Characterization of the Catalytic Domain of Human APOBEC3B and the Critical Structural Role for a Conserved Methionine.
Human APOBEC3B deaminates cytosines in DNA and belongs to the AID/APOBEC family of enzymes. These proteins are involved in innate and adaptive immunity and may cause mutations in a variety of cancers. To characterize its ability to convert cytosines into uracils, we tested several derivatives of APOBEC3B gene for their ability to cause mutations in Escherichia coli. Through this analysis, a methionine residue at the junction of the amino-terminal domain (NTD) and the carboxy-terminal domain (CTD) was found to be essential for high mutagenicity. Properties of mutants with substitutions at this position, examination of existing molecular structures of APOBEC3 family members and molecular modeling suggest that this residue is essential for the structural stability of this family of proteins. The APOBEC3B CTD with the highest mutational activity was purified to homogeneity and its kinetic parameters were determined. Size-exclusion chromatography of the CTD monomer showed that it is in equilibrium with its dimeric form and matrix-assisted laser desorption ionization time-of-flight analysis of the protein suggested that the dimer may be quite stable. The partially purified NTD did not show intrinsic deamination activity and did not enhance the activity of the CTD in biochemical assays. Finally, APOBEC3B was at least 10-fold less efficient at mutating 5-methylcytosine (5mC) to thymine than APOBEC3A in a genetic assay and was at least 10-fold less efficient at deaminating 5mC compared to C in biochemical assays. These results shed light on the structural organization of APOBEC3B catalytic domain, its substrate specificity and its possible role in causing genome-wide mutations.
Department of Chemistry, Wayne State University, Detroit, MI 48202, USA.
Sachini U
SU
Siriwardena
Department of Chemistry, Wayne State University, Detroit, MI 48202, USA.
Thisari A
TA
Guruge
Department of Chemistry, Wayne State University, Detroit, MI 48202, USA; Department of Immunology and Microbiology, Wayne State University, Detroit, MI 48202, USA.
Ashok S
AS
Bhagwat
D044503:5-Methylcytosine / Q000378:metabolism; D000595:Amino Acid Sequence; D020134:Catalytic Domain; D003564:Cytidine Deaminase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003596:Cytosine / Q000378:metabolism; D006801:Humans; D008715:Methionine / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D055503:Protein Multimerization; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D011506:Proteins; D044503:5-Methylcytosine; D003596:Cytosine; D008715:Methionine; C121399:APOBEC3A protein, human; D003564:Cytidine Deaminase
APOBEC3B multimerization; breast cancer; cancer mutational signatures; demethylation; kataegis