Zα Domain Of Human ADAR1 Research Articles

Conformational exchange of the Zα domain of human RNA editing enzyme ADAR1 studied by NMR spectroscopy

Z-DNA binding proteins (ZBPs) play important roles in RNA editing, innate immune responses, and viral infections. Numerous studies have implicated a role for conformational motions during ZBPs binding upon DNA, but the quantitative intrinsic conformational exchanges of ZBP have not been elucidated. To understand the correlation between the biological function and dynamic feature of the Zα domains of human ADAR1 (hZαADAR1), we have performed the 15N backbone amide Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments on the free hZαADAR1 at two different magnetic fields at 35 °C. The robust inter-dependence of parameters in the global fitting process using multi-magnetic field CPMG profiles allows us characterizing the dynamic properties of conformational changes in hZαADAR1. This study found that free hZαADAR1 exhibited the conformational exchange with a kex of 5784 s−1 between the states “A” (89% population) and “B” (11% population). The different hydrophobic interactions among helices α1, α2, and α3 between these two states might correlate with efficient Z-DNA binding achieved by the hydrogen bonding interactions between its side-chains and the phosphate backbone of Z-DNA.

Systematic Approach to Find the Global Minimum of Relaxation Dispersion Data for Protein-Induced B-Z Transition of DNA.

Carr–Purcell–Meiboom–Gill (CPMG) relaxation dispersion spectroscopy is commonly used for quantifying conformational changes of protein in μs-to-ms timescale transitions. To elucidate the dynamics and mechanism of protein binding, parameters implementing CPMG relaxation dispersion results must be appropriately determined. Building an analytical model for multi-state transitions is particularly complex. In this study, we developed a new global search algorithm that incorporates a random search approach combined with a field-dependent global parameterization method. The robust inter-dependence of the parameters carrying out the global search for individual residues (GSIR) or the global search for total residues (GSTR) provides information on the global minimum of the conformational transition process of the Zα domain of human ADAR1 (hZαADAR1)–DNA complex. The global search results indicated that a α-helical segment of hZαADAR1 provided the main contribution to the three-state conformational changes of a hZαADAR1—DNA complex with a slow B–Z exchange process. The two global exchange rate constants, kex and kZB, were found to be 844 and 9.8 s−1, respectively, in agreement with two regimes of residue-dependent chemical shift differences—the “dominant oscillatory regime” and “semi-oscillatory regime”. We anticipate that our global search approach will lead to the development of quantification methods for conformational changes not only in Z-DNA binding protein (ZBP) binding interactions but also in various protein binding processes.

NMR Dynamics Study Reveals the Zα Domain of Human ADAR1 Associates with and Dissociates from Z-RNA More Slowly than Z-DNA.

Human RNA editing enzyme ADAR1 deaminates adenosine in pre-mRNA to yield inosine. The Zα domain of human ADAR1 (hZαADAR1) binds specifically to left-handed Z-RNA as well as Z-DNA and stabilizes the Z-conformation. To answer the question of how hZαADAR1 can induce both the B-Z transition of DNA and the A-Z transition of RNA, we investigated the structure and dynamics of hZαADAR1 in complex with 6-base-pair Z-DNA or Z-RNA. We performed chemical shift perturbation and relaxation dispersion experiments on hZαADAR1 upon binding to Z-DNA as well as Z-RNA. Our study demonstrates the unique dynamics of hZαADAR1 during the A-Z transition of RNA, in which the hZαADAR1 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 hZαADAR1 in the Z-RNA binding conformation. Our results suggest that the A-Z transition of RNA facilitated by hZαADAR1 displays unique structural and dynamic features that may be involved in targeting ADAR1 for a role in recognition of RNA substrates.

Thermodynamic Model for B-Z Transition of DNA Induced by Z-DNA Binding Proteins

Z-DNA is stabilized by various Z-DNA binding proteins (ZBPs) that play important roles in RNA editing, innate immune response, and viral infection. In this review, the structural and dynamics of various ZBPs complexed with Z-DNA are summarized to better understand the mechanisms by which ZBPs selectively recognize d(CG)-repeat DNA sequences in genomic DNA and efficiently convert them to left-handed Z-DNA to achieve their biological function. The intermolecular interaction of ZBPs with Z-DNA strands is mediated through a single continuous recognition surface which consists of an α3 helix and a β-hairpin. In the ZBP-Z-DNA complexes, three identical, conserved residues (N173, Y177, and W195 in the Zα domain of human ADAR1) play central roles in the interaction with Z-DNA. ZBPs convert a 6-base DNA pair to a Z-form helix via the B-Z transition mechanism in which the ZBP first binds to B-DNA and then shifts the equilibrium from B-DNA to Z-DNA, a conformation that is then selectively stabilized by the additional binding of a second ZBP molecule. During B-Z transition, ZBPs selectively recognize the alternating d(CG)n sequence and convert it to a Z-form helix in long genomic DNA through multiple sequence discrimination steps. In addition, the intermediate complex formed by ZBPs and B-DNA, which is modulated by varying conditions, determines the degree of B-Z transition.

NMR Study on the Preferential Binding of the Zα Domain of Human ADAR1 to CG-repeat DNA Duplex

NMR Study on the Preferential Binding of the Zα Domain of Human ADAR1 to CG-repeat DNA Duplex

Backbone Dynamics Study of the Zβ Domain of Human DAI Bound to Z‐DNA#

Innate immune response is an essential defense against infections. Human DNA-dependent activator of IFN-regulatory factor (DAI; also known as ZBP1 or DLM-1) protein activates the innate immune response in response to DNA. The DAI protein contains two tandem Z-DNA binding domains (Zα and Zβ) at the N-terminus. The two Z-DNA binding domains are thought to be essential for full activation of a DNA-dependent immune response in vivo. The crystal structure of the Zα domain of human ADAR1 (ZαADAR1) in complex with Z-DNA shows that two Zα domains bind to each strand of double-stranded DNA and have twofold symmetry with respect to the DNA's helical axis. Structural studies with the Zβ domain of human DAI (ZβDAI) in complex with Z-DNA suggest that the ZβDAI structure is similar to that of ZαADAR1, although it demonstrates an unusual Z-DNA recognition. A previous NMR study of the ZβDAI complexed with a six-base-pair (6-bp) DNA duplex, d(CG)3, suggests that ZβDAI binds to Z-DNA via an activedi B–Z transition mechanism, where two ZβDAI proteins bind to B-DNA to form the ZβDAI–B-DNA complex; the B-DNA is subsequently converted to left-handed Z-DNA. To investigate the structural and dynamic properties of ZβDAI when it binds toDNAduplex and induces the B–Z transition in a DNA duplex, we have performed NMR backbone dynamics experiments on the free ZβDAI and the ZβDAI–d (CG)3 complex. The results revealed that ZβDAI exhibits a distinct change in the dynamics during theB–Z transition ofDNA duplex compared to ZαADAR1. This study provides valuable insights into the molecular mechanism of the B–Z transition induced by ZβDAI.

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

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.

NMR study of the Z-DNA binding mode and B–Z transition activity of the Zα domain of human ADAR1 when perturbed by mutation on the α3 helix and β-hairpin

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.

NMR investigation on the DNA binding and B–Z transition pathway of the Zα domain of human ADAR1

Human ADAR1, which has two left-handed Z-DNA binding domains, preferentially binds Z-DNA rather than B-DNA with a high binding affinity. Z-DNA can be induced in long genomic DNA by Z-DNA binding proteins through the formation of two B–Z junctions with the extrusion of one base pair from each junction. We performed NMR experiments on complexes of ZαADAR1 with three DNA duplexes at a variety of protein-to-DNA molar ratios. This study confirmed that the ZαADAR1 first binds to an 8-bp CG-rich DNA segment via a unique conformation during B–Z transition and the neighboring AT-rich region becomes destabilized. We also found that, when DNA duplexes have only 6-bp CG-rich segment, the interaction with ZαADAR1 did not affect the thermal stabilities of the 6-bp CG-rich segment as well as the neighboring two A·T base pairs. These results indicate that four ZαADAR1 proteins interact with the 8-bp DNA sequence containing a 6-bp CG-repeat segment as well as a dinucleotide step, even though the dinucleotid step contains A∙T base pairs. Thus this study suggests that the length of the CG-rich region is more important than the specific DNA sequence for determining which base-pair is extruded from the B–Z junction structure. This study also found that the ZαADAR1 in complex with a 11-bp DNA duplex exhibits a Z-DNA-bound conformation distinct from that of free ZαADAR1 and the initial contact conformations of ZαADAR1 complexed with 13-bp DNA duplexes.

Population Analysis of the Intermediate Complex States During B-Z Transition of Non-CG-repeat DNA Duplexes Induced by the Zα Domain of Human ADAR1

Figure 2. (A) The kex values of the G4b imino proton for the CA6ZαADAR1 complex determined at 25 C and (B) kex values of the G2b imino proton for the TA6-ZαADAR1 complex determined at 15 C as a function of the fZ. Black solid lines are the best fit to Eq. 1, where the kex data were weighted by the inverse of their variance. The grey lines indicate their upper and lower confidence limits (95% confidence level). Z-DNA contains nucleic acid bases in alternating antiand syn-conformations along the nucleotide chain and has only one groove that is similar to the minor groove of B-DNA. Z-DNA is in a higher energy conformation than B-DNA and is stabilized by negative supercoiling generated in vivo. Human ADAR1 has two left-handed Z-DNA binding domains at its NH2-terminus, Zα and Zβ, preferentially binds Z-DNA, rather than B-DNA, with high binding affinity. The co-crystal structure of the Zα domain of human ADAR1 (ZαADAR1) bound to Z-DNA revealed that one monomeric ZαADAR1 domain binds to one strand of double-stranded DNA and a second ZαADAR1 monomer binds to the opposite strand with two-fold symmetry with respect to the DNA helical axis. A structural study showed that ZαADAR1 binds to the Z-conformation of non-CG-repeat DNA duplexes through a common structural feature rather than by a specific sequence or structural alternations. A previous NMR study on a d(CGCGCG)2-ZαADAR1 complex suggests an active-mono B-Z transition mechanism (see Fig. 1) in which the ZαADAR1 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 ZαADAR1 molecule. Recently, we have reported NMR hydrogen exchange data of complexes between ZαADAR1 and the non-CG-repeat DNA duplexes, d(CACGTG)2 [referred to as CA6] or d(CGTACG)2 [referred to as TA6], with a variety of protein-to-DNA (P/N) molar ratios. The kex data for the G4b of the CA6-ZαADAR1 complex and for the G2b of the TA6-ZαADAR1 complex showed significant changes as the Z-DNA fraction (fZ) was increased (meaning that the P/N ratio increased) (see Fig. 2). These changes of the kex data can be explained by the presence of mixtures of two imino protons from B-form DNA (referred to as B) and B-DNA-ZαADAR1 complex (referred to as BP) in the imino peaks as given by Eq. 1:

Sequence discrimination of the Zα domain of human ADAR1 during B–Z transition of DNA duplexes

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.

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, 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.