9C64 | pdb_00009c64

Structural basis for sequence context-independent single-stranded DNA cytosine deamination by the bacterial toxin SsdA.

(2025) Nat Commun 16: 8841-8841

• PubMed: 41044082 Search on PubMedSearch on PubMed Central
• DOI: https://doi.org/10.1038/s41467-025-63943-9
• Primary Citation of Related Structures:  
  9C63, 9C64


• PubMed Abstract: 
  

  DNA deaminase toxins are involved in interbacterial antagonism and the generation of genetic diversity in surviving bacterial populations. These enzymes have also been adopted as genome engineering tools. The single-stranded (ss)DNA deaminase SsdA is representative of the bacterial deaminase toxin family-2 (BaDTF2), and it deaminates ssDNA cytosines without a strong sequence context dependence, which contrasts with the AID/APOBEC family of sequence-selective ssDNA cytosine deaminases. Here we report the crystal structure of SsdA in complex with a ssDNA substrate. The structure reveals a unique mode of substrate binding, in which a cluster of aromatic residues engages ssDNA in a V-shaped conformation sharply bent across the target cytosine. The bases 5' or 3' to the target cytosine are stacked linearly and make mostly sequence non-specific protein contacts, thus explaining the broad substrate selectivity of SsdA. Unexpectedly, SsdA contains a β-amino acid isoaspartate, which is important for enzymatic activity and contributes to the stability of SsdA as a toxin. Structure-function studies helped to design SsdA mutants active in human cells, which could lead to future applications in genome engineering.

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Organizational Affiliation: 
• Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN, USA.
Institute for Molecular Virology, University of Minnesota, Minneapolis, MN, USA.
Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA.
Department of Biochemistry and Structural Biology, University of Texas Health San Antonio, San Antonio, TX, USA.
Howard Hughes Medical Institute, University of Texas Health San Antonio, San Antonio, TX, USA.
Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN, USA. aihar001@umn.edu.
Institute for Molecular Virology, University of Minnesota, Minneapolis, MN, USA. aihar001@umn.edu.
Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA. aihar001@umn.edu.
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