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Structure-functional characterization of Lactococcus AbiA phage defense system

Gapińska, Marta ; Zajko, Weronika ; Skowronek, Krzysztof ; Figiel, Małgorzata ; Krawczyk, Paweł S ; Egorov, Artyom A LU orcid ; Dziembowski, Andrzej ; Johansson, Marcus J O LU and Nowotny, Marcin (2024) In Nucleic Acids Research 52(8). p.4723-4738
Abstract

Bacterial reverse transcriptases (RTs) are a large and diverse enzyme family. AbiA, AbiK and Abi-P2 are abortive infection system (Abi) RTs that mediate defense against bacteriophages. What sets Abi RTs apart from other RT enzymes is their ability to synthesize long DNA products of random sequences in a template- and primer-independent manner. Structures of AbiK and Abi-P2 representatives have recently been determined, but there are no structural data available for AbiA. Here, we report the crystal structure of Lactococcus AbiA polymerase in complex with a single-stranded polymerization product. AbiA comprises three domains: an RT-like domain, a helical domain that is typical for Abi polymerases, and a higher eukaryotes and prokaryotes... (More)

Bacterial reverse transcriptases (RTs) are a large and diverse enzyme family. AbiA, AbiK and Abi-P2 are abortive infection system (Abi) RTs that mediate defense against bacteriophages. What sets Abi RTs apart from other RT enzymes is their ability to synthesize long DNA products of random sequences in a template- and primer-independent manner. Structures of AbiK and Abi-P2 representatives have recently been determined, but there are no structural data available for AbiA. Here, we report the crystal structure of Lactococcus AbiA polymerase in complex with a single-stranded polymerization product. AbiA comprises three domains: an RT-like domain, a helical domain that is typical for Abi polymerases, and a higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domain that is common for many antiviral proteins. AbiA forms a dimer that distinguishes it from AbiK and Abi-P2, which form trimers/hexamers. We show the DNA polymerase activity of AbiA in an in vitro assay and demonstrate that it requires the presence of the HEPN domain which is enzymatically inactive. We validate our biochemical and structural results in vivo through bacteriophage infection assays. Finally, our in vivo results suggest that AbiA-mediated phage defense may not rely on AbiA-mediated cell death.

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author
; ; ; ; ; ; ; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Bacteriophages/genetics, Lactococcus/virology, Models, Molecular, Bacterial Proteins/metabolism, Crystallography, X-Ray, Protein Domains, RNA-Directed DNA Polymerase/metabolism, Protein Multimerization, Structure-Activity Relationship
in
Nucleic Acids Research
volume
52
issue
8
pages
16 pages
publisher
Oxford University Press
external identifiers
  • scopus:85192800429
  • pmid:38587192
ISSN
1362-4962
DOI
10.1093/nar/gkae230
language
English
LU publication?
yes
id
bf11b2f4-8f6b-4efd-a44d-0b87a9b9e3a2
date added to LUP
2024-05-15 17:30:23
date last changed
2024-06-24 16:42:41
@article{bf11b2f4-8f6b-4efd-a44d-0b87a9b9e3a2,
  abstract     = {{<p>Bacterial reverse transcriptases (RTs) are a large and diverse enzyme family. AbiA, AbiK and Abi-P2 are abortive infection system (Abi) RTs that mediate defense against bacteriophages. What sets Abi RTs apart from other RT enzymes is their ability to synthesize long DNA products of random sequences in a template- and primer-independent manner. Structures of AbiK and Abi-P2 representatives have recently been determined, but there are no structural data available for AbiA. Here, we report the crystal structure of Lactococcus AbiA polymerase in complex with a single-stranded polymerization product. AbiA comprises three domains: an RT-like domain, a helical domain that is typical for Abi polymerases, and a higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domain that is common for many antiviral proteins. AbiA forms a dimer that distinguishes it from AbiK and Abi-P2, which form trimers/hexamers. We show the DNA polymerase activity of AbiA in an in vitro assay and demonstrate that it requires the presence of the HEPN domain which is enzymatically inactive. We validate our biochemical and structural results in vivo through bacteriophage infection assays. Finally, our in vivo results suggest that AbiA-mediated phage defense may not rely on AbiA-mediated cell death.</p>}},
  author       = {{Gapińska, Marta and Zajko, Weronika and Skowronek, Krzysztof and Figiel, Małgorzata and Krawczyk, Paweł S and Egorov, Artyom A and Dziembowski, Andrzej and Johansson, Marcus J O and Nowotny, Marcin}},
  issn         = {{1362-4962}},
  keywords     = {{Bacteriophages/genetics; Lactococcus/virology; Models, Molecular; Bacterial Proteins/metabolism; Crystallography, X-Ray; Protein Domains; RNA-Directed DNA Polymerase/metabolism; Protein Multimerization; Structure-Activity Relationship}},
  language     = {{eng}},
  month        = {{05}},
  number       = {{8}},
  pages        = {{4723--4738}},
  publisher    = {{Oxford University Press}},
  series       = {{Nucleic Acids Research}},
  title        = {{Structure-functional characterization of Lactococcus AbiA phage defense system}},
  url          = {{http://dx.doi.org/10.1093/nar/gkae230}},
  doi          = {{10.1093/nar/gkae230}},
  volume       = {{52}},
  year         = {{2024}},
}