Journal
JOURNAL OF MOLECULAR BIOLOGY
Volume 434, Issue 20, Pages -Publisher
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2022.167799
Keywords
viral genome-packaging motor; large terminase; small terminase; bacteriophage E217; Pseudomonas-phages
Categories
Funding
- National Institutes of Health [P30 GM124166-01A1, R01 GM100888, R35 GM140733]
- Fondazione per la ricerca sulla Fibrosi cistica-Associazione Trentina Fibrosi Cistica ODV In ricordo di Pio Nicolini grant FFC [15/2021]
- National Cancer Institute Cancer Center Support Grant [P30 CA56036]
- NIH [S10 OD030457, S10 OD017987, S10 OD023479]
- National Science Foundation [DMR-1829070]
- NYSTAR
- NIH High-End Instrumentation Grant [S10 OD018483]
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This study explores the terminase subunits of Pseudomonas phages and reveals the structure and function of TerL and TerS subunits. TerL consists of an ATPase and nuclease domain, while TerS forms a ring-like decamer structure. These findings are important for understanding the DNA packaging process of Pseudomonas phages and their application in phage therapy.
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 similar to 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 angstrom 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 angstrom 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. (C) 2022 Elsevier Ltd. All rights reserved.
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