Mesoscale DNA features impact APOBEC3A and APOBEC3B deaminase activity and shape tumor mutational landscapes.

Antiviral DNA cytosine deaminases APOBEC3A and APOBEC3B are major sources of mutations in cancer by catalyzing cytosine-to-uracil deamination. APOBEC3A preferentially targets single-stranded DNAs, with a noted affinity for DNA regions that adopt stem-loop secondary structures. However, the detailed substrate preferences of APOBEC3A and APOBEC3B have not been fully established, and the specific influence of the DNA sequence on APOBEC3A and APOBEC3B deaminase activity remains to be investigated. Here, we find that APOBEC3B also selectively targets DNA stem-loop structures, and they are distinct from those subjected to deamination by APOBEC3A. We develop Oligo-seq, an in vitro sequencing-based method to identify specific sequence contexts promoting APOBEC3A and APOBEC3B activity. Through this approach, we demonstrate that APOBEC3A and APOBEC3B deaminase activity is strongly regulated by specific sequences surrounding the targeted cytosine. Moreover, we identify the structural features of APOBEC3B and APOBEC3A responsible for their substrate preferences. Importantly, we determine that APOBEC3B-induced mutations in hairpin-forming sequences within tumor genomes differ from the DNA stem-loop sequences mutated by APOBEC3A. Together, our study provides evidence that APOBEC3A and APOBEC3B can generate distinct mutation landscapes in cancer genomes, driven by their unique substrate selectivity.

Deciphering the role of Enterococcus faecium cytidine deaminase in gemcitabine resistance of gallbladder cancer.

Gemcitabine-based chemotherapy is a cornerstone of standard care for gallbladder cancer (GBC) treatment. Still, drug resistance remains a significant challenge, influenced by factors such as tumor-associated microbiota impacting drug concentrations within tumors. Enterococcus faecium, a member of tumor-associated microbiota, was notably enriched in the GBC patient cluster. In this study, we investigated the biochemical characteristics, catalytic activity, and kinetics of the cytidine deaminase of E. faecium (EfCDA). EfCDA showed the ability to convert gemcitabine to its metabolite 2',2'-difluorodeoxyuridine. Both EfCDA and E. faecium can induce gemcitabine resistance in GBC cells. Moreover, we determined the crystal structure of EfCDA, in its apo form and in complex with 2', 2'-difluorodeoxyuridine at high resolution. Mutation of key residues abolished the catalytic activity of EfCDA and reduced the gemcitabine resistance in GBC cells. Our findings provide structural insights into the molecular basis for recognizing gemcitabine metabolite by a bacteria CDA protein and may provide potential strategies to combat cancer drug resistance and improve the efficacy of gemcitabine-based chemotherapy in GBC treatment.

Structure-guided inhibition of the cancer DNA-mutating enzyme APOBEC3A.

The normally antiviral enzyme APOBEC3A is an endogenous mutagen in human cancer. Its single-stranded DNA C-to-U editing activity results in multiple mutagenic outcomes including signature single-base substitution mutations (isolated and clustered), DNA breakage, and larger-scale chromosomal aberrations. APOBEC3A inhibitors may therefore comprise a unique class of anti-cancer agents that work by blocking mutagenesis, slowing tumor evolvability, and preventing detrimental outcomes such as drug resistance and metastasis. Here we reveal the structural basis of competitive inhibition of wildtype APOBEC3A by hairpin DNA bearing 2'-deoxy-5-fluorozebularine in place of the cytidine in the TC substrate motif that is part of a 3-nucleotide loop. In addition, the structural basis of APOBEC3A's preference for YTCD motifs (Y = T, C; D = A, G, T) is explained. The nuclease-resistant phosphorothioated derivatives of these inhibitors have nanomolar potency in vitro and block APOBEC3A activity in human cells. These inhibitors may be useful probes for studying APOBEC3A activity in cellular systems and leading toward, potentially as conjuvants, next-generation, combinatorial anti-mutator and anti-cancer therapies.

Functions and consequences of AID/APOBEC-mediated DNA and RNA deamination.

The AID/APOBEC polynucleotide cytidine deaminases have historically been classified as either DNA mutators or RNA editors based on their first identified nucleic acid substrate preference. DNA mutators can generate functional diversity at antibody genes but also cause genomic instability in cancer. RNA editors can generate informational diversity in the transcriptome of innate immune cells, and of cancer cells. Members of both classes can act as antiviral restriction factors. Recent structural work has illuminated differences and similarities between AID/APOBEC enzymes that can catalyse DNA mutation, RNA editing or both, suggesting that the strict functional classification of members of this family should be reconsidered. As many of these enzymes have been employed for targeted genome (or transcriptome) editing, a more holistic understanding will help improve the design of therapeutically relevant programmable base editors.

The optimal pH of AID is skewed from that of its catalytic pocket by DNA-binding residues and surface charge.

Activation-induced cytidine deaminase (AID) is a member of the apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) family of cytidine deaminases. AID mutates immunoglobulin loci to initiate secondary antibody diversification. The APOBEC3 (A3) sub-branch mutates viral pathogens in the cytosol and acidic endosomal compartments. Accordingly, AID functions optimally near-neutral pH, while most A3s are acid-adapted (optimal pH 5.5-6.5). To gain a structural understanding for this pH disparity, we constructed high-resolution maps of AID catalytic activity vs pH. We found AID's optimal pH was 7.3 but it retained most (>70%) of the activity at pH 8. Probing of ssDNA-binding residues near the catalytic pocket, key for bending ssDNA into the pocket (e.g. R25) yielded mutants with altered pH preference, corroborating previous findings that the equivalent residue in APOBEC3G (H216) underlies its acidic pH preference. AID from bony fish exhibited more basic optimal pH (pH 7.5-8.1) and several R25-equivalent mutants altered pH preference. Comparison of pH optima across the AID/APOBEC3 family revealed an inverse correlation between positive surface charge and overall catalysis. The paralogue with the most robust catalytic activity (APOBEC3A) has the lowest surface charge and most acidic pH preference, while the paralogue with the most lethargic catalytic rate (AID) has the most positive surface charge and highest optimal pH. We suggest one possible mechanism is through surface charge dictating an overall optimal pH that is different from the optimal pH of the catalytic pocket microenvironment. These findings illuminate an additional structural mechanism that regulates AID/APOBEC3 mutagenesis.

Two different kinds of interaction modes of deaminase APOBEC3A with single-stranded DNA in solution detected by nuclear magnetic resonance.

APOBEC3A (A3A) deaminates deoxycytidine in target motif TC in a single-stranded DNA (we termed it as TC DNA), which mortally mutates viral pathogens and immunoglobulins, and leads to the diversification and lethality of cancers. The crystal structure of A3A-DNA revealed a unique U-shaped recognition mode of target base dC0 . However, when TC DNA was titrated into 15 N-labeled A3A solution, we observed two sets of 1 H-15 N cross-peaks of A3A in HSQC spectra, and two sets of 1 H-1 H cross-peaks of DNA in two-dimensional 13 C,15 N-filtered TOCSY spectra, indicating two different kinds of conformers of either A3A or TC DNA existing in solution. Here, mainly by NMR, we demonstrated that one DNA conformer interacted with one A3A conformer, forming a specific complex A3AS -DNAS in a way almost similar to that observed in the reported crystal A3A-DNA structure, where dC0 inserted into zinc ion binding center. While the other DNA conformer bound with another A3A conformer, but dC0 did not extend into the zinc-binding pocket, forming a nonspecific A3ANS -DNANS complex. The NMR solution structure implied three sites Asn61 , His182 and Arg189 were necessary to DNA recognition. These observations indicate a distinctive way from that reported in X-ray crystal structure, suggesting an unexpected mode of deaminase APOBEC3A to identify target motif TC in DNA in solution.

A split cytosine deaminase architecture enables robust inducible base editing.

Directed base substitution with base editing technology enables efficient and programmable conversion of C:G or A:T base pairs to T:A or G:C in the genome. Although this technology has shown great potentials in a variety of basic research, off-target editing is among one of the biggest challenges toward its way to clinical application. Base editing tools, especially the tools converting C to T, caused unpredictable off-target editing throughout the genome, which raise the concern that long-term application of these tools would induce genomic instability or even tumorigenesis. To overcome this challenge, we designed an inducible base editing tool that was active only in the presence of a clinically safe chemical, rapamycin. In the guidance of structural information, we designed four split-human APOBEC3A (A3A) -BE3 base editors in which these A3A deaminase enzymes were split at sites that were opposite to the protein-nucleotide interface. We showed that by inducible deaminase reconstruction with a rapamycin responsible interaction system (FRB and FKBP); three out of four split-A3A-derived base editors showed robust inducible base editing. However, in the absence of rapamycin, their editing ability was dramatically inhibited. Among these split editors, splicing at Aa85 of A3A generated the most efficient inducible editing. In addition, compared to the full-length base editor, the splitting did not obviously alter the editing window and motif preference, but slightly increased the product purity. We also expanded this strategy to another frequently used cytosine deaminase, rat APOBEC1 (rA1), and observed a similar induction response. In summary, these results demonstrated the concept that splitting deaminases is a practicable method for timely controlling of base editing tools.

Elucidation of the Complicated Scenario of Primate APOBEC3 Gene Evolution.

APOBEC3 proteins play pivotal roles in defenses against retroviruses, including HIV-1, as well as retrotransposons. Presumably due to the evolutionary arms race between the hosts and retroelements, APOBEC3 genes have rapidly evolved in primate lineages through sequence diversification, gene amplification and loss, and gene fusion. Consequently, modern primates possess a unique set or "repertoire" of APOBEC3 genes. The APOBEC3 gene repertoire of humans has been well investigated. There are three types of catalytic domains (Z domain; A3Z1, A3Z2, and A3Z3), 11 Z domains, and 7 independent genes, including 4 genes encoding double Z domains. However, the APOBEC3 gene repertoires of nonhuman primates remain largely unclear. Here, we characterize APOBEC3 gene repertoires among primates and investigated the evolutionary scenario of primate APOBEC3 genes using phylogenetic and comparative genomics approaches. In the 21 primate species investigated, we identified 145 APOBEC3 genes, including 69 double-domain type APOBEC3 genes. We further estimated the ages of the respective APOBEC3 genes and revealed that APOBEC3B, APOBEC3D, and APOBEC3F are the youngest in humans and were generated in the common ancestor of Catarrhini. Notably, invasion of the LINE1 retrotransposon peaked during the same period as the generation of these youngest APOBEC3 genes, implying that LINE1 invasion was one of the driving forces of the generation of these genes. Moreover, we found evidence suggesting that sequence diversification by gene conversions among APOBEC3 paralogs occurred in multiple primate lineages. Together, our analyses reveal the hidden diversity and the complicated evolutionary scenario of APOBEC3 genes in primates.IMPORTANCE In terms of virus-host interactions and coevolution, the APOBEC3 gene family is one of the most important subjects in the field of retrovirology. APOBEC3 genes are composed of a repertoire of subclasses based on sequence similarity, and a paper by LaRue et al. provides the standard guideline for the nomenclature and genomic architecture of APOBEC3 genes. However, it has been more than 10 years since this publication, and new information, including RefSeq, which we used in this study, is accumulating. Based on accumulating knowledge, APOBEC3 genes, particularly those of primates, should be refined and reannotated. This study updates knowledge of primate APOBEC3 genes and their genomic architectures. We further inferred the evolutionary scenario of primate APOBEC3 genes and the potential driving forces of APOBEC3 gene evolution. This study will be a landmark for the elucidation of the multiple aspects of APOBEC3 family genes in the future.

Small-Angle X-ray Scattering Models of APOBEC3B Catalytic Domain in a Complex with a Single-Stranded DNA Inhibitor.

In normal cells APOBEC3 (A3A-A3H) enzymes as part of the innate immune system deaminate cytosine to uracil on single-stranded DNA (ssDNA) to scramble DNA in order to give protection against a range of exogenous retroviruses, DNA-based parasites, and endogenous retroelements. However, some viruses and cancer cells use these enzymes, especially A3A and A3B, to escape the adaptive immune response and thereby lead to the evolution of drug resistance. We have synthesized first-in-class inhibitors featuring modified ssDNA. We present models based on small-angle X-ray scattering (SAXS) data that (1) confirm that the mode of binding of inhibitor to an active A3B C-terminal domain construct in the solution state is the same as the mode of binding substrate to inactive mutants of A3A and A3B revealed in X-ray crystal structures and (2) give insight into the disulfide-linked inactive dimer formed under the oxidizing conditions of purification.

Structural plasticity of substrate selection by activation-induced cytidine deaminase as a regulator of its genome-wide mutagenic activity.

Activation-induced cytidine deaminase (AID) mediates somatic hypermutation and class-switch recombination of antibodies. Computational-biochemical and crystallography analyses of AID have identified three surface grooves for binding single-stranded DNA (ssDNA). Functional studies have also found evidence for RNA-binding motifs on AID. Although AID and the related apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) enzymes share a conserved core, AID uniquely features multiple substrate-binding motifs on its surface. Here we suggest that combinatorial deployment of AID's multiple ssDNA- or RNA-binding motifs yields many substrate-binding modes that can accommodate ssDNA, RNA, or DNA/RNA substrates of diverse structures. We also suggest that AID oligomerization generates yet additional novel substrate-binding modes. We propose that this plasticity in substrate choice is an evolved aspect of AID's structure that contributes to the regulation of its differential mutagenic activity at various loci.

Alternative splicing of APOBEC3D generates functional diversity and its role as a DNA mutator.

The apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) protein family members have cytidine deaminase activity and can induce cytosine to uracil transition in nucleic acid. The main function of APOBEC3 (A3) proteins is to trigger an innate immune response to viral infections. Recent reports have shown that several APOBEC family proteins such as A3B can induce somatic mutations into genomic DNA and thus promote cancer development. However, the role of A3D on somatic mutations is unclear. Here, we identified the alternative splicing of A3D, and investigated each splice variant's subcellular localization and role in DNA mutagenesis. We identified four A3D variants, which all have one or two cytidine deaminase domains. The full-length form of A3D (variant 1) and truncated forms of A3D (variant 2, 6, 7) showed the ability to induce C/G to T/A transitions in foreign DNA and genomic DNA and retained antiretroviral activity. Furthermore, we demonstrated that A3D and A3B could induce deletions that are possibly repaired by microhomology-mediated end joining (MMEJ). Taken together, our experiments illustrated that alternative splicing generates functional diversity of A3D, and some variants can act as DNA mutators in genomic DNA.

APOBEC3C Tandem Domain Proteins Create Super Restriction Factors against HIV-1.

Humans encode proteins, called restriction factors, that inhibit replication of viruses such as HIV-1. The members of one family of antiviral proteins, apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3; shortened here to A3), act by deaminating cytidines to uridines during the reverse transcription reaction of HIV-1. The A3 locus encodes seven genes, named A3A to A3H These genes have either one or two cytidine deaminase domains, and several of these A3s potently restrict HIV-1. A3C, which has only a single cytidine deaminase domain, however, inhibits HIV-1 only very weakly. We tested novel double domain protein combinations by genetically linking two A3C genes to make a synthetic tandem domain protein. This protein created a "super restriction factor" that had more potent antiviral activity than the native A3C protein, which correlated with increased packaging into virions. Furthermore, disabling one of the active sites of the synthetic tandem domain protein resulted in an even greater increase in the antiviral activity-recapitulating a similar evolution seen in A3F and A3G (double domain A3s that use only a single catalytically active deaminase domain). These A3C tandem domain proteins do not have an increase in mutational activity but instead inhibit formation of reverse transcription products, which correlates with their ability to form large higher-order complexes in cells. Finally, the A3C-A3C super restriction factor largely escaped antagonism by the HIV-1 viral protein Vif.IMPORTANCE As a part of the innate immune system, humans encode proteins that inhibit viruses such as HIV-1. These broadly acting antiviral proteins do not protect humans from viral infections because viruses encode proteins that antagonize the host antiviral proteins to evade the innate immune system. One such example of a host antiviral protein is APOBEC3C (A3C), which weakly inhibits HIV-1. Here, we show that we can improve the antiviral activity of A3C by duplicating the DNA sequence to create a synthetic tandem domain and, furthermore, that the proteins thus generated are relatively resistant to the viral antagonist Vif. Together, these data give insights about how nature has evolved a defense against viral pathogens such as HIV.

Deamination hotspots among APOBEC3 family members are defined by both target site sequence context and ssDNA secondary structure.

The human apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3 (APOBEC3, A3) family member proteins can deaminate cytosines in single-strand (ss) DNA, which restricts human immunodeficiency virus type 1 (HIV-1), retrotransposons, and other viruses such as hepatitis B virus, but can cause a mutator phenotype in many cancers. While structural information exists for several A3 proteins, the precise details regarding deamination target selection are not fully understood. Here, we report the first parallel, comparative analysis of site selection of A3 deamination using six of the seven purified A3 member enzymes, oligonucleotides having 5'TC3' or 5'CT3' dinucleotide target sites, and different flanking bases within diverse DNA secondary structures. A3A, A3F and A3H were observed to have strong preferences toward the TC target flanked by A or T, while all examined A3 proteins did not show a preference for a TC target flanked by a G. We observed that the TC target was strongly preferred in ssDNA regions rather than dsDNA, loop or bulge regions, with flanking bases influencing the degree of preference. CT was also shown to be a potential deamination target. Taken together, our observations provide new insights into A3 enzyme target site selection and how A3 mutagenesis impacts mutation rates.

An insight into the dependence of the deamination rate of human APOBEC3F on the length of single-stranded DNA, which is affected by the concentrations of APOBEC3F and single-stranded DNA.

BACKGROUND: APOBEC3F (A3F), a member of the human APOBEC3 (A3) family of cytidine deaminases, acts as an anti-HIV-1 factor by deaminating deoxycytidine in the complementary DNA of the viral genome. A full understanding of the deamination behavior of A3F awaits further investigation. METHODS: The real-time NMR method and uracil-DNA glycosylase assay were used to track the activities of the C-terminal domain (CTD) of A3F at different concentrations of A3F-CTD and ssDNA. The steady-state fluorescence anisotropy measurement was used to examine the binding between A3F-CTD and ssDNA with different lengths. The use of the A3F-CTD N214H mutant, having higher activity than the wild-type, facilitated the tracking of the reactions. RESULTS: A3F-CTD was found to efficiently deaminate the target deoxycytidine in long ssDNA in lower ssDNA concentration conditions ([A3F-CTD] ≫ [ssDNA]), while the target deoxycytidine in short ssDNA is deaminated efficiently in higher ssDNA concentration conditions ([A3F-CTD] ≪ [ssDNA]). This property is quite different from that of the previously studied A3 family member, A3B; the concentrations of the proteins and ssDNA had no effect. CONCLUSIONS: The concentrations of A3F-CTD and ssDNA substrates affect the ssDNA-length-dependence of deamination rate of the A3F-CTD. This unique property of A3F is rationally interpreted on the basis of its binding characteristics with ssDNA. GENERAL SIGNIFICANCE: The discovery of the unique property of A3F regarding the deamination rate deepens the understanding of its counteraction against HIV-1. Our strategy is applicable to investigate the other aspects of the A3 activities, such as those involved in the cancer development.

Structural perspectives on HIV-1 Vif and APOBEC3 restriction factor interactions.

Human immunodeficiency virus (HIV) is a retroviral pathogen that targets human immune cells such as CD4+ T cells, macrophages, and dendritic cells. The human apolipoprotein B mRNA- editing catalytic polypeptide 3 (APOBEC3 or A3) cytidine deaminases are a key class of intrinsic restriction factors that inhibit replication of HIV. When HIV-1 enters the cell, the immune system responds by inducing the activation of the A3 family proteins, which convert cytosines to uracils in single-stranded DNA replication intermediates, neutralizing the virus. HIV counteracts this intrinsic immune response by encoding a protein termed viral infectivity factor (Vif). Vif targets A3 to an E3 ubiquitin ligase complex for poly-ubiquitination and proteasomal degradation. Vif is unique in that it can recognize and counteract multiple A3 restriction factor substrates. Structural biology studies have provided significant insights into the overall architectures and functions of Vif and A3 proteins; however, a structure of the Vif-A3 complex has remained elusive. In this review, we summarize and reanalyze experimental data from recent structural, biochemical, and functional studies to provide key perspectives on the residues involved in Vif-A3 protein-protein interactions.

AID, APOBEC3A and APOBEC3B efficiently deaminate deoxycytidines neighboring DNA damage induced by oxidation or alkylation.

BACKGROUND: AID/APOBEC3 (A3) enzymes instigate genomic mutations that are involved in immunity and cancer. Although they can deaminate any deoxycytidine (dC) to deoxyuridine (dU), each family member has a signature preference determined by nucleotides surrounding the target dC. This WRC (W = A/T, R = A/G) and YC (Y = T/C) hotspot preference is established for AID and A3A/A3B, respectively. Base alkylation and oxidation are two of the most common types of DNA damage induced environmentally or by chemotherapy. Here we examined the activity of AID, A3A and A3B on dCs neighboring such damaged bases. METHODS: Substrates were designed to contain target dCs either in normal WRC/YC hotspots, or in oxidized/alkylated DNA motifs. AID, A3A and A3B were purified and deamination kinetics of each were compared between substrates containing damaged vs. normal motifs. RESULTS: All three enzymes efficiently deaminated dC when common damaged bases were present in the -2 or -1 positions. Strikingly, some damaged motifs supported comparable or higher catalytic efficiencies by AID, A3A and A3B than the WRC/YC motifs which are their most favored normal sequences. Based on the resolved interactions of AID, A3A and A3B with DNA, we modeled interactions with alkylated or oxidized bases. Corroborating the enzyme assay data, the surface regions that recognize normal bases are predicted to also interact robustly with oxidized and alkylated bases. CONCLUSIONS: AID, A3A and A3B can efficiently recognize and deaminate dC whose neighbouring nucleotides are damaged. GENERAL SIGNIFICANCE: Beyond AID/A3s initiating DNA damage, some forms of pre-existing damaged DNA can constitute favored targets of AID/A3s if encountered.

APOBEC3A Loop 1 Is a Determinant for Single-Stranded DNA Binding and Deamination.

The apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3 (APOBEC3 or A3) family of proteins functions in the innate immune system. The A3 proteins are interferon inducible and hypermutate deoxycytidine to deoxyuridine in foreign single-stranded DNA (ssDNA). However, this deaminase activity cannot discriminate between foreign and host ssDNA at the biochemical level, which presents a significant danger when A3 proteins gain access to the nucleus. Interestingly, this A3 capability can be harnessed when coupled with novel CRISPR-Cas9 proteins to create a targeted base editor. Specifically, A3A has been used in vitro to revert mutations associated with disease states. Recent structural studies have shown the importance of loop regions of A3A and A3G in ssDNA recognition and positioning for deamination. In this work, we further examined loop 1 of A3A to determine how it affects substrate selection, as well as the efficiency of deamination, in the hopes of advancing the potential of A3A in base editing technology. We found that mutating residue H29 enhanced deamination activity without changing substrate specificity. Also interestingly, we found that increasing the length of loop 1 decreases substrate specificity. Overall, these results lead to a better understanding of substrate recognition and deamination by A3A and the A3 family of proteins.

Conformational Dynamics of the HIV-Vif Protein Complex.

Human immunodeficiency virus-1 viral infectivity factor (Vif) is an intrinsically disordered protein responsible for the ubiquitination of the APOBEC3 (A3) antiviral proteins. Vif folds when it binds Cullin-RING E3 ligase 5 and the transcription cofactor CBF-ß. A five-protein complex containing the substrate receptor (Vif, CBF-ß, Elongin-B, Elongin-C (VCBC)) and Cullin5 (CUL5) has a published crystal structure, but dynamics of this VCBC-CUL5 complex have not been characterized. Here, we use molecular dynamics (MD) simulations and NMR to characterize the dynamics of the VCBC complex with and without CUL5 and an A3 protein bound. Our simulations show that the VCBC complex undergoes global dynamics involving twisting and clamshell opening of the complex, whereas VCBC-CUL5 maintains a more static conformation, similar to the crystal structure. This observation from MD is supported by methyl-transverse relaxation-optimized spectroscopy NMR data, which indicates that the VCBC complex without CUL5 is dynamic on the µs-ms timescale. Our NMR data also show that the VCBC complex is more conformationally restricted when bound to the antiviral APOBEC3F (one of the A3 proteins), consistent with our MD simulations. Vif contains a flexible linker region located at the hinge of the VCBC complex, which changes conformation in conjunction with the global dynamics of the complex. Like other substrate receptors, VCBC can exist alone or in complex with CUL5 and other proteins in cells. Accordingly, the VCBC complex could be a good target for therapeutics that would inhibit full assembly of the ubiquitination complex by stabilizing an alternate VCBC conformation.

Determinants of Oligonucleotide Selectivity of APOBEC3B.

APOBEC3B (A3B) is a prominent source of mutation in many cancers. To date, it has been difficult to capture the native protein-DNA interactions that confer A3B's substrate specificity by crystallography due to the highly dynamic nature of wild-type A3B active site. We use computational tools to restore a recent crystal structure of a DNA-bound A3B C-terminal domain mutant construct to its wild type sequence, and run molecular dynamics simulations to study its substrate recognition mechanisms. Analysis of these simulations reveal dynamics of the native A3Bctd-oligonucleotide interactions, including the experimentally inaccessible loop 1-oligonucleotide interactions. A second series of simulations in which the target cytosine nucleotide was computationally mutated from a deoxyribose to a ribose show a change in sugar ring pucker, leading to a rearrangement of the binding site and revealing a potential intermediate in the binding pathway. Finally, apo simulations of A3B, starting from the DNA-bound open state, experience a rapid and consistent closure of the binding site, reaching conformations incompatible with substrate binding. This study reveals a more realistic and dynamic view of the wild type A3B binding site and provides novel insights for structure-guided design efforts for A3B.

Structural Analysis of the Active Site and DNA Binding of Human Cytidine Deaminase APOBEC3B.

APOBEC3 (A3) proteins, a family of human cytidine deaminases, protect the host from endogenous retro-elements and exogenous viral infections by introducing hypermutations. However, overexpressed A3s can modify genomic DNA to promote tumorigenesis, especially A3B. Despite their overall similarity, A3 proteins have distinct deamination activity. Recently determined A3 structures have revealed the molecular determinants of nucleotide specificity and DNA binding. However, for A3B, the structural basis for regulation of deamination activity and the role of active site loops in coordinating DNA had remained unknown. Using advanced molecular modeling followed by experimental mutational analysis and dynamics simulations, we investigated the molecular mechanism of DNA binding by A3B-CTD. We modeled fully native A3B-DNA structure, and we identified Arg211 in loop 1 as the gatekeeper coordinating DNA and critical residue for nucleotide specificity. We also identified a unique autoinhibited conformation in A3B-CTD that restricts access and binding of DNA to the active site. Our results reveal the structural basis for DNA binding and relatively lower catalytic activity of A3B and provide opportunities for rational design of specific inhibitors to benefit cancer therapeutics.