NMR Exchange Study of Z-DNA Duplexes Induced by the Zα Domain of Human ADAR1 Containing Mutation on the β-Hairpin

Abstract

Left-handed Z-DNA forms in polymers of alternating d(CG)n sequence and is a higher energy conformation than B-DNA. Z-DNA is stabilized by high salt, negative supercoiling, and complex formation with Z-DNA binding proteins. The human double-stranded RNA deaminase I (ADAR1) contains two Z-DNA binding domains (Zα and Zβ) at the NH2-terminus. 4,5 The crystal structure of the Zα domain of human ADAR1 (ZαADAR1) complexed to Z-DNA showed that each of two Zα domains binds to one strand of a double-stranded DNA, yielding two-fold symmetry with respect to the DNA helical axis. This study also revealed that the intermolecular interaction is mediated by 5 residues in the α3 helix and 4 residues in the β-hairpin (Fig. 1). This is confirmed in solution by the significant backbone chemical shift changes of most residues in the α3 helix and β2-loop-β3 region upon binding to Z-DNA. These residues play important roles in Zα function and thus replacement of them with an alanine caused a dramatic decrease in Z-DNA binding affinity compared to wild-type ZαADAR1 (wt-ZαADAR1). 5 Previous NMR study suggested 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. This study also found that the K170A and R174A mutants significantly destabilize the G2·C5 base pair compared to wt-ZαADAR1, even though these mutants could efficiently change B-DNA to left-handed Z-DNA. However, the question of how the mutants on the β-hairpin affect the base pair stabilities of the Z-DNA remains. Here, we determined hydrogen exchange rate constants (kex) for the imino protons of the d(CG)3 in the complexes with ZαADAR1 containing mutation on the β-hairpin. Comparison of these results to previous studies of the mutant ZαADAR1-d(CG)3 complexes 8 provides the information required to identify the role of each residue in the β-hairpin during BZ transition of DNA duplexes.