APOBEC3A Catalytic Inactivity Mutation Induces Tertiary Structure Destabilization

Katherine F.M. Jones, Mohamed Shehata, Michael A. Carpenter, Rommie E. Amaro, Daniel A. Harki

Research output: Contribution to journalArticlepeer-review


APOBEC3A (A3A)-catalyzed DNA cytosine deamination is implicated in virus and cancer mutagenesis, and A3A is a target for small molecule drug discovery. The catalytic glutamic acid (E72) is frequently mutated in biochemical studies to characterize deamination-dependent versus deamination-independent A3A functions. Additionally, catalytically active A3A is toxic in bacterial expression systems, which adversely affects yield during recombinant A3A expression. Here, we demonstrate that mutating the catalytic glutamic acid to an isosteric glutamine (E72Q) significantly decreases the thermal stability of the protein, compared to the alanine-inactivating mutation (E72A). Differential scanning fluorimetry and mass spectrometry reveal that A3A E72Q is less thermally stable than A3A E72A or wild-type A3A. Strikingly, A3A E72Q is partially denatured at 37 °C and binds single-stranded DNA with significantly poorer affinity compared to A3A E72A. This study constitutes an important cautionary note on A3A protein design and informs that A3A E72A is the preferred catalytic inactivation mutation for most applications.

Original languageEnglish (US)
Pages (from-to)338-343
Number of pages6
JournalACS Medicinal Chemistry Letters
Issue number3
StatePublished - Mar 9 2023

Bibliographical note

Funding Information:
This work was supported by a grant from the National Institutes of Health (NIH P01 CA234228). Funding for K.F.M.J. was also provided by the National Science Foundation Graduate Research Fellowship Program (NSF GRFP) and the University of Minnesota Doctoral Dissertation Fellowship (UMN DDF) Program.

Publisher Copyright:
© 2023 American Chemical Society.


  • catalytic inactivation
  • protein stability

PubMed: MeSH publication types

  • Journal Article


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