Structural Characterization of a Minimal Antibody against Human APOBEC3B

Abstract

APOBEC3B (A3B) is one of seven human APOBEC3 DNA cytosine deaminases that restrict viral infections as part of the overall innate immune response, but it also plays a major role in tumor evolution by mutating genomic DNA. Given the importance of A3B as a restriction factor of viral infections and as a driver of multiple human cancers, selective antibodies against A3B are highly desirable for its specific detection in various research and possibly diagnostic applications. Here, we describe a high-affinity minimal antibody, designated 5G7, obtained via a phage display screening against the C-terminal catalytic domain (ctd) of A3B. 5G7 also binds APOBEC3A that is highly homologous to A3Bctd but does not bind the catalytic domain of APOBEC3G, another Z1-type deaminase domain. The crystal structure of 5G7 shows a canonical arrangement of the heavy and light chain variable domains, with their complementarity-determining region (CDR) loops lining an antigen-binding cleft that accommodates a pair of α-helices. To understand the mechanism of A3Bctd recognition by 5G7, we used the crystal structures of A3Bctd and 5G7 as templates and computationally predicted the A3B-5G7 complex structure. Stable binding poses obtained by the simulation were further tested by site-directed mutagenesis and in vitro binding analyses. These studies mapped the epitope for 5G7 to a portion of C-terminal α6 helix of A3Bctd, with Arg374 playing an essential role. The same region of A3Bctd was used previously as a peptide antigen for generating a rabbit monoclonal antibody (mAb 5210-87-13), suggesting that this region is particularly immunogenic and that these antibodies from very different origins may share similar binding modes. Our studies provide a platform for the development of selective antibodies against A3B and other APOBEC3 family enzymes.

Special Issue “APOBECs and Virus Restriction”

“…APOBEC3B, similar to APOBEC3G, has two Zinc-dependent deaminase domains, but only the C-terminal domain is catalytically active. Tang et al describe the development of a high-affinity minimal antibody obtained from phage display screening that specifically binds the C-terminal domain of APOBEC3B [ 20 ]. A crystal structure of the APOBEC3B C-terminal domain in complex with the antibody was solved [ 20 ].…”

“…Tang et al describe the development of a high-affinity minimal antibody obtained from phage display screening that specifically binds the C-terminal domain of APOBEC3B [ 20 ]. A crystal structure of the APOBEC3B C-terminal domain in complex with the antibody was solved [ 20 ]. The antibody was found to bind to the same region as other antibodies that had been raised against APOBEC3B, suggesting that this region is highly immunogenic.…”

“…The antibody was found to bind to the same region as other antibodies that had been raised against APOBEC3B, suggesting that this region is highly immunogenic. The developed platform can be used to design other selective antibodies to be used as research or therapeutic tools [ 20 ]. Barzak et al present models of the APOBEC3B C-terminal domain obtained by small-angle X-ray scattering (SAXS) [ 21 ].…”