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Human RNA editing enzyme ADAR1 deaminates adenosine in pre-mRNA to yield inosine. The Zα domain of human ADAR1 (hZα ADAR1 ) binds specifically to left-handed Z-RNA as well as Z-DNA and stabilizes the Z-conformation. To answer the question of how hZα ADAR1 can induce both the B-Z transition of DNA and the A-Z transition of RNA, we investigated the structure and dynamics of hZα ADAR1 in complex with 6-base-pair Z-DNA or Z-RNA. We performed chemical shift perturbation and relaxation dispersion experiments on hZα ADAR1 upon binding to Z-DNA as well as Z-RNA. Our study demonstrates the unique dynamics of hZα ADAR1 during the A-Z transition of RNA, in which the hZα ADAR1 protein forms a thermodynamically stable complex with Z-RNA, similar to Z-DNA, but kinetically converts RNA to the Z-form more slowly than DNA. We also discovered some distinct structural features of hZα ADAR1 in the Z-RNA binding conformation. Our results suggest that the A-Z transition of RNA facilitated by hZα ADAR1 displays unique structural and dynamic features that may be involved in targeting ADAR1 for a role in recognition of RNA substrates.

The Zα domains of human ADAR1 (ZαADAR1) bind to Z-DNA via interaction mediated by the α3-core and β-hairpin. Five residues in the α3 helix and four residues in the β-hairpin play important roles in Zα function, forming direct or water-mediated hydrogen bonds with DNA backbone phosphates or interacting hydrophobically with DNA bases. To understand the roles of these residues during B-Z transition of duplex DNA, we performed NMR experiments on complexes of various ZαADAR1 mutants with a 6-bp DNA duplex at various protein-to-DNA molar ratios. Our study suggests that single mutations at residues K169, N173, or Y177 cause unusual conformational changes in the hydrophobic faces of helices α1, α2, and α3, which dramatically decrease the Z-DNA binding affinity. 1D imino proton spectra and chemical shift perturbation showed that single mutations at residues K170, R174, T191, P192, P193, or W195 slightly affected the Z-DNA binding affinity. A hydrogen exchange study proved that the K170A- and R174A-ZαADAR1 proteins could efficiently change B-DNA to left-handed Z-DNA via an active B-Z transition pathway, whereas the G2·C5 base pair was significantly destabilized compared to wild-type ZαADAR1., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The Z-DNA binding domain of human ADAR1 (Zα(ADAR1)) preferentially binds Z-DNA rather than B-DNA with high binding affinity. Here, we have carried out chemical shift perturbation and backbone dynamics studies of Zα(ADAR1) in the free form and in complex with three DNA duplexes, d(CGCGCG)(2), d(CACGTG)(2), and d(CGTACG)(2). This study reveals that Zα(ADAR1) initially binds to d(CGCGCG)(2) through the distinct conformation, especially in the unusually flexible β1-loop-α2 region, from the d(CGCGCG)(2)-(Zα(ADAR1))(2) complex. This study also suggests that Zα(ADAR1) exhibits a distinct conformational change during the B-Z transition of non-CG-repeat DNA duplexes with low binding affinities compared to the CG-repeat DNA duplex., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Z-DNA is produced in a long genomic DNA by Z-DNA binding proteins, through formation of two B-Z junctions with the extrusion of one base pair from each junction. To answer the question of how Z-DNA binding proteins induce B-Z transitions in CG-rich segments while maintaining the B-conformation of surrounding segments, we investigated the kinetics and thermodynamics of base-pair openings of a 13-bp DNA in complex with the Z-DNA binding protein, Zα(ADAR1). We also studied perturbations in the backbone of Zα(ADAR1) upon binding to DNA. Our study demonstrates the initial contact conformation as an intermediate structure during B-Z junction formation induced by Zα(ADAR1), in which the Zα(ADAR1) protein displays unique backbone conformational changes, but the 13-bp DNA duplex maintains the B-form helix. We also found the unique structural features of the 13-bp DNA duplex in the initial contact conformation: (i) instability of the AT-rich region II and (ii) longer lifetime for the opening state of the CG-rich region I. Our findings suggest a three-step mechanism of B-Z junction formation: (i) Zα(ADAR1) specifically interacts with a CG-rich DNA segment maintaining B-form helix via a unique conformation; (ii) the neighboring AT-rich region becomes very unstable, and the CG-rich DNA segment is easily converted to Z-DNA; and (iii) the AT-rich regions are base-paired again, and the B-Z junction structure is formed.

The Zα domain of human ADAR1 (Zα(ADAR1)) preferentially binds Z-DNA rather than B-DNA with high binding affinity. Zα(ADAR1) binds to the Z-conformation of both non-CG-repeat DNA duplexes and a d(CGCGCG)(2) duplex similarly. We performed NMR experiments on complexes between the Zα(ADAR1) and non-CG-repeat DNA duplexes, d(CACGTG)(2) or d(CGTACG)(2), with a variety of protein-DNA molar ratios. Comparison of these results with those from the analysis of d(CGCGCG)(2) in the previous study suggests that Zα(ADAR1) exhibits the sequence preference of d(CGCGCG)(2)≫d(CACGTG)(2)>d(CGTACG)(2) through multiple sequence discrimination steps during the B-Z transition., (Copyright © 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

The human RNA editing enzyme ADAR1 (double-stranded RNA deaminase I) deaminates adenine in pre-mRNA to yield inosine, which codes as guanine. ADAR1 has two left-handed Z-DNA binding domains, Z alpha and Z beta, at its NH(2)-terminus and preferentially binds Z-DNA, rather than B-DNA, with high binding affinity. The cocrystal structure of Z alpha(ADAR1) complexed to Z-DNA showed that one monomeric Z alpha(ADAR1) domain binds to one strand of double-stranded DNA and a second Z alpha(ADAR1) monomer binds to the opposite strand with 2-fold symmetry with respect to DNA helical axis. It remains unclear how Z alpha(ADAR1) protein specifically recognizes Z-DNA sequence in a sea of B-DNA to produce the stable Z alpha(ADAR1)-Z-DNA complex during the B-Z transition induced by Z alpha(ADAR1). In order to characterize the molecular recognition of Z-DNA by Z alpha(ADAR1), we performed circular dichroism (CD) and NMR experiments with complexes of Zalpha(ADAR1) bound to d(CGCGCG)(2) (referred to as CG6) produced at a variety of protein-to-DNA molar ratios. From this study, we identified the intermediate states of the CG6-Z alpha(ADAR1) complex and calculated their relative populations as a function of the Z alpha(ADAR1) concentration. These findings support an active B-Z transition mechanism in which the Z alpha(ADAR1) protein first binds to B-DNA and then converts it to left-handed Z-DNA, a conformation that is then stabilized by the additional binding of a second Z alpha(ADAR1) molecule.