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Structure of Lactococcal Phage P2 Baseplate and Its Mechanism of Activation

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Structure of Lactococcal Phage P2 Baseplate and Its Mechanism of Activation 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 Viruses, 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, Escherichia coli-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 ∣ bacteriophage 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 (Caudovirales 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 Myoviridae 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.
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