Mutational analysis of apolipoprotein B mRNA editing enzyme (APOBEC1): Structure-function relationships of RNA editing and dimerization

APOBEC1 is the catalytic subunit of an enzyme complex that mediates apolipoprotein (apo) B mRNA editing. It dimerizes in vitro and requires complementation factor(s) for its editing activity. We have performed a systematic analysis of the structure-functional relationship of APOBEC1 by targeted mutagenesis of various sequence motifs within the protein. Using in vitro RNA editing assay, we found that basic amino acid clusters at the amino-terminal region R¹⁵R¹⁶R¹⁷ and R³³K³⁴, are essential for apoB mRNA editing. Mutation of R¹⁵R¹⁶R¹⁷ to K¹⁵K¹⁶K¹⁷ and mutation of R³³K³⁴ simultaneously to A³³A³⁴ almost completely abolished in vitro editing activity. The carboxy-terminal region of APOBEC1 contains a leucine- rich motif. Deletion analysis of this region indicates that residues 181 to 210 are important for in vitro apoB mRNA editing. Single amino acid substitutions demonstrate that L¹⁸², I¹⁸⁵, and L¹⁸⁹ are important residues required for normal editing function. Furthermore, the double mutant P190A/P191A also lost >90% of editing activity which suggests that a β turn in this region of the molecule may be essential for proper functioning of APOBEC1. It was suggested that dimerization of APOBEC1 creates an active structure for deamination of apoB mRNA. When we examined the dimerization potential of truncated APOBEC1s using both amino and carboxy termini deletion mutants, we found that amino-terminal deletions up to residue A¹¹⁷ did not impair dimerization activity whereas carboxy-terminal deletions showed diminished dimerization. The systematic and extensive mutagenesis experiments in this study provide information on the role of various sequence motifs identified in APOBEC1 in enzyme catalysis and dimerization.