The in vitro Biochemical Characterization of an HIV-1 Restriction Factor APOBEC3F: Importance of Loop 7 on Both CD1 and CD2 for DNA Binding and Deamination

•Highly purified full-length and various mutational APOBEC3F proteins from E. coli expression•The N-terminal domain (CD1) of A3F greatly enhances the deamination activity of the C-terminal domain (CD2).•The CD1 of A3F is catalytically inactive but greatly enhances the ssDNA binding affinity of the CD2 domain.•The loop 7 of A3F CD1 is critical for ssDNA binding by the CD1 domain.•The loop 7 of A3F CD2 is important for ssDNA binding of CD2 domain.•The conserved tryptophan residue on the loop 7 of both CD1 and CD2 regulates the substrate DNA binding and deaminase activities.

APOBEC3F (A3F) is a member of the apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) family of proteins that can deaminate cytosine (C) to uracil (U) on nucleic acids. A3F is one of the four APOBEC members with two Zn-coordinated homologous cytosine deaminase (CD) domains, with the others being A3G, A3D, and A3B. Here we report the in vitro characterization of DNA binding and deaminase activities using purified wild-type and various mutant proteins of A3F from an Escherichia coli expression system. We show that even though CD1 is catalytically inactive and CD2 is the active deaminase domain, presence of CD1 on the N-terminus of CD2 enhances the deaminase activity by over an order of magnitude. This enhancement of CD2 catalytic activity is mainly through the increase of substrate single-stranded (ss) DNA binding by the N-terminal CD1 domain. We further show that the loop 7 of both CD1 and CD2 of A3F plays an important role for ssDNA binding for each individual domain, as well as for the deaminase activity of CD2 domain in the full-length A3F.

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