Architecture of the Complex Formed by Large and Small Terminase Subunits from Bacteriophage P22
| Autor: | McNulty, Reginald, Lokareddy, Ravi Kumar, Roy, Ankoor, Yang, Yang, Lander, Gabriel C., Heck, Albert J R, Johnson, John E., Cingolani, Gino, Sub Biomol.Mass Spect. and Proteomics, Sub Biomol.Mass Spectrometry & Proteom., Biomolecular Mass Spectrometry and Proteomics |
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| Přispěvatelé: | Sub Biomol.Mass Spect. and Proteomics, Sub Biomol.Mass Spectrometry & Proteom., Biomolecular Mass Spectrometry and Proteomics |
| Rok vydání: | 2015 |
| Předmět: |
Models
Molecular small terminase bacteriophage P22 Protein Conformation ATPase Protein subunit Viral terminase complex Crystallography X-Ray Article Bacteriophage Viral Proteins chemistry.chemical_compound Protein structure large terminase Structural Biology DNA Packaging Salmonella virus viral genome-packaging motor Endodeoxyribonucleases Molecular Biology Bacteriophage P22 electron microscopy biology Virus Assembly biology.organism_classification Protein Structure Tertiary Protein Subunits Crystallography chemistry DNA Viral biology.protein Biophysics DNA Binding domain |
| Zdroj: | Journal of Molecular Biology, 427(20), 3285. Academic Press Inc. |
| ISSN: | 0022-2836 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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