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• W2907623449 abstract "Article2 January 2019free access Transparent process Structure and transformation of bacteriophage A511 baseplate and tail upon infection of Listeria cells Ricardo C Guerrero-Ferreira orcid.org/0000-0002-3664-8277 Laboratory of Structural Biology and Biophysics, School of Basic Sciences, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland Search for more papers by this author Mario Hupfeld Institute of Food, Nutrition and Health, ETH Zurich, Zurich, Switzerland Search for more papers by this author Sergey Nazarov Laboratory of Structural Biology and Biophysics, School of Basic Sciences, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland Search for more papers by this author Nicholas MI Taylor orcid.org/0000-0003-0761-4921 Laboratory of Structural Biology and Biophysics, School of Basic Sciences, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland Search for more papers by this author Mikhail M Shneider orcid.org/0000-0002-3768-3541 Laboratory of Structural Biology and Biophysics, School of Basic Sciences, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Laboratory of Molecular Bioengineering, Moscow, Russia Search for more papers by this author Jagan M Obbineni Laboratory of Biomolecular Research, Paul Scherrer Institute, Villigen PSI, Switzerland Centre for Agricultural Innovations and Advanced Learning (VAIAL), Vellore Institute of Technology, Vellore, India Search for more papers by this author Martin J Loessner Institute of Food, Nutrition and Health, ETH Zurich, Zurich, Switzerland Search for more papers by this author Takashi Ishikawa Laboratory of Biomolecular Research, Paul Scherrer Institute, Villigen PSI, Switzerland Search for more papers by this author Jochen Klumpp Corresponding Author [email protected] orcid.org/0000-0003-3410-2702 Institute of Food, Nutrition and Health, ETH Zurich, Zurich, Switzerland Search for more papers by this author Petr G Leiman Corresponding Author [email protected] orcid.org/0000-0002-9091-0918 Laboratory of Structural Biology and Biophysics, School of Basic Sciences, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland Search for more papers by this author Ricardo C Guerrero-Ferreira orcid.org/0000-0002-3664-8277 Laboratory of Structural Biology and Biophysics, School of Basic Sciences, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland Search for more papers by this author Mario Hupfeld Institute of Food, Nutrition and Health, ETH Zurich, Zurich, Switzerland Search for more papers by this author Sergey Nazarov Laboratory of Structural Biology and Biophysics, School of Basic Sciences, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland Search for more papers by this author Nicholas MI Taylor orcid.org/0000-0003-0761-4921 Laboratory of Structural Biology and Biophysics, School of Basic Sciences, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland Search for more papers by this author Mikhail M Shneider orcid.org/0000-0002-3768-3541 Laboratory of Structural Biology and Biophysics, School of Basic Sciences, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Laboratory of Molecular Bioengineering, Moscow, Russia Search for more papers by this author Jagan M Obbineni Laboratory of Biomolecular Research, Paul Scherrer Institute, Villigen PSI, Switzerland Centre for Agricultural Innovations and Advanced Learning (VAIAL), Vellore Institute of Technology, Vellore, India Search for more papers by this author Martin J Loessner Institute of Food, Nutrition and Health, ETH Zurich, Zurich, Switzerland Search for more papers by this author Takashi Ishikawa Laboratory of Biomolecular Research, Paul Scherrer Institute, Villigen PSI, Switzerland Search for more papers by this author Jochen Klumpp Corresponding Author [email protected] orcid.org/0000-0003-3410-2702 Institute of Food, Nutrition and Health, ETH Zurich, Zurich, Switzerland Search for more papers by this author Petr G Leiman Corresponding Author [email protected] orcid.org/0000-0002-9091-0918 Laboratory of Structural Biology and Biophysics, School of Basic Sciences, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland Search for more papers by this author Author Information Ricardo C Guerrero-Ferreira1,†, Mario Hupfeld2, Sergey Nazarov1,†, Nicholas MI Taylor1,†, Mikhail M Shneider1,3, Jagan M Obbineni4,5, Martin J Loessner2, Takashi Ishikawa4, Jochen Klumpp *,2 and Petr G Leiman *,1,† 1Laboratory of Structural Biology and Biophysics, School of Basic Sciences, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland 2Institute of Food, Nutrition and Health, ETH Zurich, Zurich, Switzerland 3Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Laboratory of Molecular Bioengineering, Moscow, Russia 4Laboratory of Biomolecular Research, Paul Scherrer Institute, Villigen PSI, Switzerland 5Centre for Agricultural Innovations and Advanced Learning (VAIAL), Vellore Institute of Technology, Vellore, India †Present address: Center for Cellular Imaging and NanoAnalytics (C-CINA), Biozentrum University of Basel, Basel, Switzerland †Present address: Focal Area Infection Biology, Biozentrum University of Basel, Basel, Switzerland †Present address: Structural Biology of Molecular Machines Group, Protein Structure & Function Programme, Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark †Present address: Department of Biochemistry and Molecular Biology, Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch, Galveston, TX, USA *Corresponding author. Tel: +41 446323855; E-mail: [email protected] *Corresponding author. Tel: +1 832 908 6635; E-mail: [email protected] EMBO J (2019)38:e99455https://doi.org/10.15252/embj.201899455 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Contractile injection systems (bacteriophage tails, type VI secretions system, R-type pyocins, etc.) utilize a rigid tube/contractile sheath assembly for breaching the envelope of bacterial and eukaryotic cells. Among contractile injection systems, bacteriophages that infect Gram-positive bacteria represent the least understood members. Here, we describe the structure of Listeria bacteriophage A511 tail in its pre- and post-host attachment states (extended and contracted, respectively) using cryo-electron microscopy, cryo-electron tomography, and X-ray crystallography. We show that the structure of the tube-baseplate complex of A511 is similar to that of phage T4, but the A511 baseplate is decorated with different receptor-binding proteins, which undergo a large structural transformation upon host attachment and switch the symmetry of the baseplate-tail fiber assembly from threefold to sixfold. For the first time under native conditions, we show that contraction of the phage tail sheath assembly starts at the baseplate and propagates through the sheath in a domino-like motion. Synopsis The attachment and host envelope penetration mechanism of bacteriophages that employ a rigid tube/contractile sheath complex for infection of Gram-positive bacteria is poorly understood. This study describes the structure of the Listeria phage A511 contractile tail in the pre- and post-host attachment state. The A511 baseplate-tail fiber complex undergoes a massive conformational change and switches from threefold to sixfold symmetry upon attachment to the host cell. The distal tail fiber protein gp108 attaches to the host cell wall before the sheath contracts. The proximal part of the tail fiber carries two pyramids that are formed by gp106 trimers. The gp106 pyramids reorient to point toward the cell surface, change their conformation to protrude attachment domain, and bind to the cell wall. Contraction of the phage tail sheath assembly starts at the baseplate and propagates through the sheath in a wave-like motion. Introduction A511 is a lytic bacteriophage that infects almost all strains of Listeria monocytogenes and L. ivanovii (Zink & Loessner, 1992; Klumpp et al, 2008; Habann et al, 2014). It belongs to the subfamily Spounavirinae which also includes phages SPO1 (Stewart et al, 2009), Twort (Kwan et al, 2005), ISP (Vandersteegen et al, 2011), LP65 (Chibani-Chennoufi et al, 2004), and many others (Klumpp et al, 2010). These viruses have a similar genome organization, and despite their host diversity (Bacillus, Staphylococcus, Lactobacillus, Enterococcus, and other Gram-positive bacteria), their virion proteins display high similarity at the amino acid level (e.g., the capsid proteins have greater than 60% sequence identity; Habann et al, 2014). Many of these viruses are used as tools in biotechnology, e.g., bacterial typing (Loessner, 1991; van der Mee-Marquet et al, 1997), detection (Loessner et al, 1996; Hagens et al, 2011), and elimination (Guenther et al, 2009), as well as in medical applications, e.g., elimination of staphylococcal infections (Vandersteegen et al, 2011; Kazmierczak et al, 2014). However, the mechanism by which these viruses recognize and attach to their host cells remains poorly understood. Bioinformatic analysis shows that tail assembly proteins of A511 are encoded by a continuous cluster of genes 93 through 109 (Habann et al, 2014). The baseplate and tail fiber/receptor-binding protein genes are downstream from the tail sheath and tail tube genes 93 and 94, respectively. In this aspect, the organization of the A511 tail gene cluster and the amino acid sequences of its tail proteins are both similar to the so-called “simple” contractile tail phages, e.g., Mu, P2, in which the baseplate is composed of small proteins and the corresponding genes display synteny (Buttner et al, 2016). The A511 baseplate undergoes a major structural transformation upon sheath contraction (Habann et al, 2014) although the details of this process remain unclear. Recently, Novacek et al (2016) reported a cryo-electron microscopy (cryoEM) study of the structure of phage phi812, an A511 relative that infects Staphylococcus aureus. However, the baseplate cryoEM map was of insufficient quality to identify the location of component proteins. Here, we show that the baseplate-tail fiber complex of A511 is threefold symmetric in the extended state of the tail and switches to be nearly sixfold symmetric in the contracted state. We assign electron densities to all baseplate proteins and visualize the transformation of the baseplate upon host cell binding. We also report the crystal structure of A511 gene product 105 (gp105), which greatly helped with the interpretation of the cryoEM density. Finally, we captured the A511 particle in a partially contracted state with the baseplate attached to the Listeria cell wall, the baseplate-proximal part of the sheath in the contracted state and the distal part still in the extended state. This observation demonstrates that host cell-binding associated changes in the baseplate structure trigger sheath contraction, which then propagates through the sheath as a wave. Several elements of this complex process were observed earlier using phage particles in which the contraction was triggered by a non-physiological treatment of the sample (urea, high temperature, low pH, etc.) which was then fixed and stained with heavy atom salts, but to our knowledge no other report showed the process in its entirety and in near-native conditions for any contractile tail bacteriophage (Eiserling, 1967; Simon & Anderson, 1967a,b; Donelli et al, 1972; Benz & Goldberg, 1973; Moody, 1973). Results and Discussion CryoEM photographs of freshly prepared A511 sample showed that about 13% of particles had contracted tails (Table 1, Fig EV1A) indicating that the A511 tail is a metastable structure that can spontaneously contract resulting in a post-host cell attachment conformation, a property it shares with other contractile systems (Leiman & Shneider, 2012; Brackmann et al, 2018). Borrowing from the T4 phage system (Leiman et al, 2004; Fokine et al, 2013), we used urea to switch all particles to the contracted tail state. We then employed the single particle cryoEM image reconstruction procedure (Cheng et al, 2015) to analyze the structure of the baseplate and the baseplate-proximal part of the A511 sheath in the pre-host attachment extended state (Fig 1A, D and F), in the post-host attachment spontaneously contracted state (Fig 1B, E and H), and in the urea-induced contracted state (Fig 1C, F and I). The corresponding cryoEM maps have resolutions of 14 Å, 16 Å, and 14 Å, respectively (Table 1). We have also studied the transformation of the structure of the A511 particle upon binding to Listeria cell wall surface with the help of cryo-electron tomography (cryoET). Table 1. Data collection and processing statistics for single particle image reconstruction of A511 baseplate Extended tails (Pre-attachment) Spontaneously contracted tails (Post-attachment) Urea-contracted tails (Post-attachment) Number of initial micrographs 3,975 2,221 Micrographs after CTF screening 3,127 2,221 Number of particles extracted 14,438 2,234 10,122 Particles after 2D alignment and classification 13,334 2,209 9,218 After 3D classification 11,331 1,853 9,218 Resolution after 3D refinement (Å) 16 18 16 Resolution after post-processing (Å) 14 16 14 EMDB accession code EMD-7560 EMD-7561 EMD-7559 Click here to expand this figure. Figure EV1. Structure and composition of the A511 particle A cryoEM micrograph taken with a defocus value of –2.5 μm and a magnification of 19,000× that shows a typical freshly purified A511 sample. Note the presence of particles with extended and contracted tails and the curvature of the extended tails. Scale bar: 100 nm. The dashed boxes in panels (A–D) show the region of the tail that was included in the image reconstruction procedure: (A, B) the extended sheath and pre-host attachment conformation of the baseplate; (C, D) the contracted sheath and post-host attachment conformation of the baseplate. Coomassie-stained sodium dodecyl sulfate–polyacrylamide electrophoresis of a freshly prepared and urea-contracted A511 sample. Download figure Download PowerPoint Figure 1. CryoEM reconstructions of the baseplate region of the A511 tail in the pre- and post-host cell attachment states A–C. CryoEM reconstructions of the extended, spontaneously contracted, and urea-contracted tails colored according to the distance to the axis of the reconstruction. The color code bar shown in panel (A) and which is given in Angstroms is the same for all the three panels. D–F. The central axial sections of the reconstructions. Note the C-terminal domain of gp104 and other low-density features that do not visualize well in the isosurface representation [panels (A–C)]. Letters G, H, and I indicate the position of the cross sections shown in the homonymously labeled panels below. G. A 69 Å-thick slice of the reconstruction of the extended sheath cryoEM map orthogonal to the sixfold axis. The proximal and distal parts of one of the three fibers that run at 82° to the sixfold axis are indicated with a semitransparent cyan and magenta lines, respectively. H, I. Sections of the spontaneously and urea-contracted sheath structures that cut through the baseplate/proximal part of the fiber plane, whose position in the reconstructions is indicated with a red line in panels (E and F). The putative direction of the distal part of the fiber, which is completely disordered in the reconstructions, is indicated with a solar cross symbol. Download figure Download PowerPoint The composition and structure of the A511 baseplate is similar to that of T4 The resolution of the A511 cryoEM maps did not allow for ab initio segmentation of the electron density into the component proteins. Bioinformatic analysis that employs structure-based hidden Markov's model (HMM) profiles (Table 2) showed that most A511 tail proteins or their domains have orthologs in the well-studied phage T4 for which the atomic structure of the tube-baseplate complex is available (Taylor et al, 2016). However, many T4 tail proteins are larger than their A511 counterparts and the use of the T4 baseplate structure in the interpretation of A511 cryoEM maps required removing some parts of the model. We trimmed the T4 tail tube-baseplate structure to leave the domains that have definitive orthologs in the A511 tail (Table 2). This structure matched the conserved “tube-baseplate core complex”, which was proposed to be present in most contractile injection systems and which was described in our earlier studies (Taylor et al, 2016, 2018). It consists of the tube protein (T4 gp19 and gp54), baseplate hub protein 1 (BH1, gp48), baseplate hub 2 (BH2, gp27), baseplate spike (BS, gp5), and four baseplate wedge proteins (BW1, BW2, BW3 and BW4—gp25, gp6, gp7, and gp53, respectively, although only some domains of gp6, gp7, and gp53 are conserved). Only a small part of T4 BW4 gp53 protein, namely, a LysM-like domain (Maxwell et al, 2013), is conserved in A511 (residues 4–41 of BW1 gp102 are predicted to form a LysM domain). The small size and lower degree of conservation make placement of the LysM domain of the BW1 gp102 protein into the 14 Å resolution A511 cryoEM map uncertain. We, therefore, removed this protein from the T4-based model of the universally conserved tube-baseplate core complex. Table 2. Putative functions and their acronyms of tail-associated genes of A511 Gene name Length of gene product, a.a. Function (acronym), additional functional details T4 or other phage ortholog PDB (with chain ID) and/or PFAM code of best match Residue range of A511 protein Residue range of best match E-value 93 562 Sheath Gp18 3HXL 1–545 1–427 2.8e-44 3LML 2–559 3–455 2.1e-43 3J9Q 12–540 3–359 7e-25 5LI2 1–562 1–587 4.0E-65 94 140 Tube Gp19, gp54, gp3 2GUJ 2–133 1–134 0.0066 4W64 8–132 4–133 1.9 95 147 Tape Measure Chaperone (TMC), putative Gp28 (putative) 96 197 Unknown, possible assembly chaperone (α-helical) Unknown 97 1,242 Tape Measure Protein (TMP) Gp29 COG3941 230–865 10–586 0.002 98 795 Baseplate Hub 2 (BH2) Gp27 3D37 1–586 1–337 0.0042 1WRU 38–516 16–267 0.0096 3CDD 18–555 4–310 0.024 Peptidoglycan endopeptidase (gp5 lysozyme domain) 4Q4G 624–794 320–472 7.8E-13 99 510 Baseplate Spike (BS) Gp5 2Z6B 26–256 4–205 0.028 4MTK 29–121 364–454 0.08 4RU3 27–106 30–104 0.078 100 237 Baseplate Hub 1 (BH1), tube basal disk Gp48 PF06995 22–150 1–120 0.51 COG3499 22–161 6–136 0.13 101 177 Unknown, possible assembly chaperone (α-helical) Unknown 102 236 Baseplate Wedge 1 (BW1), tail sheath-baseplate attachment Gp25 5IV5_DG 115–231 11–131 1.4E-13 Baseplate Wedge 4 (BW4), baseplate core bundle clamp Gp53 5IV5_V 4–41 45–82 0.97 103 348 Baseplate Wedge 2 (BW2), baseplate circularization Gp6 3H2T 187–323 1–151 1.1E-8 5IV5_A 1–276 21–436 3.1E-28 104 1,309 Baseplate Wedge 3 (BW3), baseplate trifurcation unit and tail fiber attachment Gp7 PF09684 30–169 6–136 6.4E-4 105 173 Baseplate Wedge 3 Tail Fiber Network (BW3TFN) component Gp8 5IV5_E 2–173 10–334 1.1E-8 106 1,151 Receptor-Binding Protein (RBP) or Tail Fiber (TF), previously called VrlC Phage 1358 RBP 4L99 41–110 11–92 78.0 PF16075 (DUF4815) 9–613 1–580 1.3E-128 107 73 Tail Fiber Assembly chaperone (TFA), putative Prefoldin beta subunit 2ZQM 1–66 49–110 0.21 108 430 Receptor-Binding Protein (RBP) or Tail Fiber (TF) Phage phi11 orf56 5EFV 38–283 438–644 8.5E-14 Zebrafish Caprin-2 C1q domain 4OUS 284–425 6–137 1.3E-12 109 136 Tail Fiber Assembly chaperone (TFA), putative PBSX protein XkdW 2HG7 16–131 6–101 0.0026 The conserved tube-baseplate core complex of T4 was then fitted as a rigid body into the A511 extended tail cryoEM map with the help of the automatic rigid body fit algorithms as implemented in the graphics programs Coot (Emsley et al, 2010) and UCSF Chimera (Pettersen et al, 2004) (Fig 2, Table 3). We also split this structure into two parts—the central tube/spike complex and the baseplate core—and fitted them separately with the help of the Coot and UCSF Chimera rigid body fit procedures. The resulting composite structure was essentially the same as the one comprising one rigid body (Table 3). This shows that neither of the two components dominates the overall fitting procedure and indirectly confirms the validity of such interpretation. Figure 2. Fitting of the pre-attachment T4 tail tube-baseplate core complex into the A511 extended tail cryoEM map A–F. Atomic structures of T4 proteins are shown as ribbon diagrams of various colors and labeled with T4 and A511 gene product names as well as with their functional orthologs common to all contractile-like systems (e.g., BH1, see Table 2). The putative endopeptidase domain of A511 gp98 is modeled by the crystal structure of RipA of Mycobacterium tuberculosis (colored purple). Red lines and labels in panel (A) indicate the position of end-on views shown in panels (B through F) (looking from the capsid). Download figure Download PowerPoint Table 3. Local correlation coefficients—as defined and calculated by the UCSF Chimera program (Pettersen et al, 2004)—characterizing the fit of crystal structures into cryoEM maps. UCSF Chimera was used to render the crystal structures as density maps by describing each atom as a Gaussian distribution with a width proportional to map resolution (see Table 1) and amplitude proportional to the atomic number. The values in parentheses are correlation coefficients that take into account only the strong features of the maps derived from atoms Protein Fit into the extended map Fit into the contracted map BW3TFN gp105 dimer 1 (baseplate proximal) 0.62 (0.94) 0.59 (0.89) BW3TFN gp105 dimer 2 (middle) 0.62 (0.94) 0.58 (0.90) BW3TFN gp105 dimer 3 (baseplate distal) 0.64 (0.93) 0.53 (0.87) Three gp105 dimers from the extended map fitted into the contracted map as a unit 0.63 (0.88) Tube-spike complex 0.59 (0.89) Conserved part of the wedge (six wedges as a unit) 0.56 (0.86) 0.44 (0.69) Tube-spike-wedges as a unit 0.62 (0.88) Phi11 RBP residues 437–537 (N-terminal bulge of RBP gp108) 0.77 (0.96) Phi11 RBP residues 550–635 (C-terminal bulge of RBP gp108) 0.67 (0.95) C1q trimer (C-terminal domain of RBP gp108) 0.53 (0.90) The A511 cryoEM map agrees with the conserved core part of the T4 baseplate-tube structure remarkably well (Table 3, e.g., Fig 2E) considering that the corresponding proteins display about 20% sequence identity at best. Notably, no adjustment was made to the position of individual components of the T4 baseplate-derived model that comprises 49 different polypeptide chains belonging to 8 different proteins in multiple copies and yet the T4 baseplate matched the A511 cryoEM density with a precision of individual α-helices or α-helical bundles as described below. The main component of the conserved core part of the T4 baseplate is the (gp6)2-gp7 heterotrimer, which consists of an α-helical core bundle and a trifurcation unit. These heterotrimers interact with each other via the C-terminal domains of two BW2 gp6 proteins that extend from the trifurcation unit in the opposite tangential directions and form dimers. The third (radial) extension of the trifurcation unit is formed by the BW3 gp7 protein and connects the unit to the tail fiber network. The BW1 gp25 protein is located at the tip of the core bundle and comprises an integral part of the sheath. All these proteins and domains have counterparts in the A511 cryoEM density—there are densities corresponding to the α-helical core bundle, to the trifurcation unit, to the dimeric domains connecting the neighboring trifurcation units, and to the BW1 baseplate-sheath connecting protein (Fig 2A, D, E and F). These features are therefore conserved in the A511 baseplate: The A511 BW1 protein gp102 links the baseplate with the sheath (Fig 2A and D); the BW2 and BW3 proteins gp103 and gp104 form the (gp103)2-gp104 trifurcation unit (Fig 2E); the neighboring units are connected to each other via dimerization of the C-terminal domain of the BW2 gp103 protein (Fig 2F); and finally, the BW3 gp104 protein is responsible for attachment of the tail fiber network (Fig 2A and F). The central spike complexes of A511 and T4 have similar overall architecture despite targeting different cell envelopes Bioinformatic analysis shows that the tail tube-spike complex of A511 is formed by the gp94 tube protein (T4 gp19 ortholog), gp100 baseplate hub 1 (BH1, T4 gp48 ortholog), gp98 baseplate hub 2 (BS2, T4 gp27 ortholog), and gp99 baseplate spike (BS, T4 gp5 ortholog) (Table 2). In addition to these proteins, the T4 tail tube-spike complex contains gp54 (which forms the first disk of the tube and whose structure is identical to that of the main tail tube protein gp19) and the baseplate spike tip (BST) protein gp5.4. The complete structure of the T4 tail tube-spike complex (gp19-gp54-gp48-gp27-gp5-gp5.4) accounts for most of the A511 tube-spike complex cryoEM density with certain differences in the region of the BH2-BS complex (Fig 2A–C). The cryoEM density shows that the A511 central spike BH2-BS complex (gp98–gp99) is more massive and has a larger diameter than its T4 ortholog (Fig 2A). The BH2 gp98 protein is twice as long as T4 BH2 gp27 (795 vs. 391 residues) and contains a large mostly α-helical domain whose function is unknown (residues 340–520) and an additional C-terminal domain (residues 624–794) with a predicted peptidoglycan hydrolase function. The cryoEM density shows two additional domains (per BH2 gp98 monomer) connected to the T4 BH2 gp27-like hub structure. The crystal structure of the peptidoglycan endopeptidase RipA of Mycobacterium tuberculosis (PDB code 4Q4G) (Squeglia et al, 2014; Fig 2A), which is predicted by HHpred to have a similar fold (Table 2; Soding et al, 2005; Alva et al, 2016), can be fitted into one of these extra densities. This domain is likely to lacerate the host murein layer during infection. Notably, the enzymatic activity associated with the central spike complex and targeting the cell wall is conserved in both T4 and A511. The function of the central spike complex is to create a passage through the cell envelope of the target bacterium to allow the tail tube to reach the plasma membrane during sheath contraction. T4 and A511 infect hosts with markedly different cell envelope organizations (Gram-negative E. coli and Gram-positive Listeria, respectively), so the similarities in the overall organization of their central spike complexes are rather remarkable. Not only are the lengths of the two nearly identical [the lack of the spike tip protein in A511 is compensated by a longer C-terminal domain of the gp99 spike (Fig 2A)], but the location of the peptidoglycan hydrolase domain in the complex is the same. While no enzymatic domain is predicted in the BS gp99 spike of A511, the site of the T4 BS spike protein gp5 lysozyme domain (Kanamaru et al, 2002) is occupied by the putative peptidase domain of A511 BH2 gp98 hub protein giving the BS-BH2 spike complexes of T4 and A511 nearly identical shape (Fig 2A). The high degree of structural conservation of T4 and A511 central spike complexes and the fact that Listeria does not contain an outer membrane suggest that the central spike or at least some part of it in phages of Gram-negative bacteria is translocated into the cytoplasm of the host cell. This is consistent with the function of the related T6SS organelle that is known to translocate the spike complex directly into the cytoplasm of bacterial cells (Vettiger & Basler, 2016). At the same time, the envelope-attacking part of the A511 BS gp99 spike (residues 328–510) is predicted to form a coiled coil structure whereas this domain is a triple-stranded β-helix in T4 gp5 (Browning et al, 2012), which is further capped by the gp5.4 BST protein. The coiled coil domain is conserved in BS spike proteins of all A511-like phages suggesting a functional requirement for the interaction with the cell envelope of Gram-positive bacteria. The A511 baseplate-tail fiber complex has threefold symmetry in the pre-host attachment state The baseplate and the tail fiber network structure of A511 are dominated by twelve large pyramid-shaped densities that are situated around the periphery of the baseplate above and below its plane (Fig 3). The pyramids display clear threefold symmetry that makes it possible to segment them out into separate entities. The pyramids are attached with their apexes to six fibers (two pyramids per fiber) in opposite orientations—with a base pointing roughly toward and away from the phage head (Figs 1A, D and G, and 3). The fibers form rays of a star-like structure: each fiber starts at the baseplate, extends ~260 Å away from it, makes a 157° turn, and returns to the baseplate (Fig 1G). Adjacent rays are not co-planar, and the planes that run through them form angles of 82° and 60° relative to the tail's axis, resulting in overall threefold symmetry (Fig 1D). The alternate fibers start and end at the same place at the periphery of the conserved core part of the baseplate. The structures of all the pyramids are very similar, although the distal pyramid of the 60° fiber is very disordered because it" @default.
• W2907623449 created "2019-01-11" @default.
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• W2907623449 date "2019-01-02" @default.
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• W2907623449 title "Structure and transformation of bacteriophage A511 baseplate and tail upon infection of <i>Listeria</i> cells" @default.
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• W2907623449 doi "https://doi.org/10.15252/embj.201899455" @default.
• W2907623449 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/6356063" @default.
• W2907623449 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/30606715" @default.
• W2907623449 hasPublicationYear "2019" @default.
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