APOBEC2 Is a Monomer in Solution: Implications for APOBEC3G Models

APOBEC2 (Apolipoprotein B mRNA Editing Catalytic-polypeptide-like 2; A2) is a muscle specific family member of the APOBEC/AID (Activation Induced Deaminase) family of cytidine deaminases (1). This family includes AID, a protein responsible for CU deamination at distinct regions in immunoglobin genes, resulting in antibody diversification (2); APOBEC1, which catalyzes the C-U conversion of cytidine 6666 in apolipoprotein B RNA, leading to the translation of a truncated form of the protein (3, 4); the APOBEC3s (A, B, C, DE,F,G, and H) that play a variety of roles in the restriction of retroelements and retroviruses via C-U deamination of the target genome, leading to genome hypermutation (5-12); and the newly identified APOBEC4 protein, whose functional role is currently unknown (13). A2 is a highly conserved protein among vertebrates (1) whose biological function is largely unknown. In muscle tissue, protein expression is correlated with orderly aging and the maintenance of correct fiber ratios, although gene loss is not lethal (14-16). There is increasing evidence to suggest that A2 may act at the transcriptional level: A2 expression is upregulated during early stem cell differentiation (17), and expression in Xenopus mesoderm is correlated with the orderly development of the left-right body axis (18). Furthermore, simultaneous overexpression of A2 as well as various APOBEC/AID family members, a thymidine glycosylase, and Gadd45α results in gene demethylation (19-21), a phenomenon intimately related to transcriptional regulation (22-25). The coupling of APOBEC-driven methlyated cytidine deamination to thymidine, followed by base excision repair via a thymidine glycosylase has been proposed as a possible mechanism (19-21). Unfortunately, the field is currently ambivalent regarding the deaminase activity of A2: in vivo studies in higher organisms suggest that A2 deaminates cytidines in DNA (19, 26) whereas E. coli mutator studies as well as in vitro deamination assays, using purified recombinant A2 protein, have not been able to detect any deamination activity (14, 27, 28). While numerous APOBEC/AID family members are difficult to purify in a biochemically pure and physically homogeneous form, a truncated version of A2 was purified and crystallized by Prochnow et al. (29). The X-ray structure revealed a novel, extended V-shaped homotetramer, contrasting earlier findings for other deaminases that exhibited square/rectangular homodimeric or homotetrameric assemblies with the Zn coordinated active-sites of all subunits at the center (30-32). Since little was known about the function of A2, rendering structure-function studies unfeasible, the major value of the A2 structure was its utility as a surrogate for other APOBEC/AID family members. All APOBEC/AID family members exhibit a high degree of primary sequence similarity, for one-domain as well as two-domain variants (33). Among the APOBECs, A3G has received a great deal of attention given its ability to restrict HIV in a ΔVif background (5, 34). Both, monomeric as well as dimeric models of APOBEC3G (A3G) (35, 36) were created, based on the A2 structure. In the A2 crystal structure, each wing of the V is comprised of two A2 monomers and thus two deaminase domains. As a result, the majority of researchers in the A3G field have adopted the view that the double deaminase domain, monomeric A3G can be represented by the dimeric wing of the A2 structure and that the functional form, the A3G dimer (37), can be represented by the A2 tetramer. In this report, nuclear magnetic resonance spectroscopy (NMR) and light scattering measurements have been used unambiguously demonstrate that purified A2 is a monomer in solution. This holds true for both the full-length A2 protein as well as the N-terminal truncation construct that was used for crystallography. In addition, the N-terminal region in full-length A2 contributes to its thermodynamic stability and suppresses aggregation, although it does not alter the fold of the catalytic domain. These findings suggest that, in the absence of a bona-fide high-resolution A3G structure, the current views with respect to A3G structure/function relationships that are based on the A2 structure need to be re-examined.