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• W1985070147 abstract "Siphophage SPP1 infects the Gram-positive bacterium Bacillus subtilis using its long non-contractile tail and tail-tip. Electron microscopy (EM) previously allowed a low resolution assignment of most orf products belonging to these regions. We report here the structure of the SPP1 distal tail protein (Dit, gp19.1). The combination of x-ray crystallography, EM, and light scattering established that Dit is a back-to-back dimer of hexamers. However, Dit fitting in the virion EM maps was only possible with a hexamer located between the tail-tube and the tail-tip. Structure comparison revealed high similarity between Dit and a central component of lactophage baseplates. Sequence similarity search expanded its relatedness to several phage proteins, suggesting that Dit is a docking platform for the tail adsorption apparatus in Siphoviridae infecting Gram-positive bacteria and that its architecture is a paradigm for these hub proteins. Dit structural similarity extends also to non-contractile and contractile phage tail proteins (gpVN and XkdM) as well as to components of the bacterial type 6 secretion system, supporting an evolutionary connection between all these devices. Siphophage SPP1 infects the Gram-positive bacterium Bacillus subtilis using its long non-contractile tail and tail-tip. Electron microscopy (EM) previously allowed a low resolution assignment of most orf products belonging to these regions. We report here the structure of the SPP1 distal tail protein (Dit, gp19.1). The combination of x-ray crystallography, EM, and light scattering established that Dit is a back-to-back dimer of hexamers. However, Dit fitting in the virion EM maps was only possible with a hexamer located between the tail-tube and the tail-tip. Structure comparison revealed high similarity between Dit and a central component of lactophage baseplates. Sequence similarity search expanded its relatedness to several phage proteins, suggesting that Dit is a docking platform for the tail adsorption apparatus in Siphoviridae infecting Gram-positive bacteria and that its architecture is a paradigm for these hub proteins. Dit structural similarity extends also to non-contractile and contractile phage tail proteins (gpVN and XkdM) as well as to components of the bacterial type 6 secretion system, supporting an evolutionary connection between all these devices. More than 95% of bacterial viruses (bacteriophages or phages) belong to the Caudovirales order, i.e. phages with a tail. Their vast majority is Siphoviridae, characterized by the presence of a long non-contractile tail. The assembly pathway of this structure essential for infection was genetically dissected for Escherichia coli phage λ (1Katsura I. J. Mol. Biol. 1976; 107: 307-326Crossref PubMed Scopus (26) Google Scholar, 2Katsura I. Nature. 1987; 327: 73-75Crossref PubMed Scopus (133) Google Scholar) and for the host-adsorption apparatus of phages TP901-1 and Tuc2009 that infect the Gram-positive bacterium Lactococcus lactis (3Vegge C.S. Brøndsted L. Neve H. Mc Grath S. van Sinderen D. Vogensen F.K. J. Bacteriol. 2005; 187: 4187-4197Crossref PubMed Scopus (58) Google Scholar, 4Mc Grath S. Neve H. Seegers J.F. Eijlander R. Vegge C.S. Brøndsted L. Heller K.J. Fitzgerald G.F. Vogensen F.K. van Sinderen D. J. Bacteriol. 2006; 188: 3972-3982Crossref PubMed Scopus (67) Google Scholar). However, the molecular mechanisms underlying tail assembly remain poorly understood. Despite the diverse infection mechanisms displayed by Siphoviridae, using surface proteins and/or cell-wall saccharides as receptors, their tail architecture is rather conserved. It is characterized by a long non-contractile tube, assembled by stacking several tens of homo-hexameric major tail protein (MTP) 2The abbreviations used are: MTPmajor tail proteinTMPtape measure proteinT6SStype 6 secretion systemMALSmultiangle static light scatteringQELSquasielastic light scatteringPDBProtein data bank. rings, and a central core formed by a few copies of the tape measure protein (TMP) extending between both tail extremities and determining its length. At the proximal tail end (near the capsid), the homo-hexameric terminator that stops tube elongation during assembly is found, whereas the distal tail end (opposite to the capsid) is characterized by the presence of the tail adsorption apparatus. In phages of Gram-positive bacteria this structure is composed of the distal tail protein (Dit), the tail fiber, and eventually ancillary proteins, all forming the baseplate (4Mc Grath S. Neve H. Seegers J.F. Eijlander R. Vegge C.S. Brøndsted L. Heller K.J. Fitzgerald G.F. Vogensen F.K. van Sinderen D. J. Bacteriol. 2006; 188: 3972-3982Crossref PubMed Scopus (67) Google Scholar). Although three-dimensional structures of siphophages MTP (5Pell L.G. Kanelis V. Donaldson L.W. Howell P.L. Davidson A.R. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 4160-4165Crossref PubMed Scopus (207) Google Scholar) and tail terminator (6Pell L.G. Liu A. Edmonds L. Donaldson L.W. Howell P.L. Davidson A.R. J. Mol. Biol. 2009; 389: 938-951Crossref PubMed Scopus (43) Google Scholar) have been reported, no high-resolution structure is available for components of the adsorption device with the exception of our recently reported phage p2 baseplate structure (7Sciara G. Bebeacua C. Bron P. Tremblay D. Ortiz-Lombardia M. Lichière J. van Heel M. Campanacci V. Moineau S. Cambillau C. Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 6852-6857Crossref PubMed Scopus (125) Google Scholar). Several tail components of the Bacillus subtilis phage SPP1 show significant sequence similarity to equivalent proteins from lactococcal phages Tuc2009 and TP901-1. This is the case for the TMP (gp18), Dit (gp19.1), and tail-spike (gp21). However, these two lactococcal phages possess a large and bulky baseplate of 1 to 2 MDa anchored at the end of the tail-tube (3Vegge C.S. Brøndsted L. Neve H. Mc Grath S. van Sinderen D. Vogensen F.K. J. Bacteriol. 2005; 187: 4187-4197Crossref PubMed Scopus (58) Google Scholar, 4Mc Grath S. Neve H. Seegers J.F. Eijlander R. Vegge C.S. Brøndsted L. Heller K.J. Fitzgerald G.F. Vogensen F.K. van Sinderen D. J. Bacteriol. 2006; 188: 3972-3982Crossref PubMed Scopus (67) Google Scholar, 7Sciara G. Bebeacua C. Bron P. Tremblay D. Ortiz-Lombardia M. Lichière J. van Heel M. Campanacci V. Moineau S. Cambillau C. Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 6852-6857Crossref PubMed Scopus (125) Google Scholar, 8Campanacci V. Veesler D. Lichière J. Blangy S. Sciara G. Moineau S. van Sinderen D. Bron P. Cambillau C. J. Struct. Biol. 2010; 172: 75-84Crossref PubMed Scopus (34) Google Scholar, 9Sciara G. Blangy S. Siponen M. Mc Grath S. van Sinderen D. Tegoni M. Cambillau C. Campanacci V. J. Biol. Chem. 2008; 283: 2716-2723Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar), whereas SPP1 displays only an elongated tail-tip (10Plisson C. White H.E. Auzat I. Zafarani A. São-José C. Lhuillier S. Tavares P. Orlova E.V. EMBO J. 2007; 26: 3720-3728Crossref PubMed Scopus (107) Google Scholar). Therefore, despite conserved tail architecture among these viruses, the host-adsorption apparatus distinguishes them. major tail protein tape measure protein type 6 secretion system multiangle static light scattering quasielastic light scattering Protein data bank. The precise location of the different SPP1 tail and tail-tip proteins as well as their structures and interactions remain poorly understood. In this contribution, we report the SPP1 Dit purification, its oligomeric state, and size determination using light scattering/refractometry. We also determined the Dit structure by x-ray crystallography and electron microscopy (EM). Dit docking into EM maps of the SPP1 virion (10Plisson C. White H.E. Auzat I. Zafarani A. São-José C. Lhuillier S. Tavares P. Orlova E.V. EMBO J. 2007; 26: 3720-3728Crossref PubMed Scopus (107) Google Scholar) allowed reassigning its position in the tail tube end “cap” density. Based on structure and sequence comparisons between several Gram-positive infecting Siphoviridae, we suggest that the presence of Dit-like structures is a conserved feature in such phages, acting as a hub between the tail and the tail-tip/baseplate. Finally, a striking structural similarity was found between SPP1 Dit and Siphoviridae/Myoviridae tail components as well as with components of the bacterial type 6 secretion system (T6SS) providing additional evidence for a common origin between phage tails and T6SS. orf19.1 of B. subtilis phage SPP1 (NC_004166.2) was cloned into the pETG-20A expression vector according to standard GatewayTM protocols. The final construct encoded a N-terminal thioredoxin fusion followed by a hexahistidine tag and a TEV protease cleavage site before the gene of interest. The resulting plasmid was used to transform the E. coli T7 Express Iq pLysS strain (New England Biolabs). Cells were grown in Terrific Broth at 37 °C until the optical density reached 0.5 and protein expression was induced with 0.5 mm isopropyl β-thiogalactoside overnight at 25 °C. Selenomethionine-labeled protein was prepared following standard procedures in M9 minimal medium through blocking of the methionine biosynthesis pathway and with an induction temperature of 25 °C (11Doublié S. Methods Enzymol. 1997; 276: 523-530Crossref PubMed Scopus (798) Google Scholar). Purification was performed following procedures as described elsewhere (9Sciara G. Blangy S. Siponen M. Mc Grath S. van Sinderen D. Tegoni M. Cambillau C. Campanacci V. J. Biol. Chem. 2008; 283: 2716-2723Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 12Siponen M. Spinelli S. Blangy S. Moineau S. Cambillau C. Campanacci V. J. Bacteriol. 2009; 191: 3220-3225Crossref PubMed Scopus (21) Google Scholar). Briefly, after cell harvesting, lysis was achieved by adding 0.25 mg/ml of lysozyme, followed by a freezing/thawing cycle and sonication. Soluble proteins were separated from inclusion bodies and cell debris by a 30-min centrifugation step at 20,000 × g. We used an ÄKTA FPLC system to achieve four steps: a Ni2+-affinity chromatography (5-ml HisTrap, GE Healthcare) with a step gradient of 250 mm imidazole, an overnight TEV protease digestion at 4 °C using a 1:10 (w/w) protease:target ratio, a second Ni2+-affinity step (to remove the His-tagged TEV and digested peptides), and a preparative Superdex 200 HR 26/60 gel filtration run in 10 mm HEPES pH 7.5, 150 mm NaCl. Orf19.1 was then concentrated up to 6–8 mg/ml. Size exclusion chromatography was carried out on an Alliance 2695 HPLC system (Waters) using a KW804 column (Shodex) run in 10 mm HEPES pH 7.5, 150 mm NaCl at 0.5 ml/min. Multiangle static light scattering (MALS), UV spectrophotometry, quasielastic light scattering (QELS), and refractometry (RI) were achieved with a MiniDawn Treos (Wyatt Technology), a Photo Diode Array 2996 (Waters), a DynaPro (Wyatt Technology), and an Optilab rEX (Wyatt Technology), respectively, as described (8Campanacci V. Veesler D. Lichière J. Blangy S. Sciara G. Moineau S. van Sinderen D. Bron P. Cambillau C. J. Struct. Biol. 2010; 172: 75-84Crossref PubMed Scopus (34) Google Scholar, 13Veesler D. Blangy S. Siponen M. Vincentelli R. Cambillau C. Sciara G. Anal. Biochem. 2009; 388: 115-121Crossref PubMed Scopus (18) Google Scholar, 14Veesler D. Dreier B. Blangy S. Lichière J. Tremblay D. Moineau S. Spinelli S. Tegoni M. Plückthun A. Campanacci V. Cambillau C. J. Biol. Chem. 2009; 284: 30718-30726Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Mass and hydrodynamic radius calculation was done with ASTRA V software (Wyatt Technology) using a dn/dc value of 0.185 ml/g. Crystallization of selenomethionine and native gp19.1 was performed at 20 °C in 96-well Greiner crystallization plates using a nanodrop-dispensing robot (Cartesian Inc.). Crystals grew in a few days by mixing 300 nl of protein at 6–8 mg/ml with 100 nl of 2 m NaCl, 0.1 m Na+ acetate, pH 4.0. Crystals were cryoprotected with mother liquor supplemented with 26.5% glycerol and flash frozen in liquid nitrogen. One SAD dataset was collected at the selenium K-edge (λ = 0.97818 Å) from a single crystal at the BM14 beamline (European Synchrotron Radiation Facility, Grenoble, France) using a MarCCD detector (Table 1). The native data set was collected at the PROXIMA 1 beamline (SOLEIL, Gif-sur-Yvette, France) using an ADSC Q315r detector (Table 1). Data were processed and scaled using XDS (15Kabsch W. Acta Crystallogr. D Biol. Crystallogr. 2010; 66: 125-132Crossref PubMed Scopus (11544) Google Scholar) POINTLESS and SCALA (16Evans P. Acta Crystallogr. D Biol. Crystallogr. 2006; 62: 72-82Crossref PubMed Scopus (3794) Google Scholar). The phenix.autosol and phenix.autobuild wizards were used to solve the structure (11 out of 15 selenium sites were found) and perform initial model building (17Adams P.D. Afonine P.V. Bunkóczi G. Chen V.B. Davis I.W. Echols N. Headd J.J. Hung L.W. Kapral G.J. Grosse-Kunstleve R.W. McCoy A.J. Moriarty N.W. Oeffner R. Read R.J. Richardson D.C. Richardson J.S. Terwilliger T.C. Zwart P.H. Acta Crystallogr. D Biol. Crystallogr. 2010; 66: 213-221Crossref PubMed Scopus (16817) Google Scholar, 18Terwilliger T.C. Grosse-Kunstleve R.W. Afonine P.V. Moriarty N.W. Zwart P.H. Hung L.W. Read R.J. Adams P.D. Acta Crystallogr. D Biol. Crystallogr. 2008; 64: 61-69Crossref PubMed Scopus (1109) Google Scholar, 19Terwilliger T.C. Adams P.D. Read R.J. McCoy A.J. Moriarty N.W. Grosse-Kunstleve R.W. Afonine P.V. Zwart P.H. Hung L.W. Acta Crystallogr. D Biol. Crystallogr. 2009; 65: 582-601Crossref PubMed Scopus (688) Google Scholar). Manual model building was done with COOT (20Emsley P. Cowtan K. Acta Crystallogr. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (23628) Google Scholar) and TURBO-FRODO (21Roussel A. Cambillau C. The TURBO-FRODO Graphics Package. Silicon Graphics Geometry Partners Directory, Mountain View, CA1991Google Scholar). The native Dit structure was solved by molecular replacement using MolRep (22Vagin A. Teplyakov A. Acta Crystallogr. D Biol. Crystallogr. 2010; 66: 22-25Crossref PubMed Scopus (2758) Google Scholar) with one monomer as search model. Refinement was performed at 2.95 Å using Buster-TnT (23Blanc E. Roversi P. Vonrhein C. Flensburg C. Lea S.M. Bricogne G. Acta Crystallogr. D Biol. Crystallogr. 2004; 60: 2210-2221Crossref PubMed Scopus (604) Google Scholar). At various stages of the refinement process, molecular replacement-SAD and averaged kick maps were calculated to help modeling with Phaser (24McCoy A.J. Grosse-Kunstleve R.W. Adams P.D. Winn M.D. Storoni L.C. Read R.J. J. Appl. Crystallogr. 2007; 40: 658-674Crossref PubMed Scopus (14771) Google Scholar), Sharp (25Bricogne G. Vonrhein C. Flensburg C. Schiltz M. Paciorek W. Acta Crystallogr. D Biol. Crystallogr. 2003; 59: 2023-2030Crossref PubMed Scopus (558) Google Scholar), and Phenix (26Praaenikar J. Afonine P.V. Guncar G. Adams P.D. Turk D. Acta Crystallogr. D Biol. Crystallogr. 2009; 65: 921-931Crossref PubMed Scopus (58) Google Scholar). Structure analysis was assisted by the PISA server (27Krissinel E. Henrick K. J. Mol. Biol. 2007; 372: 774-797Crossref PubMed Scopus (6929) Google Scholar) and promotif 2 (28Hutchinson E.G. Thornton J.M. Protein Sci. 1996; 5: 212-220Crossref PubMed Scopus (999) Google Scholar). Electrostatic potential calculation was performed with PDB 2pqr (29Dolinsky T.J. Nielsen J.E. McCammon J.A. Baker N.A. Nucleic Acids Res. 2004; 32: W665-W667Crossref PubMed Scopus (2553) Google Scholar) and APBS (30Holst M. Saied F. J. Comput. Chem. 1993; 14: 105-113Crossref Scopus (245) Google Scholar). Fitting of the Dit x-ray structure into EM densities was performed with Chimera (31Pettersen E.F. Goddard T.D. Huang C.C. Couch G.S. Greenblatt D.M. Meng E.C. Ferrin T.E. J. Comput. Chem. 2004; 25: 1605-1612Crossref PubMed Scopus (29006) Google Scholar), VEDA (32Navaza J. Lepault J. Rey F.A. Alvarez-Rúa C. Borge J. Acta Crystallogr. D Biol. Crystallogr. 2002; 58: 1820-1825Crossref PubMed Scopus (114) Google Scholar), and the COLORES and COLACOR modules of the Situs package (33Chacón P. Wriggers W. J. Mol. Biol. 2002; 317: 375-384Crossref PubMed Scopus (294) Google Scholar). All figures were generated with Chimera.TABLE 1Data collection and refinement statisticsNative gp19.1Selenomethionine gp19.1Data collectionBeamlinePROXIMA 1 (SOLEIL)BM14 (ESRF)Space group/cell parametersI222/a = 107.2 Å, b = 125.7 Å, c = 189.35 ÅI222/a = 106.06 Å, b = 125.7 Å, c = 190.1 ÅWavelength (Å)0.980.9782Resolution limitsaRefers to the highest resolution bin. (Å)104.8-2.95 (3.11-2.95)81.1-3.05 (3.27-3.05)RmergeaRefers to the highest resolution bin. (%)5.8 (67.7)8.5 (51.1)No. of observationsaRefers to the highest resolution bin.98,780 (14651)381,322No. unique reflectionsaRefers to the highest resolution bin.27,345 (3933)24,635Mean ((I)/S.D.(I))aRefers to the highest resolution bin.16.2 (2.1)24.7 (5.4)CompletenessaRefers to the highest resolution bin. (%)99.6 (99.9)98.9 (98.6)MultiplicityaRefers to the highest resolution bin.3.6 (3.7)16.5 (16.9)RefinementResolutionaRefers to the highest resolution bin. (Å)34.4-2.95 (3.06-2.95)No. of reflectionsaRefers to the highest resolution bin.27,167 (2755)No. of protein atoms5,731No. of test set reflections958Rwork/Rfree (%)20.0/23.6Root mean square deviation bonds(Å)/angles (°)0.014/1.93Residues in Ramachandran favored/allowed regions (%)93.4/6.6a Refers to the highest resolution bin. Open table in a new tab 3 μl of freshly prepared protein at ∼10 μg/ml was applied on a 400-mesh glow-discharged carbon-coated copper grid. Excess Dit solution was blotted and 4 μl of 1% uranyl acetate was applied twice on the grid and incubated for 1 min. Grids were then dried and kept in a desiccator cabinet until use. The grids were observed under low-dose conditions with a Jeol 2200FS transmission electron microscope operating at 200 kV. Images were recorded at ×50,000 magnification and digitized using a Nikon coolscan 9000 ED with a step size of 10 μm. Individual particles (10,875) were extracted semiautomatically from 8 micrographs with Boxer (34Ludtke S.J. Baldwin P.R. Chiu W. J. Struct. Biol. 1999; 128: 82-97Crossref PubMed Scopus (2110) Google Scholar) and corrected for the phase-contrast transfer function by phase-flipping. Image processing and single-particle three-dimensional reconstruction were performed using IMAGIC-5 (35van Heel M. Harauz G. Orlova E.V. Schmidt R. Schatz M. J. Struct. Biol. 1996; 116: 17-24Crossref PubMed Scopus (1055) Google Scholar) according to the classic single-particle reconstruction approach. Briefly, an initial model was computed using a C1 start-up procedure, included in the ANGular-REConstitution program, followed by three iterative cycles of image alignment, class averaging, and three-dimensional reconstruction. The resolution of the final reconstruction including 9,027 particles was estimated at 21.5 Å using the Fourier shell correlation criterion with a cutting level of 0.5 (36Harauz G. van Heel M. Optik. 1986; : 146-156Google Scholar). The final density map was then filtered at 21-Å resolution. SPP1 Dit was overproduced and purified to homogeneity yielding 60 mg of purified protein per liter of culture. We used SEC/MALS/QELS/RI to characterize the Dit oligomeric state and size (8Campanacci V. Veesler D. Lichière J. Blangy S. Sciara G. Moineau S. van Sinderen D. Bron P. Cambillau C. J. Struct. Biol. 2010; 172: 75-84Crossref PubMed Scopus (34) Google Scholar, 13Veesler D. Blangy S. Siponen M. Vincentelli R. Cambillau C. Sciara G. Anal. Biochem. 2009; 388: 115-121Crossref PubMed Scopus (18) Google Scholar, 14Veesler D. Dreier B. Blangy S. Lichière J. Tremblay D. Moineau S. Spinelli S. Tegoni M. Plückthun A. Campanacci V. Cambillau C. J. Biol. Chem. 2009; 284: 30718-30726Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). The Dit measured mass and hydrodynamic radius were 325,629 ± 500 Da (supplemental Fig. S1) and 6.7 ± 0.4 nm, respectively, establishing the dodecameric nature of Dit in solution (theoretical monomer mass: 28,489 Da). The asymmetric unit of Dit crystals contains three monomers. The final model was refined at 2.95-Å resolution resulting in Rwork and Rfree values of 20.0 and 23.6%, respectively (Table 1). Applying crystallographic symmetry operators results in the completion of two hexameric rings stacked back-to-back with 12 protruding domains located at the periphery (Fig. 1, A–C). The two rings are rotated relative to each other by 10°. The crystal packing is formed by piles of dodecamers aligned along their channel axis (unit cell a axis) and contacting each other through the tip of the 2 × 6 protruding domains on both faces. Each pile laterally contacts 6 neighboring piles, shifted by translation of one hexamer, via the protruding domain sides. The dodecamer central core is a 75-Å high and 80-Å wide hollow cylinder made of a double ring (excluding the protruding domains). Taking into account the 12 protruding domains, radiating from the core rings, results in Dit overall dimensions of 105 Å (height) and 140 Å (diameter). The circular-shaped core harbors a ∼40-Å wide central channel whose inner surface is mainly covered by acidic residues and displays hence a strong negative electrostatic potential (Fig. 1, A, C, and D). This feature facilitates DNA traffic into the host cytoplasm during infection and prevents interactions with the channel wall. A similar negatively charged conduit is observed in the SPP1 head-to-tail connector channel (37Lhuillier S. Gallopin M. Gilquin B. Brasilès S. Lancelot N. Letellier G. Gilles M. Dethan G. Orlova E.V. Couprie J. Tavares P. Zinn-Justin S. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 8507-8512Crossref PubMed Scopus (94) Google Scholar) as well as in the phage λ tail-terminator channel (6Pell L.G. Liu A. Edmonds L. Donaldson L.W. Howell P.L. Davidson A.R. J. Mol. Biol. 2009; 389: 938-951Crossref PubMed Scopus (43) Google Scholar). The interface buried surface area between two neighboring monomers within a Dit hexameric ring is ∼1,300 Å2 per monomer (∼10% of the monomer surface). A total surface of ∼16,000 Å2 thus ensures the cohesion of one hexamer. The interaction between the two hexameric rings of a dodecamer is mediated by a smaller buried surface area (∼5700 Å2). Each monomer was built in the electron density between residues 9 and 253, the C-terminal amino acid. The N terminus is situated at the periphery of the cylinder. Some blobs of extra density account for the presence of the first 8 residues but their discontinuity precludes further modeling. In contrast, the C terminus is well ordered due to interactions of its negatively charged carboxyl-terminal group with the Arg60 guanidinium moiety from the same monomer and the Asn85 amide nitrogen from a neighboring monomer. Each Dit monomer can be divided in two domains corresponding to the N- and C-terminal parts of the polypeptide chain (Fig. 1, E–H). The N-domain (residues 9 to 135) is well defined in the electron density map and is composed of two layers comprising 8 β-strands organized in two β-sheets, a β-hairpin, and an α-helix (Fig. 1, E–G). The layer forming the wall of the central channel is folded as a four-stranded antiparallel β-sheet, constituted by strands 2, 5, 8, and 7b, from which extends a β-hairpin, termed the belt, encompassing strands 3 and 4 (residues 32 to 55). The belt projects toward the N-domain of a neighboring monomer and interacts with its four-stranded β-sheet ensuring the strength of the hexamer association (Fig. 1C). The outward layer forms the external wall of the cylinder and contains an α-helix (h1) and a three-stranded anti-parallel β-sheet made of strands 1, 6, and 7a (Fig. 1, E–G). The Dit C-domain (residues 136 to 253) protrudes out of the cylinder core and mediates all the crystal contacts. The C-domain folds as a nine-stranded β-sandwich organized in two β-sheets and an additional extended stretch (Fig. 1, E, F, and H). The domain starts at residue 136 with β-strand 1, belonging to a five-stranded antiparallel β-sheet that includes also strands 9, 3, 6, and 7. Strand 2 initiates a four-stranded antiparallel β-sheet formed with strands 8, 4, and 5. An elongated segment (7b) links strands 7 and 8. A preliminary EM reconstruction, from negatively stained Dit, resulted in a convincing dodecameric structure constituted by two identical hexameric rings in the absence of any enforced symmetry. As the single particle three-dimensional reconstruction converged to a structure with a clear 6-fold dihedral symmetry (D6), we imposed this symmetry to improve its quality. The resulting structure is a thick-wall hollow cylinder delimiting a central channel. Six identical globular domains protrude regularly outwards from each of the two back-to-back hexameric rings (Fig. 2). The 6-fold symmetry operator coincides with the channel axis. The overall dimensions and shape of our Dit EM reconstruction (at 21-Å resolution) are in good agreement with those observed in the crystal structure. Indeed, correlation coefficient values of 82.3 and 69.2% were obtained in fitting the dodecameric Dit structure into the EM map with COLACOR and VEDA, respectively, revealing a similar architecture for the isolated molecule. Noteworthy, the poor quality of the map in the protruding domain regions is likely due to their flexibility. A DALI search (38Holm L. Kääriäinen S. Rosenström P. Schenkel A. Bioinformatics. 2008; 24: 2780-2781Crossref PubMed Scopus (849) Google Scholar) with the Dit monomer resulted in a striking structural similarity with ORF15 (PDB code 2WZP, Z = 13.6, Fig. 3, A and B), a component of the phage p2 baseplate (7Sciara G. Bebeacua C. Bron P. Tremblay D. Ortiz-Lombardia M. Lichière J. van Heel M. Campanacci V. Moineau S. Cambillau C. Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 6852-6857Crossref PubMed Scopus (125) Google Scholar, 8Campanacci V. Veesler D. Lichière J. Blangy S. Sciara G. Moineau S. van Sinderen D. Bron P. Cambillau C. J. Struct. Biol. 2010; 172: 75-84Crossref PubMed Scopus (34) Google Scholar). ORF15 is composed of a N-terminal domain (residues 1–120), whose hexamer forms a channel, with two layers of β-sheets and an α-helix associated with the external one. The ORF15 hexamer is very similar to the SPP1 Dit one (Fig. 3, C and D) and is also maintained by six belt extensions. The ORF15 C-terminal domain (121–298) is a β-sandwich resembling remotely galectin, as is the case of the SPP1 Dit C-domain (Z = 5.9 with PDB code 2YV8). Phage p2 ORF15 galectin domain exhibits, however, a long insertion of ∼60 residues, termed the arm, attaching ORF18 (the receptor-binding protein, RBP) to the central core of the baseplate. This feature is absent in SPP1 Dit because it does not serve to assemble peripheral elements of a baseplate. Reconstructions of the SPP1 tail, cap and tail-tip, before and after DNA ejection, were previously obtained using contrast EM (10Plisson C. White H.E. Auzat I. Zafarani A. São-José C. Lhuillier S. Tavares P. Orlova E.V. EMBO J. 2007; 26: 3720-3728Crossref PubMed Scopus (107) Google Scholar). The tail-tube was observed to be formed by stacked hexameric MTP rings and to contact the density volume termed cap at its distal extremity. It was proposed that the cap is formed by the TMP C-terminal domain. Progressing toward the tail-tip, a globular density, located between the cap and the terminal density, was assigned to a putative gp19.1 trimer. However, neither the Dit structures (x-ray or EM) nor its mass corroborate this assignment. Fitting the Dit dodecamer into the cap density envelope provides a significantly better match for one hexamer (located in the middle part of the cap density), whereas most of the second one is outside the EM density (Fig. 4, A and B). A computational fitting of one Dit hexameric ring in the cap before DNA ejection, using COLORES, results in positioning it either into the last ring assigned to MTP and the upper cap density (Fig. 4, C and D) or in the middle part of the cap with an opposite orientation (as the proximal ring in the dodecameric fitting case, Fig. 4G). In the cap after DNA ejection, only the former solution was obtained (Fig. 4, E and F). We decided to perform thorough fitting experiments, using COLACOR and VEDA, to help assign the Dit orientation. Our results did not allow to unambigously distinguish which of the two orientations is correct because in both cases the Dit central ring and the six protruding domains are accommodated into the cap density before and after DNA ejection (Fig. 4, C–H, and supplemental Table S1). We thus believe that in the SPP1 virion, Dit might consist of a single hexamer positioned in the cap region in either of the two proposed orientations. We thus suspect that the observation of a dodecameric Dit in solution and in the crystal is an artifact that might result from the absence of its partners when expressed alone in E. coli. The above Dit assignment is in agreement with the observation of a ∼40-Å wide channel at the center of the protein, which matches the inner diameter of the tail-tube in the virion EM map after DNA ejection (42 Å) (10Plisson C. White H.E. Auzat I. Zafarani A. São-José C. Lhuillier S. Tavares P. Orlova E.V. EMBO J. 2007; 26: 3720-3728Crossref PubMed Scopus (107) Google Scholar). We also observed that the electrostatic character of the continuous channel forming the DNA ejection pathway is conserved: most components (head-to-tail connector, tail terminator, and Dit) display a negatively charged surface in the tunnel. We expect that this property could also be observed in the tail-tube interior because the MTP of phage SPP1 has an acidic pI. Finally, we demonstrated that Dit remains associated to the tail after SPP1 DNA ejection that leads to loss of the tail-spike (10Plisson C. White H.E. Au" @default.
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• W1985070147 date "2010-11-01" @default.
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• W1985070147 title "Crystal Structure of Bacteriophage SPP1 Distal Tail Protein (gp19.1)" @default.
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