Terminase Subunits from the Pseudomonas-Phage E217. Issue 20 (30th October 2022)

• Record Type: Journal Article
• Authors: Lokareddy, Ravi K.; Hou, Chun-Feng David; Doll, Steven G.; Li, Fenglin; Gillilan, Richard E.; Forti, Francesca; Horner, David S.; Briani, Federica; Cingolani, Gino
• Is Part Of: Journal of molecular biology. Volume 434:Issue 20(2022)
• Publication Date: 2022-10-30
• Subjects: viral genome-packaging motor -- large terminase -- small terminase -- bacteriophage E217 -- Pseudomonas-phages; cryo-EM cryogenic electron microscopy -- P. aeruginosa Pseudomonas aeruginosa -- TerS small terminase -- TerL large terminase -- FL-portal full-length portal protein -- SAXS Small Angle X-ray Scattering -- SEC size exclusion chromatography -- SPA single-particle analysis -- CC correlation coefficient -- M.W. molecular weight -- RMSD room-mean square deviation -- dsDNA double-stranded DNA -- SSM secondary structure superimposition -- SDS-PAGE sodium dodecyl sulfate–polyacrylamide gel electrophoresis -- pfu plaque-forming unit -- HTH helix-turn-helix -- FSC Fourier Shell Correlation; 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.2022.167799
• Languages: English
• ISSNs: 0022-2836

Abstract

Graphical abstract: Highlights: TerL and TerS are encoded by genes ∼58 kbs away in the E217 genome. E217 TerL adopts a two-domain architecture shaped like a bean. TerL nuclease domain contains two metal ions in the active site. E217 TerS folds into a decameric oligomer. Overexpression of TerS causes a 4-fold reduction of E217 burst size. Abstract: Pseudomonas phages are increasingly important biomedicines for phage therapy, but little is known about how these viruses package DNA. This paper explores the terminase subunits from the Myoviridae E217, a Pseudomonas- phage used in an experimental cocktail to eradicate P. aeruginosa in vitro and in animal models. We identified the large (TerL) and small (TerS) terminase subunits in two genes ∼58 kbs away from each other in the E217 genome. TerL presents a classical two-domain architecture, consisting of an N-terminal ATPase and C-terminal nuclease domain arranged into a bean-shaped tertiary structure. A 2.05 Å crystal structure of the C-terminal domain revealed an RNase H-like fold with two magnesium ions in the nuclease active site. Mutations in TerL residues involved in magnesium coordination had a dominant-negative effect on phage growth. However, the two ions identified in the active site were too far from each other to promote two-metal-ion catalysis, suggesting a conformational change is required for nuclease activity. We also determined a 3.38 Å cryo-EM reconstruction of E217 TerS that revealed a ring-like decamer, departing from the most common nonameric quaternary structure observed thus far. E217 TerS contains both N-terminal helix-turn-helix motifs enriched in basic residues and a central channel lined with basic residues large enough to accommodate double-stranded DNA. Overexpression of TerS caused a more than a 4-fold reduction of E217 burst size, suggesting a catalytic amount of the protein is required for packaging. Together, these data expand the molecular repertoire of viral terminase subunits to Pseudomonas -phages used for phage therapy.