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Shigeki Takeda - One of the best experts on this subject based on the ideXlab platform.
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Expression and Characterization of a Baseplate Protein for BacterioPhage Mu, gp44
2020Co-Authors: Daisuke Kitazawa, Masashi Tomihara, Shigeki Takeda, Yasuhiro Kageyama, Harumi FukadaAbstract:The gene product of gene 44 of Mu Phage (gp44) is an essential protein for baseplate assembly and has been designated as gpP, a traditional genetic assignment. The function of gp44 during the assembly or infection process is not known. In the present study, we purified the recombinant gp44 and characterized it by analytical ultracentrifugation and differential scanning microcalorimetry. The results indicate that gp44 forms a trimer comprising a complex consisting of the 42 kDa and 40 kDa subunits that had been cleaved in the C-terminal region. Thermodynamic analysis also suggested that the C-terminal region forms a flexible domain
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observation of unexpected molecular binding activity for Mu Phage tail fibre chaperones
Journal of Biochemistry, 2019Co-Authors: Kohei Sakai, Atsushi Enomoto, Eiki Yamashita, Atsushi Nakagawa, Takuma Iwazaki, Fumiya Sakuraba, Minoru Inagaki, Shigeki TakedaAbstract:In the history of viral research, one of the important biological features of bacterioPhage Mu is the ability to expand its host range. For extending the host range, the Mu Phage encodes two alternate tail fibre genes. Classical amber Mutation experiments and genome sequence analysis of Mu Phage suggested that gene products (gp) of geneS (gpS = gp49) and gene S' (gpS' = gp52) are tail fibres and that gene products of geneU (gpU = gp50) and geneU' (gpU' = gp51) work for tail fibre assembly or tail fibre chaperones. Depending on the gene orientation, a pair of genes 49-50 or 52-51 is expressed for producing different tail fibres that enable Mu Phage to recognize different host cell surface. Since several fibrous proteins including some Phage tail fibres employ their specific chaperone to facilitate folding and prevent aggregation, we expected that gp50 or gp51 would be a specific chaperone for gp49 and gp52, respectively. However, heterologous overexpression results for gp49 or gp52 (tail fibre subunit) together with gp51 and gp50, respectively, were also effective in producing soluble Mu tail fibres. Moreover, we successfully purified non-native gp49-gp51 and gp52-gp50 complexes. These facts showed that gp50 and gp51 were fungible and functional for both gp49 and gp52 each other.
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three dimensional structures of bacterioPhage neck subunits are shared in podoviridae siphoviridae and myoviridae
Genes to Cells, 2018Co-Authors: Takuma Iwasaki, Masashi Tomihara, Atsushi Enomoto, Eiki Yamashita, Atsushi Nakagawa, Shigeki TakedaAbstract:: Tailed bacterioPhages (Caudovirales) are divided into three families: Myoviridae with long contractile tails, Siphoviridae with long noncontractile tails and Podoviridae with short noncontractile tails. All have an icosahedral head with a portal vertex connected to a neck structure followed by a tail. BacterioPhage Mu belongs to the Myoviridae family. Herein, the gp29 portal subunit and neck subunits gp35, gp36 and gp37 of the Mu Phage were purified to elucidate their arrangement in the neck. Both gp29 and gp36 were monomeric in solution, like the corresponding subunits of Podoviridae P22 and Siphoviridae SPP1. X-ray crystal structure of gp36 showed structural similarity to neck subunits of Siphoviridae and Podoviridae. The gp36 structure has a characteristic aromatic hydrophobic core, and the structure of the ring form of the Mu Phage connector deduced from the Siphoviridae and Podoviridae connector showed that this feature builds the contact surface between gp36 subunits. Structural comparison with the neck of Siphoviridae and Podoviridae also implies direct interaction between gp36 and gp29. Because gp35 and gp36 form a stable complex, we predict that the head-portal ring (gp29), the connector complex (gp36 and gp35), the tail terminator (gp37) and the tube (gp40) are arranged in the Mu Phage neck in this order.
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Crystal structure of the C-terminal domain of Mu Phage central spike and functions of bound calcium ion
Biochimica et biophysica acta, 2012Co-Authors: Kenichi Harada, Eiki Yamashita, Atsushi Nakagawa, Takamitsu Miyafusa, Kouhei Tsumoto, Takashi Ueno, Yoshiharu Toyama, Shigeki TakedaAbstract:Abstract BacterioPhage Mu, which has a contractile tail, is one of the most famous genus of Myoviridae. It has a wide host range and is thought to contribute to horizontal gene transfer. The Myoviridae infection process is initiated by adhesion to the host surface. The Phage then penetrates the host cell membrane using its tail to inject its genetic material into the host. In this penetration process, Myoviridae Phages are proposed to puncture the membrane of the host cell using a central spike located beneath its baseplate. The central spike of the Mu Phage is thought to be composed of gene 45 product (gp45), which has a significant sequence homology with the central spike of P2 Phage (gpV). We determined the crystal structure of shortened Mu gp45Δ1-91 (Arg92–Gln197) at 1.5 A resolution and showed that Mu gp45 is a needlelike structure that punctures the membrane. The apex of Mu gp45 and that of P2 gpV contained iron, chloride, and calcium ions. Although the C-terminal domain of Mu gp45 was sufficient for binding to the E. coli membrane, a Mutant D188A, in which the Asp amino acid residue that coordinates the calcium ion was replaced by Ala, did not exhibit a propensity to bind to the membrane. Therefore, we concluded that calcium ion played an important role in interaction with the host cell membrane.
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the c terminal domain is sufficient for host binding activity of the Mu Phage tail spike protein
Biochimica et Biophysica Acta, 2010Co-Authors: Hidetaka Suzuki, Yoshiharu Toyama, Seiko Yamada, Shigeki TakedaAbstract:The Mu Phage virion contains tail-spike proteins beneath the baseplate, which it uses to adsorb to the outer membrane of Escherichia coli during the infection process. The tail spikes are composed of gene product 45 (gp45), which contains 197 amino acid residues. In this study, we purified and characterized both the full-length and the C-terminal domains of recombinant gp45 to identify the functional and structural domains. Limited proteolysis resulted in a Ser64-Gln197 sequence, which was composed of a stable C-terminal domain. Analytical ultracentrifugation of the recombinant C-terminal domain (gp45-C) indicated that the molecular weight of gp45-C was about 58 kDa and formed a trimeric protomer in solution. Coprecipitation experiments and a quartz crystal microbalance (QCM) demonstrated that gp45-C irreversibly binds to the E. coli membrane. These results indicate that gp45 shows behaviors similar to tail-spike proteins of other Phages; however, gp45 did not show significant sequence homology with the other Phage tail-spike structures that have been identified.
Fidelma E Boyd - One of the best experts on this subject based on the ideXlab platform.
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crystal structure of vc1805 a conserved hypothetical protein from a vibrio cholerae pathogenicity island reveals homology to human p32
Proteins, 2008Co-Authors: Arif Sheikh, Jane A Potter, K A Johnson, Robert B Sim, Fidelma E Boyd, Garry L TaylorAbstract:The severe diarrhoeal disease cholera is caused by the gram-negative bacterium Vibrio cholerae and continues to be a major cause of morbidity and mortality. Like many Vibrio species V. cholerae inhabits an aquatic ecosystem, and most V. cholerae isolates do not possess the ability to cause cholera. Of more than 200 known serotypes, only O1 and O139 serogroups are highly pathogenic and acknowledged to cause epidemic disease.1 The O1 serogroup can be divided into two groups: classical and El Tor, with the first cholera pandemic, beginning in Asia in 1817, and the subsequent five pandemics probably caused by the classical biotype. The seventh and present pandemic began in 1961 caused by the El Tor biotype.2 In 1992 a novel O-serogroup, O-139, emerged to cause epidemic cholera.3 All V. cholerae O1 and O139 serogroups encode the major virulence factors cholera toxin and toxin coregulated pilus, the latter being encoded on a pathogenicity island, named Vibrio Pathogenicity Island-I (VPI-1).4 Several other genomic regions have been identified that occur mainly among epidemic O1 and O139 serogroup isolates, including the so-called Vibrio seventh pandemic island-I (VSP-I) encoding ORFs VC0175 to VC0185, VSP-II encoding ORFs VC0490 to VC0516 and VPI-2 encoding ORFs VC1758 to VC1809.5–7 A microarray analysis of V. cholerae El Tor isolates was used to identify VSP-I and VSP-II encompassing genes that are possibly responsible for the unique characteristics of the seventh pandemic (El Tor) strains.5 An evolutionary genetic analysis of clinical and environmental isolates suggested that pandemic strains arose from a common O1 serogroup progenitor through the successive acquisition of new virulence regions.8 VPI-2 is a 57.3 kb region which consists of 52 ORFs present in all toxigenic O1 and O139 serogroup isolates, but lacking in non-O1 and non-O139 nontoxigenic isolates.6 VPI-2 encodes a type-I restriction modification system, a nan-nag region of genes involved in sialic acid metabolism that may play a nutritional role,9 a sialidase/neuraminidase known to convert intestinal higher-order gangliosides to GM1,10 and a region with homology to Mu Phage. In addition, VPI-2 contains several genes that code for hypothetical proteins. One of these proteins, VC1805, is a 148 amino acid protein with no function revealed so far through sequence analysis. VC1805 is also encoded in the reduced 20 kb VPI-2 region present among most O139 serogroup isolates that are missing ORFs VC1761 to VC1788.6 A paralogue of VC1805 exists within VSP-II, VC0508 a 147 residue protein that shares 59% amino acid sequence identity with VC1805. A search of the sequence database using PSI-BLAST reveals homologues of VC1805 in several Vibrio species: V. vulnificus, V. splendidus, V. alginolyticus and V. fischeri, and orthologues in Altermonas macleodii, Aeromonas hydrophilia, and some Shewanella species. In addition, PSI-BLAST reveals that the adjacent hypothetical protein VC1804, with 104 residues, is a homologue of VC1805 sharing 26% sequence identity. Similarly the hypothetical protein VC0509 is a homologue of the adjacent protein VC0508. The four proteins VC0508, VC0509, VC1804, and VC1805 are therefore likely to share the same protein fold and be functionally related. As part of a structural genomics approach to understanding the function of hypothetical proteins within V. cholerae genomic islands we have determined the crystal structure of VC1805 to a resolution of 2.1 A using heavy atom isomorphous replacement. The structure reveals a similarity to the human mitochondrial protein p32 that is known to have several binding partners, including the human complement system protein C1q. This study shows that VC1805 does bind C1q, suggesting potential biological roles for the protein and its homologues.
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three pathogenicity islands of vibrio cholerae can excise from the chromosome and form circular intermediates
Journal of Bacteriology, 2008Co-Authors: R A Murphy, Fidelma E BoydAbstract:Vibrio pathogenicity island-2 (VPI-2) is a 57-kb region integrated at a transfer RNA (tRNA)-serine locus that encompasses VC1758 to VC1809 on the V. cholerae N16961 genome and is present in pandemic isolates. VPI-2 encodes a P4-like integrase, a restriction modification system, a Mu Phage-like region, and a sialic acid metabolism region, as well as neuraminidase (VC1784), which is a glycosylhydrolase known to release sialic acid from sialoglycoconjugates to unmask GM1 gangliosides, the receptor for cholera toxin. We examined the tRNA-serine locus among the sequenced V. cholerae genomes and identified five variant VPI-2 regions, four of which retained the sialometabolism region. Three variant VPI-2 regions contained a type three secretion system. By using an inverse nested PCR approach, we found that the VPI-2 region can form an extrachromosomal circular intermediate (CI) molecule after precise excision from its tRNA-serine attachment site. We constructed a knockout Mutant of VC1758 (int) with V. cholerae strain N16961 and found that no excision PCR product was produced, indicating that a functional cognate, VPI-2 integrase, is required for excision. The Vibrio seventh pandemic island-I (VSP-I) and VSP-II regions are present in V. cholerae O1 El Tor and O139 serogroup isolates. Novel regions are present at the VSP-I insertion site in strain MZO-3 and at the VSP-II insertion site in strain 623-39. VSP-II is a 27-kb region that integrates at a tRNA-methionine locus, is flanked by direct repeats, and encodes a P4-like integrase. We show that VSP-II can excise and form a CI and that the cognate VSP-II integrase is required for excision. Interestingly, VSP-I is not inserted at a tRNA locus and does encode a XerDC-like recombinase, but similar to VPI-2 and VSP-II, VSP-I does excise from the genome to form a CI. These results show that all three pathogenicity islands can excise from the chromosome, which is likely a first step in their horizontal transfer.
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molecular evolution of vibrio pathogenicity island 2 vpi 2 mosaic structure among vibrio cholerae and vibrio mimicus natural isolates
Microbiology, 2005Co-Authors: William S Jermyn, Fidelma E BoydAbstract:Vibrio cholerae is a Gram-negative rod that inhabits the aquatic environment and is the aetiological agent of cholera, a disease that is endemic in Much of Southern Asia. The 57.3 kb Vibrio pathogenicity island-2 (VPI-2) is confined predominantly to toxigenic V. cholerae O1 and O139 serogroup isolates and encodes 52 ORFs (VC1758 to VC1809), which include homologues of an integrase (VC1758), a restriction modification system, a sialic acid metabolism gene cluster (VC1773-VC1783), a neuraminidase (VC1784) and a gene cluster that shows homology to Mu Phage. In this study, a 14.1 kb region of VPI-2 comprising ORFs VC1773 to VC1787 was identified by PCR and Southern blot analyses in all 17 Vibrio mimicus isolates examined. The VPI-2 region in V. mimicus was inserted adjacent to a serine tRNA similar to VPI-2 in V. cholerae. In 11 of the 17 V. mimicus isolates examined, an additional 5.3 kb region encoding VC1758 and VC1804 to VC1809 was present adjacent to VC1787. The evolutionary history of VPI-2 was reconstructed by comparative analysis of the nanH (VC1784) gene tree with the species gene tree, deduced from the housekeeping gene malate dehydrogenase (mdh), among V. cholerae and V. mimicus isolates. Both gene trees showed an overall congruence; on both gene trees V. cholerae O1 and O139 serogroup isolates clustered together, whereas non-O1/non-O139 serogroup isolates formed separate divergent branches with similar clustering of strains within the branches. One exception was noted: on the mdh gene tree, V. mimicus sequences formed a distinct divergent lineage from V. cholerae sequences; however, on the nanH gene tree, V. mimicus clustered with V. cholerae non-O1/non-O139 isolates, suggesting horizontal transfer of this region between these species.
Angela M Gronenborn - One of the best experts on this subject based on the ideXlab platform.
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the wing of the enhancer binding domain of Mu Phage transposase is flexible and is essential for efficient transposition
Proceedings of the National Academy of Sciences of the United States of America, 1996Co-Authors: Robert T Clubb, Michiyo Mizuuchi, Jeffrey R Huth, James G Omichinski, Harri Savilahti, Kiyoshi Mizuuchi, G M Clore, Angela M GronenbornAbstract:Abstract A tetramer of the Mu transposase (MuA) pairs the recombination sites, cleaves the donor DNA, and joins these ends to a target DNA by strand transfer. Juxtaposition of the recombination sites is accomplished by the assembly of a stable synaptic complex of MuA protein and Mu DNA. This initial critical step is facilitated by the transient binding of the N-terminal domain of MuA to an enhancer DNA element within the Mu genome (called the internal activation sequence, IAS). Recently we solved the three-dimensional solution structure of the enhancer-binding domain of Mu Phage transposase (residues 1-76, MuA76) and proposed a model for its interaction with the IAS element. Site-directed Mutagenesis coupled with an in vitro transposition assay has been used to assess the validity of the model. We have identified five residues on the surface of MuA that are crucial for stable synaptic complex formation but dispensable for subsequent events in transposition. These Mutations are located in the loop (wing) structure and recognition helix of the MuA76 domain of the transposase and do not seriously perturb the structure of the domain. Furthermore, in order to understand the dynamic behavior of the MuA76 domain prior to stable synaptic complex formation, we have measured heteronuclear 15N relaxation rates for the unbound MuA76 domain. In the DNA free state the backbone atoms of the helix-turn-helix motif are generally immobilized whereas the residues in the wing are highly flexible on the pico- to nanosecond time scale. Together these studies define the surface of MuA required for enhancement of transposition in vitro and suggest that a flexible loop in the MuA protein required for DNA recognition may become structurally ordered only upon DNA binding.
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the wing of the enhancer binding domain of Mu Phage transposase is flexible and is essential for efficient transposition helix turn helixyMutagenesisynmryheteronuclear relaxation
1996Co-Authors: Robert T Clubb, Michiyo Mizuuchi, James G Omichinski, Harri Savilahti, Kiyoshi Mizuuchi, J Effrey, R Huth, Angela M GronenbornAbstract:A tetramer of the Mu transposase (MuA) pairs the recombination sites, cleaves the donor DNA, and joins these ends to a target DNA by strand transfer. Juxta- position of the recombination sites is accomplished by the assembly of a stable synaptic complex of MuA protein and Mu DNA. This initial critical step is facilitated by the transient binding of the N-terminal domain of MuA to an enhancer DNA element within the Mu genome (called the internal activation sequence, IAS). Recently we solved the three-dimensional solution structure of the enhancer-binding domain of Mu Phage transposase (residues 1-76, MuA 76 ) and proposed a model for its interaction with the IAS element. Site-directed Mutagenesis coupled with an in vitro transposition assay has been used to assess the validity of the model. We have identified five residues on the surface of MuA that are crucial for stable synaptic complex formation but dispensable for subsequent events in transposition. These Mutations are located in the loop (wing) structure and recognition helix of the MuA 76 domain of the transposase and do not seriously perturb the structure of the domain. Furthermore, in order to understand the dynamic behavior of the MuA 76 domain prior to stable synaptic complex formation, we have measured heteronuclear 15 N relaxation rates for the unbound MuA 76 domain. In the DNA free state the backbone atoms of the helix-turn-helix motif are generally immobilized whereas the residues in the wing are highly f lexible on the pico- to nanosecond time scale. Together these studies define the surface of MuA required for enhancement of transposition in vitro and suggest that a f lexible loop in the MuA protein required for DNA recognition may become structurally or- dered only upon DNA binding.
Eiki Yamashita - One of the best experts on this subject based on the ideXlab platform.
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observation of unexpected molecular binding activity for Mu Phage tail fibre chaperones
Journal of Biochemistry, 2019Co-Authors: Kohei Sakai, Atsushi Enomoto, Eiki Yamashita, Atsushi Nakagawa, Takuma Iwazaki, Fumiya Sakuraba, Minoru Inagaki, Shigeki TakedaAbstract:In the history of viral research, one of the important biological features of bacterioPhage Mu is the ability to expand its host range. For extending the host range, the Mu Phage encodes two alternate tail fibre genes. Classical amber Mutation experiments and genome sequence analysis of Mu Phage suggested that gene products (gp) of geneS (gpS = gp49) and gene S' (gpS' = gp52) are tail fibres and that gene products of geneU (gpU = gp50) and geneU' (gpU' = gp51) work for tail fibre assembly or tail fibre chaperones. Depending on the gene orientation, a pair of genes 49-50 or 52-51 is expressed for producing different tail fibres that enable Mu Phage to recognize different host cell surface. Since several fibrous proteins including some Phage tail fibres employ their specific chaperone to facilitate folding and prevent aggregation, we expected that gp50 or gp51 would be a specific chaperone for gp49 and gp52, respectively. However, heterologous overexpression results for gp49 or gp52 (tail fibre subunit) together with gp51 and gp50, respectively, were also effective in producing soluble Mu tail fibres. Moreover, we successfully purified non-native gp49-gp51 and gp52-gp50 complexes. These facts showed that gp50 and gp51 were fungible and functional for both gp49 and gp52 each other.
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three dimensional structures of bacterioPhage neck subunits are shared in podoviridae siphoviridae and myoviridae
Genes to Cells, 2018Co-Authors: Takuma Iwasaki, Masashi Tomihara, Atsushi Enomoto, Eiki Yamashita, Atsushi Nakagawa, Shigeki TakedaAbstract:: Tailed bacterioPhages (Caudovirales) are divided into three families: Myoviridae with long contractile tails, Siphoviridae with long noncontractile tails and Podoviridae with short noncontractile tails. All have an icosahedral head with a portal vertex connected to a neck structure followed by a tail. BacterioPhage Mu belongs to the Myoviridae family. Herein, the gp29 portal subunit and neck subunits gp35, gp36 and gp37 of the Mu Phage were purified to elucidate their arrangement in the neck. Both gp29 and gp36 were monomeric in solution, like the corresponding subunits of Podoviridae P22 and Siphoviridae SPP1. X-ray crystal structure of gp36 showed structural similarity to neck subunits of Siphoviridae and Podoviridae. The gp36 structure has a characteristic aromatic hydrophobic core, and the structure of the ring form of the Mu Phage connector deduced from the Siphoviridae and Podoviridae connector showed that this feature builds the contact surface between gp36 subunits. Structural comparison with the neck of Siphoviridae and Podoviridae also implies direct interaction between gp36 and gp29. Because gp35 and gp36 form a stable complex, we predict that the head-portal ring (gp29), the connector complex (gp36 and gp35), the tail terminator (gp37) and the tube (gp40) are arranged in the Mu Phage neck in this order.
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Crystal structure of the C-terminal domain of Mu Phage central spike and functions of bound calcium ion
Biochimica et biophysica acta, 2012Co-Authors: Kenichi Harada, Eiki Yamashita, Atsushi Nakagawa, Takamitsu Miyafusa, Kouhei Tsumoto, Takashi Ueno, Yoshiharu Toyama, Shigeki TakedaAbstract:Abstract BacterioPhage Mu, which has a contractile tail, is one of the most famous genus of Myoviridae. It has a wide host range and is thought to contribute to horizontal gene transfer. The Myoviridae infection process is initiated by adhesion to the host surface. The Phage then penetrates the host cell membrane using its tail to inject its genetic material into the host. In this penetration process, Myoviridae Phages are proposed to puncture the membrane of the host cell using a central spike located beneath its baseplate. The central spike of the Mu Phage is thought to be composed of gene 45 product (gp45), which has a significant sequence homology with the central spike of P2 Phage (gpV). We determined the crystal structure of shortened Mu gp45Δ1-91 (Arg92–Gln197) at 1.5 A resolution and showed that Mu gp45 is a needlelike structure that punctures the membrane. The apex of Mu gp45 and that of P2 gpV contained iron, chloride, and calcium ions. Although the C-terminal domain of Mu gp45 was sufficient for binding to the E. coli membrane, a Mutant D188A, in which the Asp amino acid residue that coordinates the calcium ion was replaced by Ala, did not exhibit a propensity to bind to the membrane. Therefore, we concluded that calcium ion played an important role in interaction with the host cell membrane.
Atsushi Nakagawa - One of the best experts on this subject based on the ideXlab platform.
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observation of unexpected molecular binding activity for Mu Phage tail fibre chaperones
Journal of Biochemistry, 2019Co-Authors: Kohei Sakai, Atsushi Enomoto, Eiki Yamashita, Atsushi Nakagawa, Takuma Iwazaki, Fumiya Sakuraba, Minoru Inagaki, Shigeki TakedaAbstract:In the history of viral research, one of the important biological features of bacterioPhage Mu is the ability to expand its host range. For extending the host range, the Mu Phage encodes two alternate tail fibre genes. Classical amber Mutation experiments and genome sequence analysis of Mu Phage suggested that gene products (gp) of geneS (gpS = gp49) and gene S' (gpS' = gp52) are tail fibres and that gene products of geneU (gpU = gp50) and geneU' (gpU' = gp51) work for tail fibre assembly or tail fibre chaperones. Depending on the gene orientation, a pair of genes 49-50 or 52-51 is expressed for producing different tail fibres that enable Mu Phage to recognize different host cell surface. Since several fibrous proteins including some Phage tail fibres employ their specific chaperone to facilitate folding and prevent aggregation, we expected that gp50 or gp51 would be a specific chaperone for gp49 and gp52, respectively. However, heterologous overexpression results for gp49 or gp52 (tail fibre subunit) together with gp51 and gp50, respectively, were also effective in producing soluble Mu tail fibres. Moreover, we successfully purified non-native gp49-gp51 and gp52-gp50 complexes. These facts showed that gp50 and gp51 were fungible and functional for both gp49 and gp52 each other.
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three dimensional structures of bacterioPhage neck subunits are shared in podoviridae siphoviridae and myoviridae
Genes to Cells, 2018Co-Authors: Takuma Iwasaki, Masashi Tomihara, Atsushi Enomoto, Eiki Yamashita, Atsushi Nakagawa, Shigeki TakedaAbstract:: Tailed bacterioPhages (Caudovirales) are divided into three families: Myoviridae with long contractile tails, Siphoviridae with long noncontractile tails and Podoviridae with short noncontractile tails. All have an icosahedral head with a portal vertex connected to a neck structure followed by a tail. BacterioPhage Mu belongs to the Myoviridae family. Herein, the gp29 portal subunit and neck subunits gp35, gp36 and gp37 of the Mu Phage were purified to elucidate their arrangement in the neck. Both gp29 and gp36 were monomeric in solution, like the corresponding subunits of Podoviridae P22 and Siphoviridae SPP1. X-ray crystal structure of gp36 showed structural similarity to neck subunits of Siphoviridae and Podoviridae. The gp36 structure has a characteristic aromatic hydrophobic core, and the structure of the ring form of the Mu Phage connector deduced from the Siphoviridae and Podoviridae connector showed that this feature builds the contact surface between gp36 subunits. Structural comparison with the neck of Siphoviridae and Podoviridae also implies direct interaction between gp36 and gp29. Because gp35 and gp36 form a stable complex, we predict that the head-portal ring (gp29), the connector complex (gp36 and gp35), the tail terminator (gp37) and the tube (gp40) are arranged in the Mu Phage neck in this order.
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Crystal structure of the C-terminal domain of Mu Phage central spike and functions of bound calcium ion
Biochimica et biophysica acta, 2012Co-Authors: Kenichi Harada, Eiki Yamashita, Atsushi Nakagawa, Takamitsu Miyafusa, Kouhei Tsumoto, Takashi Ueno, Yoshiharu Toyama, Shigeki TakedaAbstract:Abstract BacterioPhage Mu, which has a contractile tail, is one of the most famous genus of Myoviridae. It has a wide host range and is thought to contribute to horizontal gene transfer. The Myoviridae infection process is initiated by adhesion to the host surface. The Phage then penetrates the host cell membrane using its tail to inject its genetic material into the host. In this penetration process, Myoviridae Phages are proposed to puncture the membrane of the host cell using a central spike located beneath its baseplate. The central spike of the Mu Phage is thought to be composed of gene 45 product (gp45), which has a significant sequence homology with the central spike of P2 Phage (gpV). We determined the crystal structure of shortened Mu gp45Δ1-91 (Arg92–Gln197) at 1.5 A resolution and showed that Mu gp45 is a needlelike structure that punctures the membrane. The apex of Mu gp45 and that of P2 gpV contained iron, chloride, and calcium ions. Although the C-terminal domain of Mu gp45 was sufficient for binding to the E. coli membrane, a Mutant D188A, in which the Asp amino acid residue that coordinates the calcium ion was replaced by Ala, did not exhibit a propensity to bind to the membrane. Therefore, we concluded that calcium ion played an important role in interaction with the host cell membrane.