title: Direct interaction of the bacteriophage SPP1 packaging ATPase with the portal protein.
abstract: DNA packaging in tailed bacteriophages and other viruses requires assembly of a complex molecular machine at a specific vertex of the procapsid. This machine is composed of the portal protein that provides a tunnel for DNA entry, an ATPase that fuels DNA translocation (large terminase subunit), and most frequently, a small terminase subunit. Here we characterized the interaction between the terminase ATPase subunit of bacteriophage SPP1 (gp2) and the procapsid portal vertex. We found, by affinity pulldown assays with purified proteins, that gp2 interacts with the portal protein, gp6, independently of the terminase small subunit gp1, DNA, or ATP. The gp2-procapsid interaction via the portal protein depends on gp2 concentration and requires the presence of divalent cations. Competition experiments showed that isolated gp6 can only inhibit gp2-procapsid interactions and DNA packaging at gp6:procapsid molar ratios above 10-fold. Assays with gp6 carrying mutations in distinct regions of its structure that affect the portal-induced stimulation of ATPase and DNA packaging revealed that none of these mutations impedes gp2-gp6 binding. Our results demonstrate that the SPP1 packaging ATPase binds directly to the portal and that the interaction is stronger with the portal embedded in procapsids. Identification of mutations in gp6 that allow for assembly of the ATPase-portal complex but impair DNA packaging support an intricate cross-talk between the two proteins for activity of the DNA translocation motor.
authors.affiliation: Unité de Virologie Moléculaire et Structurale, Unité Mixte de Recherche, CNRS 2472, Institut Fédératif de Recherche 1157, and IFR 115, Bâtiment 14B, Avenue de la Terrasse, 91190 Gif-sur-Yvette, France. oliveira@vms.cnrs-gif.fr
authors.forename: Leonor
authors.initials: L
authors.lastname: Oliveira
authors.forename: Ana
authors.initials: A
authors.lastname: Cuervo
authors.forename: Paulo
authors.initials: P
authors.lastname: Tavares
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001435:Bacteriophages / Q000235:genetics / Q000378:metabolism*; D002213:Capsid / Q000378:metabolism; D042003:DNA Packaging; D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism; D008958:Models, Molecular; D020409:Molecular Motor Proteins / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D020409:Molecular Motor Proteins; D021122:Protein Subunits; D014764:Viral Proteins; C417248:bacteriophage SPP1 terminase; C115880:portal protein, Bacteriophage SPP1; D004706:Endodeoxyribonucleases; D000251:Adenosine Triphosphatases; D008274:Magnesium