TY - JOUR
T1 - APOBEC3A Catalytic Inactivity Mutation Induces Tertiary Structure Destabilization
AU - Jones, Katherine F.M.
AU - Shehata, Mohamed
AU - Carpenter, Michael A.
AU - Amaro, Rommie E.
AU - Harki, Daniel A.
N1 - Publisher Copyright:
© 2023 American Chemical Society.
PY - 2023/3/9
Y1 - 2023/3/9
N2 - 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.
AB - 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.
KW - APOBEC3A
KW - catalytic inactivation
KW - protein stability
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U2 - 10.1021/acsmedchemlett.2c00517
DO - 10.1021/acsmedchemlett.2c00517
M3 - Article
C2 - 36923917
AN - SCOPUS:85148501473
SN - 1948-5875
VL - 14
SP - 338
EP - 343
JO - ACS Medicinal Chemistry Letters
JF - ACS Medicinal Chemistry Letters
IS - 3
ER -