Architecture of the Complex Formed by Large and Small Terminase Subunits from Bacteriophage P22. Issue 20 (9th October 2015)

• Record Type: Journal Article
• Authors: McNulty, Reginald; Lokareddy, Ravi Kumar; Roy, Ankoor; Yang, Yang; Lander, Gabriel C.; Heck, Albert J.R.; Johnson, John E.; Cingolani, Gino
• Is Part Of: Journal of molecular biology. Volume 427:Issue 20(2015:Oct. 15)
• Page Start: 3285
• Page End: 3299
• Publication Date: 2015-10-09
• Subjects: S-terminase small terminase -- SBD S-terminase binding domain -- L-terminase large terminase -- LBD L-terminase binding domain -- dsDNA double-stranded DNA -- MBP maltose binding protein -- MS mass spectrometry -- DDM n-dodecyl-β-d-maltoside -- AMP-PNP 5′-adenylyl-β, γ-imidodiphosphate -- AUC analytical ultracentrifugation -- EM electron microscopy -- RELION Regularized Likelihood Optimization -- ISAC Iterative Stable Alignment and Clustering; viral genome-packaging motor -- large terminase -- small terminase bacteriophage P22 -- Salmonella virus -- electron microscopy; Molecular biology -- Periodicals; Biology -- Periodicals; Biochemistry -- Periodicals; Bacteriology -- Periodicals; Molecular Biology -- Periodicals; Biochemistry -- Periodicals; Biologie moléculaire -- Périodiques; Biologie -- Périodiques; Biochimie -- Périodiques; Moleculaire biologie; Biochemistry; Biology; Molecular biology; Periodicals; 572.805
• DOI: 10.1016/j.jmb.2015.08.013
• Languages: English
• ISSNs: 0022-2836

Abstract

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. Graphical abstract: Highlights: We assembled the terminase holoenzyme of the bacteriophage P22. The ATPase domain of P22 L-terminase associates with S-terminase C-terminus. P22 terminase complex consists of 1S-terminase:2L-terminase.