A Portable Hot Spot Recognition Loop Transfers Sequence Preferences from APOBEC Family Members to Activation-induced Cytidine Deaminase

Abstract

Enzymes of the AID/APOBEC family, characterized by the targeted deamination of cytosine to generate uracil within DNA, mediate numerous critical immune responses. One family member, activation-induced cytidine deaminase (AID), selectively introduces uracil into antibody variable and switch regions, promoting antibody diversity through somatic hypermutation or class switching. Other family members, including APOBEC3F and APOBEC3G, play an important role in retroviral defense by acting on viral reverse transcripts. These enzymes are distinguished from one another by targeting cytosine within different DNA sequence contexts; however, the reason for these differences is not known. Here, we report the identification of a recognition loop of 9 -11 amino acids that contributes significantly to the distinct sequence motifs of individual family members. When this recognition loop is grafted from the donor APOBEC3F or 3G proteins into the acceptor scaffold of AID, the mutational signature of AID changes toward that of the donor proteins. These loop-graft mutants of AID provide useful tools for dissecting the biological impact of DNA sequence preferences upon generation of antibody diversity, and the results have implications for the evolution and specialization of the AID/APOBEC family.The polynucleotide cytosine deaminases have been identified as key contributors to both the adaptive and innate immune responses to pathogens. This enzyme family includes activation-induced cytidine deaminase (AID), 2 which initiates antibody diversification, and the APOBEC3 enzymes, which inhibit retroviral infection (1, 2). Although the enzymes share a common chemical mechanism for catalyzing the deamination of cytosine to generate mutagenic uracil within DNA, they have distinct biological functions based on differences in their expression, localization, and DNA sequence specificity (2, 3).AID is a B-cell-specific enzyme whose catalytic action on mammalian antibody genes initiates somatic hypermutation and class switch recombination (4, 5). The uracil deamination product is processed by familiar DNA repair proteins, including uracil DNA N-glycosylase (UNG) and mismatch repair enzymes. However, the cellular environment within activated B-cells promotes mutagenic processing by these pathways, resulting in point mutations that ultimately enhance antibody affinity in somatic hypermutation or promote clustered double-strand breaks that alter antibody isotype in class switch recombination (6, 7).The activity of AID is restricted to relatively small 3-kb regions within the immunoglobulin locus around rearranged variable genes and heavy chain switch regions. Within these regions, the targeting of AID is partially dictated by its DNA sequence preferences. For example, it has been noted that the mutable complementarity determining regions from variable gene segments have an abundance of AGC codons as compared with adjacent framework regions that function in stabilizing antibody structure (8). In vivo, somatic hypermutation is focused on WRCW (W ϭ A...

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

“…For AID-WT, the local sequence preferences were in complete agreement with the canonical WRC hotspot preference (Fig. 3B), and these in vivo results correlate well with our prior investigations on in vitro sequence preferences (27). Expression of the AID loop variants generates C/G 3 T/A transition mutations with the predicted local sequence preference of the loop donor.…”

“…Relative to AID-WT, both AID-3FL and AID-3GL are altered in their CDR to non-CDR targeting (p Յ 0.05) and have increasing preference for deamination outside of the CDRs. The propensity for regional targeting of each construct correlates remarkably with the previously reported (27) preference for deamination of oligonucleotides with WRC/non-WRC sequences in vitro (supplemental Fig. S4A).…”

“…Available structures of the unliganded enzymes have led to debate over their DNA binding mode as well as a general deficit in our understanding of how they recognize cytosine in different sequence contexts (25,46). Building upon observations that identified a protein loop within the catalytic deaminase domain that appeared relevant for local sequence targeting (23)(24)(25)(26)47), we previously demonstrated that grafting this loop could transfer the sequence specificity from the A3 enzymes to AID (27). In other varied scaffolds, using either CTD-2K3A variant of A3G or "upmutant" variants of AID, the importance of this loop in local DNA sequence recognition has also been consistently seen (28,29).…”

“…Building on these observations, we recently demonstrated that local targeting could be predictably altered. By grafting the entire 9 -11-amino acid protein loops of A3G or A3F into the scaffold of AID, the resulting chimeric enzymes shifted toward the local sequence targeting preferences of the donor enzyme using either purified oligonucleotide substrates in vitro or by examining their mutagenic profiles in bacteria (27). These findings have subsequently been confirmed by several groups (28,29).…”

A Structural Basis for the Biochemical Behavior of Activation-induced Deoxycytidine Deaminase Class-switch Recombination-defective Hyper-IgM-2 Mutants

“…S83P and S85N are located next to the conserved Trp-84 that may be involved in positioning the target cytidine into the active center; therefore, mutations in this surrounding area likely inhibit deamination activity. The "specificity loop" (loop 7) has previously been shown to contain residues that determine an APOBEC-specific hot spot deamination motif within the ssDNA target substrate (32)(33)(34). Mutations in this loop (A111E, R112C, and L113P) may alter the stability of the specificity loop, leading to the inability of AID to properly position the substrate in the active center.…”