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Functional Requirements of AID's Higher Order Structures And Functional requirements of AID’s higher order PNAS PLUS structures and their interaction with RNA-binding proteins Samiran Mondala, Nasim A. Beguma, Wenjun Hua, and Tasuku Honjoa,1 aDepartment of Immunology and Genomic Medicine, Graduate School of Medicine, Kyoto University, Yoshida Sakyo-ku, Kyoto 606-8501, Japan Contributed by Tasuku Honjo, February 3, 2016 (sent for review October 27, 2015; reviewed by Atsushi Miyawaki and Kazuko Nishikura) Activation-induced cytidine deaminase (AID) is essential for the interaction, enabling AID to exert distinct physiological functions somatic hypermutation (SHM) and class-switch recombination (CSR) through its association with cofactors. Regrettably, however, there of Ig genes. Although both the N and C termini of AID have unique is little structural information available that can explain any of functions in DNA cleavage and recombination, respectively, during AID’s regulatory modes of action, including its cofactor association SHM and CSR, their molecular mechanisms are poorly understood. mechanisms, in the context of its physiological functions. Using a bimolecular fluorescence complementation (BiFC) assay Although a significant amount of structural information is available combined with glycerol gradient fractionation, we revealed that for a number of APOBEC family members, the 3D structures of A1 the AID C terminus is required for a stable dimer formation. Further- and AID are yet to be resolved (19, 20). The CDD family of enzymes more, AID monomers and dimers form complexes with distinct exists in nature in a variety of structural forms, including monomeric, dimeric, and tetrameric forms, and comparative structural modeling heterogeneous nuclear ribonucleoproteins (hnRNPs). AID monomers using the yeast CDD structure predicts a dimeric structure for both associate with DNA cleavage cofactor hnRNP K whereas AID dimers A1 and AID (21, 22). On the other hand, homology modeling with associate with recombination cofactors hnRNP L, hnRNP U, and the APOBEC2 (A2) crystal structure, which seems to be a tetramer Serpine mRNA-binding protein 1. All of these AID/ribonucleoprotein composed of two head-to-head interacting dimers, predicts that AID ’ associations are RNA-dependent. We propose that AID s structure- forms a tetramer (23). Notably, A2waslaterreportedtoexistasa specific cofactor complex formations differentially contribute to its monomer in solution (24). Similarly, an atomic force microscopic DNA-cleavage and recombination functions. (AFM) study found that AID exists in the cell predominantly as a monomer associated with a single-strand DNA substrate (25). AID | BiFC | hnRNP U | SERBP1 | APOBEC However, the same AFM dataset was interpreted differently by an- other group of investigators, who concluded that AID probably forms ’ ctivation-induced cytidine deaminase (AID), which is expressed an A2-like tetramer in solution (26). The modeling of AID scatalytic pocket in reference to eight APOBEC family members suggested Ain antigen-stimulated mature B cells, is essential for Ig somatic – hypermutation (SHM) and class-switch recombination (CSR) (1, 2). that most of the AID DNA complex remains in an inactive state due AID induces DNA breaks at the variable (V) and switch (S) regions to occlusion by the substrate DNA, which may explain its weak cat- alytic activity for cleaving DNA in vitro (27). during SHM and CSR, respectively (3, 4). Although both processes One of the limitations of the computational modeling of AID’s are initiated by AID-induced DNA cleavage, point mutations at the structure is that AID’s N-and C-terminal sequences are sub- V region are executed mostly by error-prone DNA repair whereas stantially different from those of other APOBEC members and CSR is accomplished by recombination of cleaved ends at donor thus reside outside the modeling template. Although the struc- and acceptor S regions (5, 6). However, the detailed mechanisms tural outcome of a protein can differ by a variety of reasons, by which AID carries out the two mechanistically distinct functions including the methods applied (28), none of the AID studies for SHM and CSR have yet to be uncovered (7). Studies on AID mentioned above explain why the C-terminal deletion of AID ’ INFLAMMATION mutants revealed that AID s N- and C-terminal domains are dis- leads to the loss of CSR function only. Therefore, model-based IMMUNOLOGY AND tinctly required for its DNA-cleavage and recombination functions, computational simulation may not explain the physiological respectively (8–10). Mutations at the N terminus of AID impair structure–function relationship of AID in B cells. SHM as well as CSR whereas those at the C terminus abrogate Here, we explored AID’s structure–function relationship using CSR only and show increased SHM activity. Recent studies dem- a bimolecular fluorescence complementation (BiFC) assay, onstrated that the CSR process after DNA cleavage, including the synapsis formation between cleaved ends, is impaired with the Significance C-terminally defective AID, indicating that AID’s C terminus confers a CSR-specific recombination function, independent of ’ This paper demonstrates that activation-induced cytidine de- AID s DNA cleavage function, by an unknown mechanism (11, 12). aminase (AID), an essential enzyme in antigen-induced antibody AID belongs to the APOBEC (apolipoprotein B mRNA-editing diversification, forms distinct ribonucleoprotein complexes de- enzyme catalytic polypeptide) family of cytidine deaminases pending on its structural states: namely monomers or dimers. The (CDDs) and shows high sequence homology with APOBEC1 (A1) identified RNA-binding proteins are required for the function of (1, 13, 14), which edits apolipoprotein B (APOB) mRNA. The AID: namely DNA cleavage or recombination. In addition, the APOB mRNA editing ability of A1 is highly dependent on its complex formation between AID and heterogeneous nuclear ri- cofactors, A1CF/ACF (15, 16) and RBM47 (17), both of which bonucleoproteins (hnRNPs) is RNA-dependent. belong to the heterogeneous nuclear ribonucleoprotein (hnRNP) family. Recently, two A1CF-like hnRNPs, hnRNP K and hnRNP Author contributions: S.M., N.A.B., and T.H. designed research; S.M., N.A.B., and W.H. L, were identified as the cofactors of AID and found to be in- performed research; N.A.B. contributed new reagents/analytic tools; S.M. analyzed data; volved in the cleavage and recombination of DNA, respectively and S.M., N.A.B., and T.H. wrote the paper. (18). Because the N and C termini of AID differentially regulate Reviewers: A.M., Brain Science Institute of RIKEN; and K.N., The Wistar Institute. two functions of AID—cleavage and recombination, respectively— The authors declare no conflict of interest. we speculated that the AID termini would be critical for function- 1To whom correspondence should be addressed. Email: [email protected]. coupled cofactor association. For instance, the N or C terminus of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. AID may function as a molecular switch that induces an AID–AID 1073/pnas.1601678113/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1601678113 PNAS | Published online February 29, 2016 | E1545–E1554 Downloaded by guest on September 23, 2021 which detects homo- or heteromeric protein–protein interactions expression amount of all of the four constructs (Fig. 1D). We in live cells (29, 30). For the homomeric interaction assay, the observed that none of the fusion constructs alone produced a target protein is fused to two nonfluorescent halves of a green or fluorescence signal above background. We also verified that the red fluorescent protein. An interaction between two of the target coexpression of mKG.N and mKG.C did not produce any BiFC proteins brings the two nonfluorescent halves of the fluorescent signal (Fig. S1D). Furthermore, the efficiency of the AID–AID protein into close proximity, reconstituting the fluorescence. The interaction was assessed by a direct co-IP analysis (Fig. 1E), which BiFC assay thus allows a rapid analysis of the dimerization of a mostly agreed with the BiFC MFI profile (Fig. 1 B and C). protein of interest in live cells. Fluorescence microscopic observation showed that the BiFC sig- By combining this assay with other biochemical approaches, nal of AID was localized to the cytoplasm (Fig. S1C), which was such as coimmunoprecipitation (co-IP) and glycerol gradient previously shown to be the major localization site of AID (Fig. sedimentation, we revealed the presence of both monomeric and S1C) (41). The expected subcellular localizations were observed dimeric forms of AID in analyzed cells. Intriguingly, C-terminal for the other APOBECs, confirming that the BiFC signals rep- AID mutants that lost CSR function showed a severe dimerization resent native characteristics of the APOBEC proteins. defect, suggesting that AID’s C terminus is required to stabilize the dimeric structure that is required for CSR. We also showed AID C-Terminal Mutants That Are Defective in CSR Are Unable to Form a that the AID monomer and dimer associate with different RNA- Dimer. To evaluate the functional relevance of AID dimer for- binding proteins (RBPs) to form ribonucleoprotein (RNP) com- mation, we examined the BiFC signals using AID mutants that plexes. Based on these findings, we propose that the monomeric showed clear functional defects. AID C-terminal mutants are of AID–RNP complex includes hnRNP K (18) and contributes to the particular importance because they lose CSR function but retain DNA cleavage function of AID whereas the dimeric AID–RNP strong SHM activity (Fig. 2A). Such well-characterized C-terminal complexes include hnRNP L (18), hnRNP U (31), or Serpine AID mutants include JP8Bdel (R183X), P20 (34-aa insertion at mRNA-binding protein 1 (SERBP1) (32) and contribute to the residue 182), JP41 (R190X), and JP8B (26-aa frameshift re- recombination step of CSR. placement at residue 183) (8). Remarkably, each mutant showed severely defective BiFC signal generation in all four pairwise Results combinations even though they were all expressed well (Fig. 2 B and Detection of APOBEC Homodimer Formation by the Monomeric Kusabira C).
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