NMR spectroscopic elucidation of the B-Z transition of a DNA double helix induced by the Zα domain of human ADAR1

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, Zα and Zβ, at its NH ₂-terminus and preferentially binds Z-DNA, rather than B-DNA, with high binding affinity. The cocrystal structure of Zα_ADAR1 complexed to Z-DNA showed that one monomeric Zα_ADAR1 domain binds to one strand of double-stranded DNA and a second Zα_ADAR1 monomer binds to the opposite strand with 2-fold symmetry with respect to DNA helical axis. It remains unclear how Zα_ADAR1 protein specifically recognizes Z-DNA sequence in a sea of B-DNA to produce the stable Zα_ADAR1-Z-DNA complex during the B-Z transition induced by Zα_ADAR1. In order to characterize the molecular recognition of Z-DNA by Zα_ADAR1, we performed circular dichroism (CD) and NMR experiments with complexes of Zα_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-Zα_ADAR1 complex and calculated their relative populations as a function of the Zα_ADAR1 concentration. These findings support an active B-Z transition mechanism in which the Zα_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 Zα_ADAR1 molecule.