title: Structure-function analysis of the DNA translocating portal of the bacteriophage T4 packaging machine.
abstract: Tailed bacteriophages and herpesviruses consist of a structurally well conserved dodecameric portal at a special 5-fold vertex of the capsid. The portal plays critical roles in head assembly, genome packaging, neck/tail attachment, and genome ejection. Although the structures of portals from phages φ29, SPP1, and P22 have been determined, their mechanistic roles have not been well understood. Structural analysis of phage T4 portal (gp20) has been hampered because of its unusual interaction with the Escherichia coli inner membrane. Here, we predict atomic models for the T4 portal monomer and dodecamer, and we fit the dodecamer into the cryo-electron microscopy density of the phage portal vertex. The core structure, like that from other phages, is cone shaped with the wider end containing the "wing" and "crown" domains inside the phage head. A long "stem" encloses a central channel, and a narrow "stalk" protrudes outside the capsid. A biochemical approach was developed to analyze portal function by incorporating plasmid-expressed portal protein into phage heads and determining the effect of mutations on head assembly, DNA translocation, and virion production. We found that the protruding loops of the stalk domain are involved in assembling the DNA packaging motor. A loop that connects the stalk to the channel might be required for communication between the motor and the portal. The "tunnel" loops that project into the channel are essential for sealing the packaged head. These studies established that the portal is required throughout the DNA packaging process, with different domains participating at different stages of genome packaging.
authors.affiliation: Department of Biology, The Catholic University of America, 620 Michigan Avenue Northeast, Washington, DC 20064, USA.
authors.forename: Victor
authors.initials: V
authors.lastname: Padilla-Sanchez
authors.affiliation: Department of Biology, The Catholic University of America, 620 Michigan Avenue Northeast, Washington, DC 20064, USA; Marine Drug Research Institute, Huaihai Institute of Technology, Lianyungang, Jiangsu 222001, China.
authors.forename: Song
authors.initials: S
authors.lastname: Gao
authors.affiliation: Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA.
authors.forename: Hyung Rae
authors.initials: HR
authors.lastname: Kim
authors.affiliation: Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA; Department of Computer Science, Purdue University, West Lafayette, IN 47907, USA.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Kihara
authors.affiliation: Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA.
authors.forename: Lei
authors.initials: L
authors.lastname: Sun
authors.affiliation: Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA.
authors.forename: Michael G
authors.initials: MG
authors.lastname: Rossmann
authors.affiliation: Department of Biology, The Catholic University of America, 620 Michigan Avenue Northeast, Washington, DC 20064, USA. Electronic address: rao@cua.edu.
authors.forename: Venigalla B
authors.initials: VB
authors.lastname: Rao
mesh_terms: D017122:Bacteriophage T4 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D036022:Capsid Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D042003:DNA Packaging / Q000502:physiology*; D004279:DNA, Viral / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli / Q000821:virology; D008958:Models, Molecular; D011487:Protein Conformation; D017510:Protein Folding; D014771:Virion / Q000235:genetics; D019065:Virus Assembly / Q000502:physiology*
chemical_list: D036022:Capsid Proteins; D004279:DNA, Viral; C061379:gp20 protein, bacteriophage T4
keywords: DNA packaging motor; head assembly; headful packaging; portal vertex; structural modeling