Local Sequence Targeting in the AID/APOBEC Family Differentially Impacts Retroviral Restriction and Antibody Diversification

Abstract

Nucleic acid cytidine deaminases of the activation-induced deaminase (AID)/APOBEC family are critical players in active and innate immune responses, playing roles as target-directed, purposeful mutators. AID specifically deaminates the host immunoglobulin (Ig) locus to evolve antibody specificity, whereas its close relative, APOBEC3G (A3G), lethally mutates the genomes of retroviral pathogens such as HIV. Understanding the basis for the target-specific action of these enzymes is essential, as mistargeting poses significant risks, potentially promoting oncogenesis (AID) or fostering drug resistance (A3G). AID prefers to deaminate cytosine in WRC (W ‫؍‬ A/T, R ‫؍‬ A/G) motifs, whereas A3G favors deamination of CCC motifs. This specificity is largely dictated by a single, divergent protein loop in the enzyme family that recognizes the DNA sequence. Through grafting of this substrate-recognition loop, we have created enzyme variants of A3G and AID with altered local targeting to directly evaluate the role of sequence specificity on immune function. We find that grafted loops placed in the A3G scaffold all produced efficient restriction of HIV but that foreign loops in the AID scaffold compromised hypermutation and class switch recombination. Local targeting, therefore, appears alterable for innate defense against retroviruses by A3G but important for adaptive antibody maturation catalyzed by AID. Notably, AID targeting within the Ig locus is proportionally correlated to its in vitro ability to target WRC sequences rather than non-WRC sequences. Although other mechanisms may also contribute, our results suggest that local sequence targeting by AID/APOBEC3 enzymes represents an elegant example of co-evolution of enzyme specificity with its target DNA sequence.

Crystal Structure of the DNA Deaminase APOBEC3B Catalytic Domain

“…Previous studies have demonstrated the importance of loop 7 residues in sequence selectivity of related A3 family members, AID, A3A, A3F, and A3G (53,(55)(56)(57)(58). Consistent with these reports, replacing loop 7 of A3B with that from A3G (GL7) converted the target sequence preference to 5Ј-CC-3Ј preferred by A3G (cytosine 1691 in rpoB; Fig.…”

“…Previous structural and functional studies of A3 proteins have demonstrated that loop 1 (between ␣1 and ␤1) and loop 7 (between ␤4 and ␣4) surround the active site and suggested that these flexible regions play important roles in binding singlestranded DNA substrates (30,53,(55)(56)(57)(58). The structures of A3Bctd-QM⌬loop3 in both crystal forms show a tightly closed conformation of the active site compared with those observed for other A3 proteins (Fig.…”

“…The closed conformation is stabilized by a stacking interaction between Arg-211 and Tyr-315 from loops 1 and 7, respectively. Previous studies together with the mutation analyses presented here demonstrate that these loop regions are important for catalytic activity and dinucleotide target sequence selectivity, respectively, strongly implicating direct interactions with ssDNA substrates (30,53,(55)(56)(57)(58). Based on these observations, we hypothesize that the binding of a ssDNA substrate carrying the preferred 5Ј-TCG or 5Ј-TCA target sequence triggers conformational changes of structural elements including loops 1 and 7, which in turn stabilize the open conformation of the active site and allow entry of the target cytosine base into the active site pocket.…”

A putative antiviral role of plant cytidine deaminases

“…HIV-1 hypermutated genomes show mutational hot spots as well, which are due to preference of A3G and A3F for deamination of the third C in 5’-CCC (negative-strand) and 5’-GGC, respectively 51, 52 . The context of the C complementary to G181 (5’-GGC) differs from what has been described for APOBEC3, suggesting that if At CDAs had context preference, it would be different from the one described for A3G.…”

Determinants of Substrate Specificity in APOBEC3B

“…31 In conjunction with other studies, this work established the role of loops 1, 3 and 7 in the process of DNA substrate recognition. 24,[31][32][33][34][35] Initial crystal structures of A3 deaminase domains show these loops being adjacent to the binding site. 22 Subsequent crystallization of A3 proteins in complex with substrates showed residues on loops 1 and 7 binding to oligonucleotide substrates.…”