title: Architecture of the Complex Formed by Large and Small Terminase Subunits from Bacteriophage P22.
abstract: Packaging of viral genomes inside empty procapsids is driven by a powerful ATP-hydrolyzing motor, formed in many double-stranded DNA viruses by a complex of a small terminase (S-terminase) subunit and a large terminase (L-terminase) subunit, transiently docked at the portal vertex during genome packaging. Despite recent progress in elucidating the structure of individual terminase subunits and their domains, little is known about the architecture of an assembled terminase complex. Here, we describe a bacterial co-expression system that yields milligram quantities of the S-terminase:L-terminase complex of the Salmonella phage P22. In vivo assembled terminase complex was affinity-purified and stabilized by addition of non-hydrolyzable ATP, which binds specifically to the ATPase domain of L-terminase. Mapping studies revealed that the N-terminus of L-terminase ATPase domain (residues 1-58) contains a minimal S-terminase binding domain sufficient for stoichiometric association with residues 140-162 of S-terminase, the L-terminase binding domain. Hydrodynamic analysis by analytical ultracentrifugation sedimentation velocity and native mass spectrometry revealed that the purified terminase complex consists predominantly of one copy of the nonameric S-terminase bound to two equivalents of L-terminase (1S-terminase:2L-terminase). Direct visualization of this molecular assembly in negative-stained micrographs yielded a three-dimensional asymmetric reconstruction that resembles a "nutcracker" with two L-terminase protomers projecting from the C-termini of an S-terminase ring. This is the first direct visualization of a purified viral terminase complex analyzed in the absence of DNA and procapsid.
authors: Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA. Electronic address: rmcnulty@scripps.edu.
authors: Reginald
authors: R
authors: McNulty
authors: Department of Biochemistry and Molecular Biology, Thomas Jefferson University, 233 South 10th Street Philadelphia, PA 19107, USA.
authors: Ravi Kumar
authors: RK
authors: Lokareddy
authors: Department of Biochemistry and Molecular Biology, Thomas Jefferson University, 233 South 10th Street Philadelphia, PA 19107, USA.
authors: Ankoor
authors: A
authors: Roy
authors: Biomolecular Mass Spectrometry and Proteomics, Bijvoet Centre for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, Netherlands Proteomics Center, Padualaan 8, 3584 CH Utrecht, The Netherlands.
authors: Yang
authors: Y
authors: Yang
authors: Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
authors: Gabriel C
authors: GC
authors: Lander
authors: Biomolecular Mass Spectrometry and Proteomics, Bijvoet Centre for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, Netherlands Proteomics Center, Padualaan 8, 3584 CH Utrecht, The Netherlands.
authors: Albert J R
authors: AJR
authors: Heck
authors: Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
authors: John E
authors: JE
authors: Johnson
authors: Department of Biochemistry and Molecular Biology, Thomas Jefferson University, 233 South 10th Street Philadelphia, PA 19107, USA. Electronic address: gino.cingolani@jefferson.edu.
authors: Gino
authors: G
authors: Cingolani
mesh_terms: D017100:Bacteriophage P22 / Q000378:metabolism*; D018360:Crystallography, X-Ray; D042003:DNA Packaging / Q000502:physiology*; D004279:DNA, Viral / Q000378:metabolism*; D004706:Endodeoxyribonucleases / Q000378:metabolism / Q000648:ultrastructure*; D008958:Models, Molecular; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000378:metabolism*; D014764:Viral Proteins / Q000378:metabolism; D019065:Virus Assembly / Q000502:physiology*
chemical_list: D004279:DNA, Viral; D021122:Protein Subunits; D014764:Viral Proteins; D004706:Endodeoxyribonucleases; C030894:terminase
keywords: Salmonella virus; electron microscopy; large terminase; small terminase bacteriophage P22; viral genome-packaging motor