Structure of lactococcal phage p2 baseplate and its mechanism of activation

Giuliano Sciaraa, Cecilia Bebeacuab, Patrick Bronc, Denise Tremblayd,e, Miguel Ortiz-Lombardiaa, Julie Lichièrea, Marin van Heelb, Valérie Campanaccia, Sylvain Moineaud,e, and Christian Cambillaua,1

aArchitecture et Fonction des Macromolécules Biologiques, UMR 6098 Centre National de la Recherche Scientifique and Universités d’Aix-Marseille I & II, Campus de Luminy, Case 932, 13288 Marseille Cedex 09, France; bDepartment of Biological Sciences, Imperial College London, South Kensington Campus, London SW7 2AZ, United Kingdom; cCentre de Biochimie Structurale, Institut National de la Santé et de la Recherche Médicale U554/Centre National de la Recherche Scientifique UMR 5048, 29 rue de Navacelles, 34090 Montpellier, France; dGroupe de Recherche en Écologie Buccale and Félix d’Hérelle Reference Center for Bacterial , Faculté de Médecine Dentaire, Université Laval; and eDépartement de Biochimie et de Microbiologie, Faculté des Sciences et de Génie, Université Laval, Québec City, Québec, Canada G1V 0A6

Edited* by Michael G. Rossmann, Purdue University, West Lafayette, IN, and approved March 1, 2010 (received for review January 7, 2010)

Siphoviridae is the most abundant viral family on earth which proteins have never been documented in phages with non- infects bacteria as well as archaea. All known siphophages infect- contractile tails, such as those of the Siphoviridae family. ing gram+ Lactococcus lactis possess a baseplate at the tip of their Within the baseplate are located, among other proteins, tail involved in host recognition and attachment. Here, we report several copies of the phage receptor-binding proteins (RBPs) analysis of the p2 phage baseplate structure by X-ray crystallo- which are necessary to specifically recognize the receptors at graphy and electron microscopy and propose a mechanism for the host cell surface (6–9, 13). Recently, the 3D structures of the baseplate activation during attachment to the host cell. This RBPs from three lactococcal phages (p2, bIL170, TP901-1) have ∼1 MDa, -expressed baseplate is composed of three been solved (14–17). These homotrimeric proteins are composed protein species, including six trimers of the receptor-binding pro- of three domains named shoulders, neck, and head, the latter do- tein (RBP). RBPs host-recognition domains point upwards, towards main bearing the receptor-binding area. These structures made it the capsid, in agreement with the electron-microscopy map of the possible to identify a sugar/glycerol binding site in the RBP head þ free virion. In the presence of Ca2 , a cation mandatory for infec- domain and led to postulate that they may recognize lipoteichoic BIOPHYSICS AND acids (LTAs), which are phospho-glycerol polymers. This hypo- tion, the RBPs rotated 200° downwards, presenting their binding COMPUTATIONAL BIOLOGY sites to the host, and a channel opens at the bottom of the thesis is still waiting experimental confirmation. Here, we have baseplate for DNA passage. These conformational changes reveal structurally and functionally characterized the baseplate of a si- a novel siphophage activation and host-recognition mechanism phophage. We have chosen the virulent phage p2, a represen- leading ultimately to DNA ejection. tative of the 936 group, which is the most prominent group of lactococcal phages isolated from dairy samples worldwide. ∣ ∣ ∣ crystal structure electon microscopy Lactococcus lactis Results Siphoviridae ∣ Baseplate Composition and Overall Structure. The genome of phage actococcus lactis p2 consists of a linear, double-stranded, 27 595 bp DNA molecule is a gram-positive bacterium extensively used containing 49 orfs and is very similar to the lactococcal phage Las starter cultures for the industrial production of an array of sk1 genome (18). Twelve structural proteins (ORF4–ORF11, milk fermented products, including cheeses (1). Virulent lacto- ORF14, ORF15, ORF16, ORF18) were identified by liquid chro- coccal phages are ubiquitous in dairy environments and their lytic matography coupled mass spectrometry (LCMS/MS) analysis cycle leads to bacterial cell lysis, thereby slowing the milk fermen- using the whole purified phage p2 virions as well as from bands tation process and lowering the overall quality of the manufac- cut from a preparation of p2 migrated on SDS-PAGE gels tured products. Hundreds of virulent L. lactis phages have been (Fig. S1). characterized worldwide and the vast majority of them belong to Sequence analysis as well as its size (999 aa) indicated that the the Siphoviridae family (2). ORF14 is the tape measure protein while the ORF19 (holin) and Phages of the Siphoviridae family ( order) possess ORF20 (endolysin) are involved in cell lysis. Based on their geno- a proteinacious capsid, containing a double-stranded DNA gen- mic location, we hypothesized that the four genes downstream ome, connected to a long noncontractile tail. The host-recogni- of orf14, namely, orfs 15, 16, 17, and 18 (RBP) encoded base- tion and adsorption device is located at the tip of the tail and is plate-related proteins. ORF16 and ORF17 are highly conserved used to start the phage infection process (3). Contrary to what is among lactococcal phages whereas ORF15 and ORF18 show observed in most Siphoviridae phages, such as coliphage T5 (4) some diversity. and Bacillus phage SPP1 (5), the adsorption device of most lac- The contiguous cluster of four genes was cloned and expressed tococcal phages is a large organelle of 1–2 MDa with a typical in E. coli (19). We then purified the resulting macromolecular diameter of 20–30 nm called baseplate (6–9). Still, the host- complex of ∼1 MDa by affinity chromatography (ORF15 was recognition process by phages is poorly understood in gram- His6-tagged at the N-terminus) and gel filtration. We could positive bacteria and the mechanistic details are only beginning determine that the ensemble produced is the phage p2 baseplate, to be unraveled. formed by ORFs 15, 16 and 18. ORF17 could not be detected in In contrast, the proteinacious baseplates are common in phages of the family (with contractile tail) and that Author contributions: C.C. designed research; G.S., C.B., P.B., D.T., M.O.-L., J.L., M.v.H., V.C., of coliphage T4 has been extensively studied. T4 baseplate is a S.M., and C.C. performed research; G.S., C.B., P.B., M.O.-L., M.v.H., V.C., S.M., and C.C. remarkable nano-machine able to perform movements of several analyzed data; and S.M. and C.C. wrote the paper. hundreds of Å (10, 11). These conformational changes trigger the The authors declare no conflict of interest. tail contraction, leading to the ejection of the DNA from the *This Direct Submission article had a prearranged editor. capsid through the tail tube into the host (12). This organelle 1To whom correspondence should be addressed. E-mail: [email protected]. Escherichia coli is much larger in myophage T4 than in lacto- This article contains supporting information online at www.pnas.org/cgi/content/full/ coccal phages. Moreover, such large movements of baseplate 1000232107/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1000232107 PNAS Early Edition ∣ 1of6 Downloaded by guest on September 23, 2021 this ensemble, which is in agreement with its absence in virion secondary structure matching when using this domain was the particles. PDB entry 3eaa, a Type-VI secretion system (T6SS) protein Crystals of the isolated phage p2 baseplate were obtained but (EvpC) from the enterobacteria Edwardsiella tarda. A long showed no or poor diffraction (∼20 Å). We therefore mixed the kinked extension (the “belt”) of four β-strands embraces the next p2 baseplate with an excess of the camelid antibody heavy-chain ORF15 molecule in the hexameric ring (Fig. 1 G and H and variable fragment (VHH5), which has been previously shown to Fig. S4A). The N-terminal domains form a tight ring with two bind ORF18/RBP and to inhibit phage adsorption (6, 16, 20). We layers of β-strands. This ring is ∼35 Å high and frames a channel obtained rhombohedric crystals and collected several datasets of 40 Å diameter, largely sufficient for dsDNA passage. Of note, eventually reaching 2.60 Å resolution. The structure was solved the enterobacterial T6SS protein EvpC forms hexamers very si- by molecular replacement using a template model built from milar to those formed by ORF15 of lactococcal phage p2. The C- the ORF18/RBP trimeric structure and its complex with terminal domains (residues 137–275) are located at the ring per- VHH5 (16) (Table 1). iphery and do not contact each other (Fig. 1G). They display a The baseplate-VHH5 (BP-VHH) structure is 230 Å wide and galectin fold (DALI Z score ¼ 6.0), except for a long extension 160 Å high, displays a quasi hexagonal symmetry, and from (the “arm,” residues 147–188) which plays a critical role in the bottom to top is formed of three ORF16, six ORF15, and six tri- baseplate assembly (Fig. 1H and Fig. S4B). The arm extremity mers of ORF18, as well as 18 VHH5 (Fig. 1 A–E and Fig. S2). forms a three-digit hand that grips the N-terminal domain of Strict hexameric symmetry is not observed because ORF16 is tri- the RBP (ORF18, see below). meric. Furthermore, the symmetry of ORF16 disturbs the hex- The structure of ORF18/RBP (264 amino acids) was described americ assemblies of ORF15 and ORF18. Notwithstanding, previously (14, 16). As indicated above, ORF18 is a trimer hexameric or quasihexameric symmetry seems to be a conserved (Fig. 1J) with three domains: an N-terminal β-sandwich domain feature of the baseplate of many lactococcal phages (7, 9). (the “shoulder” residues 1– 141), a central domain (the “neck” residues 142–163) forming an interlaced β-helix, and a C-terminal Structure of the Baseplate Components and Their Assembly. ORF16 double greek-key domain (the “head” residues 164–264) harbor- is a four domain protein (Fig. 1I and Fig. S3A) of 398 residues ing the receptor-binding site. The shoulder domain is relatively that adopts a fold comparable to that of gp27 of myophage T4, well conserved among RBPs of other lactococcal phages of the the latter being located within the central part of the baseplate 936 group, whereas the neck and head domains are significantly (12) [DALI (21) Z score ¼ 11.3; Fig. S3B]. In contrast with gp27, diverse. Contrary to the structures of ORF18 crystallized alone, the trimeric association ORF16 forms a dome at the terminus of the N-terminal residues 2–17 of ORF18 in the baseplate structure the baseplate of the siphophage p2, thereby closing its central are ordered and visible in the density. This ordering of the N-ter- channel (Fig. 1F). minus is due to a tight interaction with the three-digit hand from ORF15 is composed of two domains. The N-terminal domain the ORF15 galectin domain (Fig. S5). Furthermore, the most N- (“ring domain” 1–132) shows a split barrel-like fold similar to that terminal residues of ORF18 (residues 2–7) protrude from each found in FMN-binding proteins (SCOP:50475; DALI Z score ¼ subunit, forming the first strand of the shoulder domain of the 6.6). The most similar Protein Data Bank (PDB) entry found by next subunit (Fig. 1J). Finally, each ORF18 trimer is coordinated

Fig. 1. Crystal structure of the phage p2 baseplate and its components. (A) View of the baseplate surface; ORF15 is in green, ORF16 in red, and ORF18 in blue. The blue arrow indicates the position of the quasi 6-fold axis and points toward the rest of the phage tail and the capsid. (B) The baseplate has been rotated by 90° around the horizontal axis. The central channel formed by ORF15 hexamer is closed by the ORF16 trimeric dome. (C) The arrangement of ORF18 as six trimers. (D) Hexameric ORF15. (E) Trimeric ORF16. (F) View of ORF16 trimer rotated 180° relative to baseplate (B) with a different color for each subunit. (G) ORF15 hexamer is viewed in the same orientation as in (B). Each subsubunit, as well as the N- and C-terminus domains, have a different color. The central channel is ∼40 Å wide. (H) Ribbon view of ORF15 subunit. The N- and C-terminus domains have their β-strands colored dark and light blue, respectively; helices are colored red and violet, respectively; and coiled sections are gray. The striking features of the structure, the arm (which helps hexamerization) and the extension (which grips ORF18 trimer) have been identified. Note the closure of the surface at the center of the trimer. (I) Ribbon view of ORF16 subunit. The four domains have been identified by D 1 to 4 and different colors of the β-strands. Domain 4 is only helical. These domains correspond to those identified in gp27 from phage T4 (12). (J) Ribbon view of the receptor-binding protein ORF18 trimer, with a different color for each chain. The trimer domains of shoulders, neck, and head are represented. The VHHs are displayed in Fig. S2.

2of6 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1000232107 Sciara et al. Downloaded by guest on September 23, 2021 by three VHH5s. The 18 VHH5 molecules together with the head mined from the crystals formed after degradation of the domains of ORF18 build a large layer assembled through tight ORF15/ORF16 complex did not display the ORF16 protein-protein contacts, which likely led to better diffracting trimeric assembly observed in the baseplate, but was instead crystals. monomeric. 2 All in all, a total surface area (22) of 156; 000 Å is buried in Despite their large interaction surfaces, neither ORF15 nor the X-ray structure of the baseplate (excluding the VHHs), ORF16 were able to oligomerize on their own, and complexes 2 compared to a total solvent-accessible surface of 285; 000 Å . of ORF15/ORF16 with “native” stoichiometry were formed only A large part of this buried surface, however, is involved in upon coexpression (19). It is likely that one molecule of ORF16 100; 000 2 ORF18 trimerisation ( Å ) and therefore does not con- has to assemble to two molecules of ORF15 as an initial building tribute directly to the baseplate stability. Each ORF15 contacts block that will further trimerize to form the inner core of the two ORF18s and two ORF16s. Notably, there are no contacts baseplate. ORF18 would then be added to this core. It is between ORF16 and ORF18. Therefore, the ORF15 hexamer noteworthy that a freshly prepared ORF15/ORF16 complex plays the role of a central hub to which ORF16 and ORF18 are was capable to capture added ORF18 and to form a “native” attached (Fig. S5). baseplate, as assessed by gel filtration. One of the striking features of phage p2 baseplate is the grasp of the ORF15 hand on the ORF18 shoulder domain (Fig. S5 A E Structure of the p2 Baseplate by Electron Microscopy. We have inves- and ). To adapt to the trimeric ORF18, the ORF15 hand adopts tigated the structure of the phage p2 virion by negative contrast a quasi 3-fold symmetry. A bundle of six residues from ORF15 electron microscopy. We report here the baseplate structure as establishes strong hydrogen bonds with the three Asp23 residues seen in the virion (Fig. S6) and compare it with the expressed from the ORF18 trimer: Lys158 and Tyr178 with Asp23h, Tyr 160 A baseplate X-ray structure. The baseplate structure was easily re- and 168 with Asp23i, and Tyr 170 and 176 with Asp23g (Fig. S5 A B E cognizable at the end of the tail (Fig. 2 and ). Its overall size and ). Besides this strong and remarkable architecture, other ∼220 hydrogen bonds are established at the periphery between was Å wide by 180 Å high. We were able to fit as a block our main-chain atoms or main-chain/side-chain atoms. X-ray structure, without the VHHs into the electron-microscopy Other useful information about the assembly of the p2 base- (EM) structure using CHIMERA (23) and UROX (24). ORF15 plate can be derived from the expression pattern of these pro- and ORF16 could be fitted readily together, while small devia- teins. We also cloned orf15 and orf16 in tandem and purified tions appeared in the orientation of ORF18. We decided there- their complex readily. It is formed of six ORF15s and three fore to fit each of them separately. The fit was excellent and all BIOPHYSICS AND

ORF16s, as seen by multiangle light scattering/UV/refractive in- the details of the structure were clearly visible, such as the posi- COMPUTATIONAL BIOLOGY dex (MALS/UV/RI) (9), in agreement with the complete base- tion of the galectin domains and extensions, as well as the con- plate structure. However, ORF15 degraded with time, leading tacts between ORF15 and ORF18 (Fig. 2C and Table 2). to smaller fragments. Consistent with this observation, crystalli- However, a large electron density lump was left empty above zation assays on ORF15/ORF16 complex samples yielded crystals ORF15 and below the last disk of the tail (Fig. 2C). This density of ORF16 alone. To our surprise, the ORF16 structure deter- consisted of two superposed rings of different diameters, the

Fig. 2. EM structure of the baseplate of phage p2 and results of the fitting of the baseplate crystal structure. (A) EM micrograph of the virulent lactococcal phage p2. Overall EM structure of phage p2 (right) showing the capsid (blue, top), the tail, formed of rings of the major tail protein hexamers (gold), and the globular baseplate (gold, bottom). ( B) View of the EM structure of the baseplate with the first major tail protein (MTP) hexamer. The RBP positions are identified by blue arrows or a blue dot. The RBP head is pointing upwards. (C) View of the crystal structure of the baseplate fitted in the EM map. ORF15 is green, ORF16 red, and ORF18 blue. Each RBP, ORF15 hexamer, and ORF16 trimer have been fitted as rigid blocks. (D), (E), (F) Views of the cryoEM map of the expressed p2 baseplate, from side (D), up (E), and down (F), respectively. (G) Sliced view showing the fitting of a second ORF15 hexamer (brown) in the EM map, 180° rotated relative to that in the crystal structure. The ring formed by the N-terminus has been fitted as a block, and the rotated and translated galectin domains have been fitted as a second block. Note the extension touching the RBP head domain (red arrows).

Sciara et al. PNAS Early Edition ∣ 3of6 Downloaded by guest on September 23, 2021 highest possessing six protrusions joining the ORF18 head domain. We hypothesized that another hexamer of ORF15 might fill this orphan density. To verify this hypothesis, we determined the cryoEM structure of the expressed baseplate. As expected, the baseplate possess the same two extra rings at its top, as seen in the phage structure (Fig. 2 D, E, and F and Fig. S7). ORF16 and ORF18 could not account for this density, since their shapes were not compatible with it, while the shape of ORF15 was found to be compatible with the EM density. The second hexamer of ORF15 was modeled in the EM maps (virion and expressed baseplate) by placing it back to back to the hexamer observed in the crystal structure (Fig. 2G, Fig. S7, and Table 2). The fit of the N-terminal ring was excellent, while the galectin domains and extensions were almost totally out of density. However, the shape of the density suggested that it could account for galectin domains and extensions from ORF15, pro- vided that they undertook a rigid-body rearrangement. Indeed, after rigid body fitting of each of the six ORF15 galectin domains, we could obtain a satisfactory structure with these domains fitted in the second orphan ring, and their corresponding arms and hands filling in the map extensions towards ORF18 (Fig. 2G and Table 2). In contrast with their position in the lower ring, the galectin domains of the second ORF15 ring model are in con- tact with each other and form a continuous volume. Their arm and hand extensions contact the head domain of ORF18. This contact is not made over the 3-fold axis, but laterally. This ar- rangement implies that the way these extensions interact with ORF18 is very different from that of the lower ORF15 exten- sions, a feature that we did not model at the atomic level. All these data taken altogether, the native baseplate of the virulent lactococcal siphophage p2 is composed of 2 × 6 ORF15s, 3 ORF16s, and 6 × 3 ORF18s (Fig. 2G).

An “Activated” Form of the p2 Baseplate. The structure of the base- plate reported above exhibited an unexpected conformation. In- deed, one would have expected the head domains of the RBPs (ORF18), which harbor the receptor-binding site, to point “downwards,” i.e. in the direction of the host cell surface as de- termined by the position of the tip protein ORF16. Instead, the RBPs were observed in a “heads-up” conformation. Additional explanations came from two other crystal forms of the baseplate of phage p2 that were obtained in the presence of Fig. 3. The crystal structure of the “heads-down” conformations of p2 2þ 2þ baseplate. (A) Side view in ribbon representation of the “heads-down” con- Ca (sg P21)orSr (sg C2) and in the absence of VHH5 (Ta- ble 1). Calcium has been shown to be essential for lactococcal formations of p2 baseplate. (B) View from top (ORF15, green; ORF16, pink; ORF18, blue). (C) Superposition of the rings formed by the N-terminal do- phages infection (25). The new structures were determined at mains of ORF15. ORF18 trimers have undergone a 200° rotation downwards. 5.5 and 3.9 Å resolution, respectively, by molecular replacement using successively ORF18, ORF15, and ORF16 as search models (Table 1). The two structures are identical, taking into account for dsDNA passage. This opening results from an outwards rota- their resolution. Because the structure obtained in the presence tion of ORF16 cores with respect to the channel axis, and the 2þ of Sr is better defined, it will be used further in the description opening of a crevice between domains 1, 2, 4, and 3 in an iris-like of this conformation of the baseplate. The structure is composed movement. Domain 3 remains in close contact with the next of six ORF15s, three ORF16s, and 3 × 6 ORF18s (Fig. 3 A ORF16 in the trimer (Movie S2). Noteworthy is the large contact and B). The most striking feature of this baseplate structure is area between ORF16s and ORF18s, a feature absent in the a “downwards” rotation of the ORF18 trimers by ∼200°, leading BP-VHH structure. In fact, these contacts lock the ORF18s in to a “heads-down” conformation (Fig. 3C). The ring formed by their “heads-down” conformation, giving to ORF16 the role the N-terminal domain of ORF15 superposed well in the two played by the VHH5 molecules in the BP-VHH complex or by structures (r.m.s. deviation of 0.52 Å), but the galectin, arm, the second ORF15 hexamer in the native virion EM structure. and hand domains had moved significantly (Fig. S8 and Movie S1). The Sr2þ ion (or Ca2þ ion) is located at the interface Discussion between the N-terminal and the galectin domains of ORF15 and Host recognition by bacterial viruses is often a two-step process is coordinated by side chains of residues Asn10, Asp12, Asn241, (26, 27). Coliphage T4 (Myoviridae) recognizes first its host Asp246 and main-chain carbonyl of Leu11. The hand domains saccharidic receptors via its long tail fibers. This first interaction have rotated so that they are oriented in opposite directions as induces a conformational change of the baseplate that exposes compared to the “heads-up” structure (Fig. S8 and Movie S1). the short tail fibers and ensures a definitive docking of the phage The ORF16 trimer was strongly affected, resulting in the opening onto the host cell wall (10). In parallel, the tail contracts and the of the dome with the concomitant formation of a channel of dsDNA is ejected. For noncontractile-tailed siphophages, such ∼32 Å diameter (Fig. 3B and Movies S1 and S2), large enough as E. coli T5, or Bacillus subtilis SPP1, saccharidic chains are

4of6 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1000232107 Sciara et al. Downloaded by guest on September 23, 2021 and to place it in a more favorable position for DNA ejection. Furthermore, this large movement might represent a firing signal, which, transmitted through the tail to the phage portal protein would trigger portal opening and dsDNA release and its passage through the tail and the open ORF16 trimer in the baseplate. Although functional similarities have been underlined between coliphage T4 and lactococcal phage p2, the Myoviridae baseplate is notably more complex than the one found in Siphoviridae. Aside from their distinct tail morphology and some structural si- milarity in some of their components, they have completely dif- ferent mechanisms for cell-wall puncturing and DNA ejection. The above-mentioned similarities should therefore be the result of convergent evolution towards an efficient system of adsorp- Fig. 4. Schematic representation of the putative adsorption mechanism of tion, instead of a reminiscence of a common ancient adsorption phage p2 to its host. The rest form (left) of the baseplate is activated by Ca2þ mechanism. cations and by the traction of lipoteichoic acids bound to a few receptor- binding sites which destabilize the RBPs. They subsequently rotate by Materials and Methods 200° and become available for a complete binding. Concomitantly, the tip Purification and Crystallization. The baseplate orfs 15–18 of the lactococcal of the baseplate opens, giving way to dsDNA. phage p2 were cloned into the Gateway pDEST147 vector (His6-tag at N-terminal of ORF15), overexpressed in E. coli and purified by Ni affinity column and gel filtration (19). Crystallization and structure determination recognized nonspecifically by phage components in a first rever- are reported in Table 1 and SI Text. sible step, followed by the irreversible specific docking of the tail fiber protein onto the host receptor, the FhuA porin (4) or the X-Ray Structure Determination and Refinement. Several datasets were extramembrane domain of YueB (28), respectively. In contrast collected at Swiss Light Source (SLS) (Villigen, Switzerland), Soleil (Saint Au- with the latter phages, and despite using several different strate- bin, France), and European Synchrotron Radiation Facility (ESRF) (Grenoble, gies, no proteinaceous receptor has been identified for the lacto- France). Data were integrated and reduced with XDS and SCALA (29). The coccal phages belonging to the 936 group. structures were solved by molecular replacement using an ORF18 trimer BIOPHYSICS AND The conformational difference observed with the ORF18/RBP (RBP) as a search model with the program PHASER (29). Model building was performed with COOT (30), and refinement was begun with COMPUTATIONAL BIOLOGY in our two crystal forms of the baseplate suggests also that, as for REFMAC5 (31) and completed with AutoBuster v 1.7.2 (32). Data are pre- phage T4, host recognition could be based on a two-step mecha- sented in Table 1 and SI Text. nism. When free in the environment, lactococcal phage p2 has its “ ” “ RBPs in a heads-up conformation, forming the baseplate rest Electron Microscopy. Virion structure. Samples were adsorbed onto a freshly form.” In this conformation, the RBP trimers could still recognize glow-discharged carbon-coated grid and stained with 2% uranyl acetate. saccharidic receptors laterally, but with less affinity, because they About 1,000 CCD images were recorded on a CM200 operating at 200 kV use a limited number of their binding sites. This recognition and 38 K magnification. The digitized images were then coarsened by 2 × might be sufficient to destabilize the baseplate and provoke 2 pixel averaging, thereby resulting in a pixel size of 2.32 Å/pixel (Fig the RBPs rotation (Fig. 4). In this context, it is noteworthy that .S6A). Images were collected at different defoci in the range of 500– due to the presence of numerous phosphate groups, LTAs are 1,500 nm (Fig. S8A) and contrast transfer function (CTF) corrected using IMA- well known as “Ca2þ sponges” (25). Thus, it is possible that GIC-5 FINDCTF2D program. Approximately 10,000 particles were manually selected from CTF-cor- the binding of the first LTA molecules to the RBPs may help 2þ rected images and processed using IMAGIC-5 (33). The particles were wind- the release of Ca cations from the phosphate groups, which 100 × 100 “ owed into pixels, band-pass filtered, and subjected to multivariate in turn might facilitate the conformational change to the acti- statistical analysis (34) and classified with approximately 10 images per class. vated form” of the base plate. In a subsequent step, the 18 recep- A class average was selected and aligned to have the 6-fold axis aligned to tor-binding sites of the “heads-down” activated form would the z axis. An initial three-dimensional model was calculated from the become available to firmly anchor the phage to the receptors aligned class averages imposing 6-fold symmetry. The final model was

Table 1. Data collection and refinement statistics Data collection Form 1 (rest) Form 2 (active) Form 3 (active) Molecular content (asymmetric unit) 1∕3 baseplate 2 baseplates 1 baseplate Space group R 3 2 (H 3 2) P21 C2 Cell dimensions a,b,c (Å), ß(°) 202.9,202.9, 760.5 219.5,219.3, 392.4 β ¼ 90° 300.3,239.5, 274.8, β ¼ 124.4° Beam line, wavelength (Å) ESRF, ID23-1, 0.9708 Soleil, Proxima 1, 0.980 Soleil, Proxima 1, 0.980 Resolution (Å) 48.7–2.6 (2.7–2.6) 44.6–5.5 (5.8–5.5) 120–3.9 (4.1–3.9) R sym (last shell) 0.130 (0.476) 0.073 (0.458) 0.160 (0.483) Mean I∕σI 8.1 (2.6) 12.3 (2.8) 5.4 (2.3) Completeness (%) 99.3 (99.9) 91.4 (87.2) 97.0 (97.5) Redundancy 5.0 (5.0) 4.2 (3.9) 3.0 (3.0) No. of reflections 184,640 112,351 (15,601) 141,413 Refinement Resolution (Å) 38.1–2.60 (2.67–2.60) 44.56–5.46 (5.66–5.46) 39.31–3.90 (4.00–3.90) No. of reflections 183,300 (13268) 112,191 (9789) 141,150 (10511) R R work; free 17.8; 20.9 (25.9; 29.2) 29.1; 29.7 (34.7; 35.9) 22.9; 24.2 (25.3; 26.0) No. of protein atoms/water 25,638/2009 119,226/6 Ca 59,613/6 Sr 2 B-factors protein (Å ) 67.7* 205 80.0* R.m.s. deviations Bonds (Å)/angles (°) 0.009/1.15 0.01/1.67 0.007/1.03 Last shell data are in parenthesis. *Residual B-factor after TLS refinement.

Sciara et al. PNAS Early Edition ∣ 5of6 Downloaded by guest on September 23, 2021 Table 2. Percentage of atoms and correlation coefficient resulting from the fitting of molecules or ensembles of the p2 baseplate in the EM map of the virion Molecule or complex Atoms Fit % atoms Correlation coeff. (20 Å) ORF15 hexamer lower (all) 14,554 13,454 92.5 0.52 ORF16 trimer 9,069 8,595 94.8 0.70 ORF18 monomer * 6,024 5,783 96.0 0.84 ORF15 hexamer upper C-term † 8,034 7,883 90.1 0.71 *Fitting has been done for the six monomers, yielding close values. †Fitting is the best in this instance, because each galectin domain has been preliminarily positioned manually in the EM map to account for their conformational change.

calculated to a resolution of 22 Å determined by Fourier shell correlation respectively. The virion baseplate EM map has been deposited at Electron with the ½ bit correlation criterion (Fig. S6C) (35). Microscopy Data Bank, with accession code EMD-1699. The expressed base- plate EM map has been deposited at EMDB, with accession code EMD-1706. CryoEM of the expressed baseplate. Expressed p2 baseplates were cross- linked by glutaraldehyde, followed by freezing in liquid ethane, and imaged ACKNOWLEDGMENTS. We would like to thank Adeline Goulet for help with using a JEOL 2200 FS electron microscope. Micrographs were digitized using a the cryoEM studies. We would like to thank the synchrotrons ESRF, Soleil, Nikon Coolscan 9000 ED; 10,732 individual views of baseplates were and SLS for providing us with beam time access. This work was supported, semiautomatically extracted and submitted to image processing using in part, by grants from the Marseille-Nice Génopole (to J.L.), the Centre IMAGIC V package (Fig. S7). National de la Recherche Scientifique, the Natural Sciences and Engineering Research Council of Canada (to S.M.), the novel alternatives to antibiotics Models fitting. All models fittings in the EM maps were done in two steps, research initiative of the Canadian Institutes of Health Research (to S.M.), with Chimera (23) followed by UROX (24). and the Agence Nationale de la Recherche (Grant ANR-07-BLAN-0095, “siphophages”). Molecular graphics images were produced using the Univer- sity of California, San Francisco (UCSF) Chimera package from the Resource Accession Codes. The genome sequence of phage p2 has been deposited at for Biocomputing, Visualization, and Informatics at the University of GenBank (GQ979703). The structures of the baseplate in complex with VHH 2þ 2þ California, San Francisco (supported by National Institutes of Health P41 form and the Ca and Sr activated forms have been submitted to the RR-01081). Protein Data Bank with accession codes 2WZP, 2X54 þ 2X5A, and 2X53,

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