vogensen F.K. Virology. 2001; 283: 93-109Crossref PubMed Scopus (76) Google Scholar). Phage TP901-1 has a long noncontractile tail with a distal baseplate (19Vegge C.S. Brondsted L. Neve H. McGrath S. van Sinderen D. Vogensen F.K. J. Bacteriol. 2005; 187: 4187-4197Crossref PubMed Scopus (58) Google Scholar). Recently, one of its ORFs (ORF49 or BppL) has been identified as being the phage RBP (11Dupont K. Janzen T. Vogensen F.K. Josephsen J. Stuer-Lauridsen B. Appl. Environ. Microbiol. 2004; 70: 5825-5832Crossref PubMed Scopus (63) Google Scholar, 20Vegge C.S. Vogensen F.K. Mc Grath S. Neve H. van Sinderen D. Brondsted L. J. Bacteriol. 2006; 188: 55-63Crossref PubMed Scopus (58) Google Scholar). The mechanism of assembly of the baseplate of phage TP901-1 was also recently deciphered, indicating that BppL forms the lower baseplate and is the last component to be assembled (19Vegge C.S. Brondsted L. Neve H. McGrath S. van Sinderen D. Vogensen F.K. J. Bacteriol. 2005; 187: 4187-4197Crossref PubMed Scopus (58) Google Scholar). In the present study, we have solved the second RBP structure of a lactococcal phage, namely the RBP (BppL) of TP901-1 (20Vegge C.S. Vogensen F.K. Mc Grath S. Neve H. van Sinderen D. Brondsted L. J. Bacteriol. 2006; 188: 55-63Crossref PubMed Scopus (58) Google Scholar), using molecular replacement with the head domain of p2 RBP as a starting model. As with the latter, the RBP of phage TP901-1 harbors a glycerol molecule located in a saccharide binding site of the C-terminal domain. Tryptophan fluorescence quenching experiments performed with a F145W mutant indicated that, in solution, the RBP of TP901-1 binds strongly to glycerol, phosphoglycerol, N-acetylmuramic acid, and muramyl-dipeptide. Native RBP Production and Crystallization—The ORF49 (RBP) of phage TP901-1 was cloned in the Gateway™ pDEST17 vector (21Walhout A.J. Temple G.F. Brasch M.A. Hartley J.L. Lorson M.A. van den Heuvel S. Vidal M. Methods Enzymol. 2000; 328: 575-592Crossref PubMed Google Scholar) according to standard procedures as described previously. A tobacco etch virus protease cleavage site was inserted between the ATTB1 and the gene of interest. The resulting vector was transformed in Escherichia coli Rosetta-pLysS strains. Cells were grown at 28 °C in terrific broth medium in agitated flasks (4 × 750 ml) until the OD reached 0.5 and then induced with isopropyl 1-thio-β-d-galactopyranoside (0.5 mm). After cell centrifugation, lysis was performed in 150 ml of lysis solution (50 mm Tris, 150 mm NaCl, 10 mm imidazole, pH 8.0, 0.25 mg/ml lysozyme, 1 mm PMSF). Purification was performed in two steps, a Ni2+-NTA column, followed by gel filtration (HiLoad S200) on a GE Δkta fast protein liquid chromatograph. The protein was concentrated to 5.6 mg/ml (2.5 mg total) and subjected to crystallization screening with a Cartesian nanodrop-dispensing robot (22Sulzenbacher G. Gruez A. Roig-Zamboni V. Spinelli S. Valencia C. Pagot F. Vincentelli R. Bignon C. Salomoni A. Grisel S. Maurin D. Huyghe C. Johansson K. Grassick A. Roussel A. Bourne Y. Perrier S. Miallau L. Cantau P. Blanc E. Genevois M. Grossi A. Zenatti A. Campanacci V. Cambillau C. Acta Crystallogr. Sect. D Biol. Crystallogr. 2002; 58: 2109-2115Crossref PubMed Scopus (101) Google Scholar). Crystals were obtained at 20 °C by mixing 300 nl of protein (5 mg/ml in 1.8 mm KH2PO4, 10.1 mm mm mm NaCl, pH with nl of solution and Crystals 1 were using a and in a solution of crystallization medium glycerol as Crystals belong to the with cell a and They a in the J. Mol. Biol. PubMed Scopus Google F145W BppL amino was using the by L. J.L. 2004; PubMed Scopus Google Scholar) and from the the vector was by the using two the was to using the and The were by gel and were with to the of 1 was transformed and on an and were performed as for the Fluorescence experiments were on a using a in a was and 1 for the and The interaction of RBP with saccharides was by the quenching of the protein fluorescence the of was and were in the The was the was 20 for a protein of 1 A was with a of were at with 1 protein in 10 mm mm NaCl, pH The fluorescence at the of for different of were for the and The affinity was by the of fluorescence at the as × the is the of fluorescence of the protein is the fluorescence the of is the fluorescence at of The were using by for a binding site with the × is the binding and is the of to of TP901-1 1.65 Å was at A of were on an were and using and Acta Crystallogr. Sect. D. 1994; PubMed Scopus Google Scholar). replacement was performed with J. Acta Sect. A. 1994; Scopus Google Scholar) using the phage p2 RBP head as residues The and at Å were and for the were lower and was performed with G. Acta Crystallogr. Sect. D. PubMed Scopus Google Scholar) with using A. Cambillau C. The Scholar). After and the head and were obtained this a better electron to the of the molecule (residues to from a 3-fold the first 20 residues to be in each of the The first residues in are in the electron to and of the residues are in the R. M. D. J. J. Appl. Crystallogr. 1993; Google Scholar) and are in the of and are in and of on in a RBP RBP of phage TP901-1 is 163 amino acids which is the RBP of the lactococcal phage p2 its C-terminal domain (residues to be of the as the head domain of p2 RBP (residues and that their be In the domain of the RBP of TP901-1 residues as with amino acids of p2 RBP, and can be between them be that these two phages and the L. lactis and their RBPs have their The RBP of phage TP901-1 is formed of three in a homotrimer of × × Å The three monomers are related by a noncrystallographic 3-fold with the long of the in and The first or and C amino acids of the chain are in the electron the of and forms of RBP with a of This is with a of residues comprising from the vector the the and the tobacco etch virus protease cleavage site the first residues of the RBP with of and The and the that RBP can be on Ni2+-NTA to that several forms of one or two with two or one The N of these are in the to the of or interactions. that the residues in the and with RBP The RBP is three an an interlaced the and a C-terminal This is in or RBPs from T. J. 2002; PubMed Scopus Google Scholar, D. S. G. N. J. 2004; PubMed Scopus Google Scholar) as well as from (16Spinelli S. Desmyter A. Verrips C.T. de Haard H.J. Moineau S. Cambillau C. Nat. Struct. Mol. Biol. 2006; 13: 85-89Crossref PubMed Scopus (107) Google Scholar, G. M.R. S. J. Mol. Biol. 2001; PubMed Scopus Google Scholar). The three of the of Å for The head are within the with Å The are at the three of the RBP interactions a large of surface of in a large to the interlaced The surface in the domain of the and the head domain surface is for each which of its surface The residues is assembled through chain as in these A. S. van J. Struct. Biol. 2002; PubMed Scopus Google Scholar). The residues are also to have a helical structure by K. D.T. 2000; PubMed Scopus Google Scholar). The in the are to that the of each with of the two other domains, by to This domain is its in phage p2 RBP, the which amino acids and has a in p2 RBP, the assembly of the three the is by three (residues located to the 3-fold axis, in a as of TP901-1 RBP and This domain other helical in A well is the a structure A. S. van J. Struct. Biol. 2002; PubMed Scopus Google Scholar, Y. Full Text Full Text PDF PubMed Scopus Google Scholar). is with the protein of the formed of three by three external Y. Y. H. P. G.F. J. Biol. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). The well of Å for 10 with the RBP domain This of the is also in other proteins in the mechanism Mol. Biol. 2004; PubMed Scopus (76) Google Scholar). the a structure (residues the domain (residues and the (residues A of this from a in this binds to residues from the and residues from the chain is to the chain its chain and are to and The domain three residues of each and of the three of the is of from the three monomers a interlaced have been by A. S. van J. Struct. Biol. 2002; PubMed Scopus Google in the protein tail of G. M.R. S. J. Mol. Biol. 2001; PubMed Scopus Google Scholar), in the RBP of lactococcal phage p2 (16Spinelli S. Desmyter A. Verrips C.T. de Haard H.J. Moineau S. Cambillau C. Nat. Struct. Mol. Biol. 2006; 13: 85-89Crossref PubMed Scopus (107) Google Scholar) and in a A.M. Sci. S. A. 2005; PubMed Scopus Google Scholar). The of this is to that of of phage to phage p2 neck, significant were The exhibits a acid, This is in or in the RBP of lactococcal phage The first is a followed by a of two a and a with a the last is by a This the last of the the chain to the in a in the RBP of phage p2 (16Spinelli S. Desmyter A. Verrips C.T. de Haard H.J. Moineau S. Cambillau C. Nat. Struct. Mol. Biol. 2006; 13: 85-89Crossref PubMed Scopus (107) Google Scholar). The RBP head domain of TP901-1 (residues is a formed of It from a to the of two that and This domain is the of RBPs and this head of Å for the in between the of of the RBP head domain of TP901-1 from that of p2 RBP in a to the RBP of phage TP901-1 has three residues in its are and in The large between formed between them the possibility of a The to the glycerol molecule was to the head domain. This molecule is better in with monomers A or C. this their are and in monomers and A and are and with and As from the of RBPs three of these residues are to of phage p2 RBP, and the is a in p2 from the glycerol molecule are strongly to the RBP In the is and to the in RBPs and This strongly suggests that the binding site harbors the of the receptor the in the of the of the receptor to the The of the binding of glycerol in RBPs is in amino acids are in the second binding which the binding surface and the between different phage and strains comparison between TP901-1 and p2 in a Tryptophan Fluorescence to the of p2 RBP (15Tremblay D.M. Tegoni M. Spinelli S. Campanacci V. Blangy S. Huyghe C. Desmyter A. Labrie S. Moineau S. Cambillau C. J. Bacteriol. 2006; 188: 2400-2410Crossref PubMed Scopus (92) Google Scholar), the of as of or in the of the site from binding using fluorescence quenching with glycerol or other F145W mutant has been and Fluorescence quenching of was performed with glycerol, phosphoglycerol, N-acetylmuramic acid, muramyl-dipeptide, and In the the of three other the at was The first of the of This also the affinity of these for BppL The binds strongly to TP901-1 RBP, followed by N-acetylmuramic acid, glycerol, and by of several saccharides for TP901-1 BppL and p2 RBP or p2 RBP head domain (15Tremblay D.M. Tegoni M. Spinelli S. Campanacci V. Blangy S. Huyghe C. Desmyter A. Labrie S. Moineau S. Cambillau C. J. Bacteriol. 2006; 188: 2400-2410Crossref PubMed Scopus (92) Google TP901-1 p2 (15Tremblay D.M. Tegoni M. Spinelli S. Campanacci V. Blangy S. Huyghe C. Desmyter A. Labrie S. Moineau S. Cambillau C. J. Bacteriol. 2006; 188: 2400-2410Crossref PubMed Scopus (92) Google p2 head (15Tremblay D.M. Tegoni M. Spinelli S. Campanacci V. Blangy S. Huyghe C. Desmyter A. Labrie S. Moineau S. Cambillau C. J. Bacteriol. 2006; 188: 2400-2410Crossref PubMed Scopus (92) Google in a with of the baseplate of phage TP901-1 with a electron that TP901-1 baseplate is of two the and lower (19Vegge C.S. Brondsted L. Neve H. McGrath S. van Sinderen D. Vogensen F.K. J. Bacteriol. 2005; 187: 4187-4197Crossref PubMed Scopus (58) Google Scholar). The lower has been to the assembly of several RBP (BppL) The lower as a of the through the size of the baseplate as determined by electron is to and to the RBP structure the electron The size of the head domain well with that of the the and the the This that the nature of the host receptor of lactococcal phages is The that glycerol binds to the RBP of phage p2 (15Tremblay D.M. Tegoni M. Spinelli S. Campanacci V. Blangy S. Huyghe C. Desmyter A. Labrie S. Moineau S. Cambillau C. J. Bacteriol. 2006; 188: 2400-2410Crossref PubMed Scopus (92) Google Scholar) strongly that the host receptor molecule be a or also bind to the RBP of phage TP901-1, the It be be of the host or acids are to They of a of the cell wall with a chain of J. T. M. E. P. PubMed Scopus Google Scholar). The is or by a or a a that be in the different strains of L. lactis by p2 and TP901-1 phages lactis strains and their glycerol binding are (15Tremblay D.M. Tegoni M. Spinelli S. Campanacci V. Blangy S. Huyghe C. Desmyter A. Labrie S. Moineau S. Cambillau C. J. Bacteriol. 2006; 188: 2400-2410Crossref PubMed Scopus (92) Google Scholar). This suggests that be in the recognition or that the different of the first the recognition and increase the affinity for TP901-1 The RBP structure of the temperate lactococcal phage TP901-1, a member of the P335 species, exhibits a in a as that in the RBP of the lytic lactococcal p2 phage of the 936 species (16Spinelli S. Desmyter A. Verrips C.T. de Haard H.J. Moineau S. Cambillau C. Nat. Struct. Mol. Biol. 2006; 13: 85-89Crossref PubMed Scopus (107) Google Scholar). The head receptor recognition are in phages We have shown previously that the head domain of phage p2 RBP has a to that of RBPs of (16Spinelli S. Desmyter A. Verrips C.T. de Haard H.J. Moineau S. Cambillau C. Nat. Struct. Mol. Biol. 2006; 13: 85-89Crossref PubMed Scopus (107) Google Scholar), T. J. 2002; PubMed Scopus Google Scholar), and D. S. G. N. J. 2004; PubMed Scopus Google Scholar). This to genetically distinct phage of the Siphoviridae In the the RBP in the phage baseplate are different between p2 and The which the and the head domains, is in an interlaced in their of residues and for the RBPs of p2 and TP901-1, the RBP of TP901-1 has a with that of phage TP901-1 RBP exhibits a which is the for the RBP of phage Recently, the for host recognition in the lactococcal phages and of to species 936 were also identified (11Dupont K. Janzen T. Vogensen F.K. Josephsen J. Stuer-Lauridsen B. Appl. Environ. Microbiol. 2004; 70: 5825-5832Crossref PubMed Scopus (63) Google Scholar). These phages have a high level of different host Functional phages RBP gene from were and the phages were to the host of phage The comparison of the RBPs of phages with that of a in the of the The of phages are the and the head The being the in with other proteins of the phage their (8Dupont K. Vogensen F.K. Neve H. Bresciani J. Josephsen J. Appl. Environ. Microbiol. 2004; 70: 5818-5824Crossref PubMed Scopus (69) Google Scholar) to the RBPs between phages and their the been the would have led to phages. A RBP was recently performed with phages to the P335 species. phages TP901-1 and a large in the for proteins in phage (20Vegge C.S. Vogensen F.K. Mc Grath S. Neve H. van Sinderen D. Brondsted L. J. Bacteriol. 2006; 188: 55-63Crossref PubMed Scopus (58) Google Scholar). RBPs, the as between and of the 936 species. The (residues are the head domain and different a of the by the structure solved here and structure to the RBP to the phage baseplate (19Vegge C.S. Brondsted L. Neve H. McGrath S. van Sinderen D. Vogensen F.K. J. Bacteriol. 2005; 187: 4187-4197Crossref PubMed Scopus (58) Google the receptor recognition in with their distinct host The structural of the phage domain in phages TP901-1 and to the RBPs and the host the head recognition of the p2 and TP901-1 RBPs, to two different lactococcal phage species, are in in RBPs from phages of the species and TP901-1 are to this Phages are to a They the of the proteins in assembly and Biol. 2002; PubMed Scopus Google Scholar) as well as at a fast the proteins in host recognition in to the of their host or to host strains. Lactococcal phages to this by three of structural in their The domain is of the phage and to the RBP to the of the phage It is between phages that are genetically A and domain to the phage domain and the the neck, the head domain is in the recognition of one of the receptor of the different L. lactis host strains. the head domain to a saccharide-binding the of a residues of the second binding be in this

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