Terminase Subunits from the Pseudomonas-Phage E217.

Autor: Lokareddy RK; Department of Biochemistry and Molecular Biology, Thomas Jefferson University, 1020 Locust Street, Philadelphia, PA 19107, USA., Hou CD; Department of Biochemistry and Molecular Biology, Thomas Jefferson University, 1020 Locust Street, Philadelphia, PA 19107, USA., Doll SG; Department of Biochemistry and Molecular Biology, Thomas Jefferson University, 1020 Locust Street, Philadelphia, PA 19107, USA., Li F; Department of Biochemistry and Molecular Biology, Thomas Jefferson University, 1020 Locust Street, Philadelphia, PA 19107, USA., Gillilan RE; Macromolecular Diffraction Facility, Cornell High Energy Synchrotron Source (MacCHESS), Cornell University, 161 Synchrotron Drive, Ithaca, NY 14853, USA., Forti F; Dipartimento di Bioscienze, Università degli Studi di Milano, Milan, Italy., Horner DS; Dipartimento di Bioscienze, Università degli Studi di Milano, Milan, Italy., Briani F; Dipartimento di Bioscienze, Università degli Studi di Milano, Milan, Italy. Electronic address: federica.briani@unimi.it., Cingolani G; Department of Biochemistry and Molecular Biology, Thomas Jefferson University, 1020 Locust Street, Philadelphia, PA 19107, USA. Electronic address: gino.cingolani@jefferson.edu.
Jazyk: angličtina
Zdroj: Journal of molecular biology [J Mol Biol] 2022 Oct 30; Vol. 434 (20), pp. 167799. Date of Electronic Publication: 2022 Aug 22.
DOI: 10.1016/j.jmb.2022.167799
Abstrakt: 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.
Competing Interests: Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
(Copyright © 2022 Elsevier Ltd. All rights reserved.)
Databáze: MEDLINE
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