Ronda Bransteitter

Activation-induced cytidine deaminase deaminates deoxycytidine on single-stranded DNA but requires the action of RNase.

The expression of activation-induced cytidine deaminase (AID) is prerequisite to a “trifecta” of key molecular events in B cells: class-switch recombination and somatic hypermutation in humans and mice and gene conversion in chickens. Although this critically important enzyme shares common sequence motifs with apolipoprotein B mRNA-editing enzyme, and exhibits deaminase activity on free deoxycytidine in solution, it has not been shown to act on either RNA or DNA. Recent mutagenesis data in Escherichia coli suggest that AID may deaminate dC on DNA, but its putative biochemical activities on either DNA or RNA remained a mystery. Here, we show that AID catalyzes deamination of dC residues on single-stranded DNA in vitro but not on double-stranded DNA, RNA–DNA hybrids, or RNA. Remarkably, it has no measurable deaminase activity on single-stranded DNA unless pretreated with RNase to remove inhibitory RNA bound to AID. AID catalyzes dC → dU deamination activity most avidly on double-stranded DNA substrates containing a small “transcription-like” single-stranded DNA bubble, suggesting a targeting mechanism for this enigmatic enzyme during somatic hypermutation.

Processive AID-catalysed cytosine deamination on single-stranded DNA simulates somatic hypermutation

Activation-induced cytidine deaminase (AID) is a protein required for B cells to undergo class switch recombination and somatic hypermutation (SHM)—two processes essential for producing high-affinity antibodies. Purified AID catalyses the deamination of C to U on single-stranded (ss)DNA. Here, we show in vitro that AID-catalysed C deaminations occur preferentially on 5′ WRC sequences in accord with SHM spectra observed in vivo. Although about 98% of DNA clones suffer no mutations, most of the remaining mutated clones have 10–70 C to T transitions per clone. Therefore, AID carries out multiple C deaminations on individual DNA strands, rather than jumping from one strand to another. The avid binding of AID to ssDNA could result from its large net positive charge (+11) at pH 7.0, owing to a basic amino-terminal domain enriched in arginine and lysine. Furthermore, AID exhibits a 15-fold preference for C deamination on the non-transcribed DNA strand exposed by RNA polymerase than the transcribed strand protected as a RNA–DNA hybrid. These deamination results on ssDNA bear relevance to three characteristic features of SHM: preferential mutation at C sites within WRC hotspot sequences, the broad clonal mutagenic heterogeneity of antibody variable regions targeted for mutation, and the requirement for active transcription to obtain mutagenesis.

Crystal structure of the anti-viral APOBEC3G catalytic domain and functional implications.

The APOBEC family members are involved in diverse biological functions. APOBEC3G restricts the replication of human immunodeficiency virus (HIV), hepatitis B virus and retroelements by cytidine deamination on single-stranded DNA or by RNA binding. Here we report the high-resolution crystal structure of the carboxy-terminal deaminase domain of APOBEC3G (APOBEC3G-CD2) purified from Escherichia coli. The APOBEC3G-CD2 structure has a five-stranded β-sheet core that is common to all known deaminase structures and closely resembles the structure of another APOBEC protein, APOBEC2 (ref. 5). A comparison of APOBEC3G-CD2 with other deaminase structures shows a structural conservation of the active-site loops that are directly involved in substrate binding. In the X-ray structure, these APOBEC3G active-site loops form a continuous ‘substrate groove’ around the active centre. The orientation of this putative substrate groove differs markedly (by 90 degrees) from the groove predicted by the NMR structure. We have introduced mutations around the groove, and have identified residues involved in substrate specificity, single-stranded DNA binding and deaminase activity. These results provide a basis for understanding the underlying mechanisms of substrate specificity for the APOBEC family.

The APOBEC-2 crystal structure and functional implications for the deaminase AID.

APOBEC-2 (APO2) belongs to the family of apolipoprotein B messenger RNA-editing enzyme catalytic (APOBEC) polypeptides, which deaminates mRNA and single-stranded DNA. Different APOBEC members use the same deamination activity to achieve diverse human biological functions. Deamination by an APOBEC protein called activation-induced cytidine deaminase (AID) is critical for generating high-affinity antibodies, and deamination by APOBEC-3 proteins can inhibit retrotransposons and the replication of retroviruses such as human immunodeficiency virus and hepatitis B virus. Here we report the crystal structure of APO2. APO2 forms a rod-shaped tetramer that differs markedly from the square-shaped tetramer of the free nucleotide cytidine deaminase, with which APOBEC proteins share considerable sequence homology. In APO2, two long α-helices of a monomer structure prevent the formation of a square-shaped tetramer and facilitate formation of the rod-shaped tetramer via head-to-head interactions of two APO2 dimers. Extensive sequence homology among APOBEC family members allows us to test APO2 structure-based predictions using AID. We show that AID deamination activity is impaired by mutations predicted to interfere with oligomerization and substrate access. The structure suggests how mutations in patients with hyper-IgM-2 syndrome inactivate AID, resulting in defective antibody maturation.

Biochemical analysis of hypermutational targeting by wild type and mutant activation-induced cytidine deaminase.

The synthesis of high affinity antibodies requires activation-induced cytidine deaminase (AID) to initiate somatic hypermutation and class-switch recombination. Here we investigate AID-catalyzed deamination of C → U on single-stranded DNA and on actively transcribed closed circular double-stranded DNA. Mutations are initially favored at canonical WRC (W = A or T, R = A or G) somatic hypermutation hot spot motifs, but over time mutations at neighboring non-hot spot sites increase creating random clusters of mutated regions in a seemingly processive manner. N-terminal AID mutants R35E and R35E/R36D appear less processive and have altered mutational specificity compared with wild type AID. In contrast, a C-terminal deletion mutant defective in CSR in vivo closely resembles wild type AID. A mutational spectrum generated during transcription of closed circular double-stranded DNA indicates that wild type AID retains its specificity for WRC hot spot motifs within the confines of a moving transcription bubble while introducing clusters of multiple deaminations predominantly on the nontranscribed strand.

The current structural and functional understanding of APOBEC deaminases

The apolipoprotein B mRNA-editing enzyme catalytic polypeptide (APOBEC) family of cytidine deaminases has emerged as an intensively studied field as a result of their important biological functions. These enzymes are involved in lipid metabolism, antibody diversification, and the inhibition of retrotransposons, retroviruses, and some DNA viruses. The APOBEC proteins function in these roles by deaminating single-stranded (ss) DNA or RNA. There are two high-resolution crystal structures available for the APOBEC family, Apo2 and the C-terminal catalytic domain (CD2) of Apo3G or Apo3G-CD2 [Holden et al. (Nature 456:121–124, 2008); Prochnow et al. (Nature 445:447–451, 2007)]. Additionally, the structure of Apo3G-CD2 has also been determined using NMR [Chen et al. (Nature 452:116–119, 2008); Furukawa et al. (EMBO J 28:440–451, 2009); Harjes et al. (J Mol Biol, 2009)]. A detailed structural analysis of the APOBEC proteins and a comparison to other zinc-coordinating deaminases can facilitate our understanding of how APOBEC proteins bind nucleic acids, recognize substrates, and form oligomers. Here, we review the recent development of structural and functional studies that apply to Apo3G as well as the APOBEC deaminase family.

APOBEC deaminases-mutases with defensive roles for immunity.

In recent years, tremendous progress has been made in the elucidation of the biological roles and molecular mechanisms of the apolioprotein B mRNA-editing enzyme catalytic polypeptide (APOBEC) family of enzymes. The APOBEC family of cytidine deaminases has important functional roles within the adaptive and innate immune system. Activation induced cytidine deaminase (AID) plays a central role in the biochemical steps of somatic hypermutation and class switch recombination during antibody maturation, and the APOBEC 3 enzymes are able to inhibit the mobility of retroelements and the replication of retroviruses and DNA viruses, such as the human immunodeficiency virus type-1 and hepatitis B virus. Recent advances in structural and functional studies of the APOBEC enzymes provide new biochemical insights for how these enzymes carry out their biological roles. In this review, we provide an overview of these recent advances in the APOBEC field with a special emphasis on AID and APOBEC3G.

Lentivirus restriction by diverse primate APOBEC3A proteins

Rhesus macaque APOBEC3A (rhA3A) is capable of restricting both simian-human immunodeficiency virus (SHIVΔvif) and human immunodeficiency virus (HIV-1Δvif) to a greater extent than hA3A. We constructed chimeric A3A proteins to define the domains required for differential lentivirus restriction. Substitution of amino acids 25-33 from rhA3A into hA3A was sufficient to restrict HIVΔvif to levels similar to rhA3A restriction of SHIVΔvif. We tested if differential lentivirus restriction is conserved between A3A from Old World monkey and hominid lineages. A3A from African green monkey restricted SHIVΔvif but not HIV-1Δvif and colobus monkey A3A restricted both wild type and SHIVΔvif and HIV-1Δvif. In contrast, the gibbon ape A3A restricted neither SHIVΔvif nor HIV-1Δvif. Restriction of SHIVΔvif and HIV-1Δvif by New World monkey A3A proteins was not conserved as the A3A from the squirrel monkey but not the northern owl monkey restricted SHIVΔvif. Finally, the colobus A3A protein appears to restrict by a novel post-entry mechanism.

Somatic hypermutation: a mutational panacea.

Publisher Summary This chapter discusses activation-induced deaminase (AID), which was discovered using a subtractive hybridization screen for genes activated on induction of class switch recombination (CSR). AID expression is restricted to activated B cells and is required for somatic hypermutation (SHM) in mice. AID was the cause of human immune disorder, Hyper-IgM-2 syndrome, in which patients exhibited AID deficiency, resulting in the abolishment of both CSR and SHM. Hyper-IgM syndrome (HIGM) is characterized by normal or elevated serum IgM levels associated with the absence of IgG, IgA, and IgE isotypes as a result of defective CSR. Mutations in AID suggests that it functions as a multimer of two or four identical subunits and that specific domains in each peptide may be responsible for recruiting factors that mediate the outcome of SHM and CSR. Attempts to reconstitute the entire SHM process in a test tube are in their infancy. Transcription of AID is highly regulated during normal B-cell development. In humans and mice, its expression is restricted to centroblast cells within germinal centers, and mRNA production in naive B-cells, the cells that have not yet encountered antigen, occurs predominantly within 48 hours after in vitro stimulation. AID is homologous to the mRNA editing enzyme Apobec-1, sharing 34% of amino acid identity. By generating a diverse set of antibodies, the immune response is capable of providing ample protection.

The prospect of APOBEC3G for the future of HIV therapy

APOBEC3G (Apo3G), a cellular protein that has the ability to inhibit HIV, offers a new hope for fighting against HIV-1 infection. Recent advances in Apo3G structural and functional studies provide an opportunity for structurebased drug design and development to unleash the potent anti-HIV activity of Apo3G for AIDS prevention and therapy. Although the availability of antiretroviral regimens in the USA has significantly improved the life expectancy of patients presently receiving treatment, viral strains are emerging that are resistant to at least one or more of the six major classes of HIV drugs, which include inhibitors of viral entry, fusion, integrases, reverse transcriptase (nucleoside/nucleotide analogs and non-nucleoside inhibitors) and proteases [1]. Also troubling is the prevalent number of new infections that involve the transmission of drugresistant viral strains [1]. As HIV patients live longer, the emergence of drug-resistant HIV viral strains has become a more common occurrence. There is an urgent need for novel classes of HIV drugs that can inactivate the highly mutagenic HIV virus with high efficiency and less toxicity.