NMR Dynamics Study Reveals the ZÎ± Domain of Human ADAR1

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NMR Dynamics Study Reveals the Z# Domain of Human ADAR1 Associates with and Dissociates from Z-RNA more slowly than Z-DNA Ae-Ree Lee, Jihyun Hwang, Jeong Hwan Hur, Kyoung-Seok Ryu, Kyeong Kyu Kim, Byong-Seok Choi, Nak-Kyoon Kim, and Joon-Hwa Lee ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.8b00914 • Publication Date (Web): 28 Dec 2018 Downloaded from http://pubs.acs.org on December 31, 2018

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NMR Dynamics Study Reveals the Zα Domain of Human ADAR1 Associates with and Dissociates from Z-RNA More Slowly than Z-DNA Ae-Ree Lee,† Jihyun Hwang,‡ Jeong Hwan Hur,§ Kyoung-Seok Ryu,# Kyeong Kyu Kim,§ Byong-Seok Choi,‡ Nak-Kyoon Kim,*,¶ and Joon-Hwa Lee*,† †Department

of Chemistry and RINS, Gyeongsang National University, Gyeongnam 52828, South Korea of Chemistry, Korea Advanced Institute of Science and Technology, Daejeon 34141, South Korea §Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Gyeonggi 16419, South Korea #Protein Structure Research Team, Korea Basic Science Institute, Chungbuk 28119, South Korea ¶Advanced Analysis Center, Korea Institute of Science and Technology, Seoul 02792, South Korea KEYWORDS. Z-DNA, Z-RNA, NMR, A-Z transition, Z-DNA binding protein, RNA-protein interaction, Relaxation dispersion, Protein dynamics. ‡Department

Supporting Information ABSTRACT: 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.

INTRODUCTION Left-handed Z-DNA forms in polymers of alternating d(CG)n and is stabilized in vitro by high-salt conditions and in vivo by negative supercoiling occurring during transcription.1-6 A distinct biological function of Z-DNA is suggested by the conservation of the sequence and structural features of Z-DNA binding proteins (ZBPs), including human double-stranded (ds) RNA-specific adenosine deaminase I (ADAR1),7,8 mammalian DNA-dependent activator of interferon-regulatory factor (DAI, also known as DLM-1, ZBP1),9,10 poxviral E3L protein,12-14 and fish ZBP-containing protein kinase (PKZ)15 (Figure 1a). The crystal structures of the Zα domains of human ADAR1 (hZαADAR1),16 mouse DAI (mZαDLM1),9 yatapoxvirus E3L (yabZαE3L),17 and goldfish PKZ (caZαPKZ)18 in complex with 6base-pair (6-bp) DNA showed that one Zα protein binds to each strand of dsZ-DNA, while a second protein binds to opposite strand, yielding two-fold symmetry with respect to the DNA helical axis. The intermolecular interactions of ZBPs with ZDNA are mediated by 5 residues in the α3 helix and 4 residues in the β-hairpin (β2-loop-β3) (Figure 1b). A previous NMR study suggested an active B–Z transition mechanism for a 6-bp dsDNA, in which i) one molecule of Zα

(denoted as P) first binds to B-DNA (denoted as N); ii) the BDNA in the complex (NP) is converted to Z-form and thus the ZP complex forms (Z-DNA denoted as Z); iii) finally, the stable ZP2 complex is produced by the addition of another P to ZP (Figure 1c).19-21 Recently, global analysis of chemical shift perturbations of caZαPKZ upon binding to 6-bp Z-DNA suggested that the structure of the intermediate complex formed by caZαPKZ and B-DNA can be modulated by varying the salt concentration and determines the degree of B–Z transition.21 This study also reported the determination of dissociation/association rate constants of caZαPKZ binding to ZDNA by NMR relaxation dispersion experiments.21 Human ADAR1 deaminates adenosine in pre-mRNA to yield inosine, which codes as a guanine residue in mRNA.7,8 ADAR1 edits dsRNA in vitro at significantly higher levels when dsRNA contains the purine-pyrimidine repeats that could easily form a left-handed Z-RNA helix.22 It was discovered that hZαADAR1 would bind to Z-RNA by circular dichroism.23 Protein kinase PKR, which is a functional analog of PKZ containing two dsRNA-binding domains instead of two Zα domains,15 initiates the activation of the interferon-response pathway by the detection of viral dsRNA.24 Thus, it is thought that some ZBPs such as ADAR1 and PKZ are targeted to RNA in addition to ZDNA.

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The A-form RNA duplex can be transformed into a lefthanded Z-RNA structure.25 Structural information on Z-RNA has been obtained from crystallographic analyses or solution studies involving CD, Raman, and NMR spectroscopy.26-30 It was also reported that hZαADAR1 complexes with 6-bp RNA in such a way that the Z-RNA helix is associated with a unique solvent pattern that distinguishes it from the similar conformation of Z-DNA (Figure 1b).31 However, the detailed molecular mechanism by which the Zα protein converts a righthanded A-form structure in an RNA duplex to a left-handed Zform is not well understood. In this study, we performed NMR experiments on hZαADAR1 complexed with 6-bp duplex DNA [d(CG)3] or RNA [r(CG)3]

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at a variety of [P]t/[N]t ratios, where [P]t and [N]t are total concentrations of hZαADAR1 and DNA or RNA duplex, respectively. We studied the hZαADAR1–DNA and hZαADAR1– RNA interactions using imino proton and heteronuclear singlequantum correlation (HSQC) titrations. We also performed the 15N relaxation dispersion and 15N -exchange experiments to z study the kinetics of Z-DNA and Z-RNA binding of hZαADAR1. This study provides information about the conformational differences in hZαADAR1 between the free and DNA/RNA-bound states as well as between A/B-form-bound and Z-form-bound states, which play important roles in the B–Z transition in DNA or A–Z transition in RNA.

Figure 1. Conformational transition to Z-form helix of nucleic acid by hZαADAR1. (a) Multiple sequence alignment of Z-DNA binding proteins. Numbering and secondary structure elements for hZαADAR1 are shown on top of the sequence. Yellow and gray bars indicate residues important for the Z-DNA recognition and protein folding, respectively. (b) Residues of hZαADAR1 involved in intermolecular interaction with d(CG)3 (left) and r(CG)3 (right) reported in previous studies.16,31 Intermolecular H-bonds and van der Waals contacts are indicated by solid lines and open circles, respectively. The water molecules in key positions within the protein-DNA interface are indicated by ovals. (c) Mechanism for facilitation of the B–Z transition of DNA and the A–Z transition of RNA by two hZαADAR1 proteins. N indicates the B form of DNA or A form of RNA, Z indicates the Z form of DNA/RNA, and P indicates hZαADAR1. (d) 1D imino proton spectra of d(CG)3 (left) and r(CG)3 (right) at 35 °C upon titration with hZαADAR1. The resonances from B-form DNA are labeled as dG2b and dG4b, those from Aform RNA are labeled as rG2a and rG4a, and those from Z-form DNA and RNA are labeled as dG2z, dG4z, rG2z and rG4z, respectively. (e) Relative Z-form populations (fZ) of d(CG)3 (green squares) and r(CG)3 (orange circles) induced by hZαADAR1 as a function of [P]t/[N]t ratio.

RESULTS Titration of hZαADAR1 into d(CG)3 and r(CG)3. Figure 1d shows changes in the imino proton spectra of d(CG)3 and r(CG)3 upon titration with hZαADAR1 as a function of [P]t/[N]t at 35 °C. The imino proton resonances of the free r(CG)3 and its complex with hZαADAR1 were assigned by analysis of their NOESY spectra (data not shown). As previously reported,19 the new resonances [dG2z and dG4z of d(CG)3 and rG2z and rG4z of r(CG)3] are indicative of the Z-form helix induced by hZαADAR1. The relative populations of Z-form (fZ) in DNA/RNA duplexes were determined by integration of these new resonances using eq 3 as a function of the [P]t/[N]t ratio (Figure 1e). As shown in

Figure 1e, essentially all of d(CG)3 and r(CG)3 were in the Zconformation at [P]t/[N]t > 2.0. This result indicates that hZαADAR1 induces an A–Z transition in CG-repeat duplex RNA to an extent similar to the B–Z transition it induces in CG-repeat duplex DNA. In order to compare the relative binding affinity of hZαADAR1 for Z-DNA and Z-RNA, we prepared a one-to-one mixture of d(CG)3 and r(CG)3 and then performed the titration experiment with hZαADAR1 at 35 °C. As the [P]t/[N]t ratio increased, the intensities of imino proton resonances of both B-form DNA (dG2b and dG4b) and A-form RNA (rG2a and rG4a) gradually decreased (Supplementary Figure S6). Simultaneously, the imino proton resonances of Z-form helix (dG2z, dG4z, rG2z, and rG4z) emerged (Figure S6). These results demonstrate that hZαADAR1 have a similar binding affinity for Z-RNA and Z-


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DNA. In addition, the biolayer interferometry experiments found that the hZαADAR1 has a 2-fold smaller Kd for a 12-bp RNA [r(CG)6] than the corresponding DNA [d(CG)6] (Figure S7). Comparison of binding modes of hZαADAR1 to Z-DNA and Z-RNA. A superimposition of the 1H/15N-HSQC spectra of free hZαADAR1 and hZαADAR1 bound to d(CG)3 and r(CG)3 is shown in Figure S8. The weighted average 1H/15N backbone chemical shift differences between the free and complex hZαADAR1 (Δδavg) were determined for each residue by using eq 4 (Figure 2a). In the hZαADAR1–r(CG)3 complex, significant chemical shift changes upon binding to the RNA substrate were observed in the α3 helix as well as in the β1-α2 and β-hairpin regions, similar to Z-DNA binding (Figure 2a,b). Interestingly, several residues of hZαADAR1 exhibited significantly different chemical shift perturbation results when bound to Z-RNA versus Z-DNA

(Figure 2c). The most striking feature was that T191 in the βhairpin exhibited a significantly larger upfield shift of its 15N resonance upon binding to Z-RNA than Z-DNA (Figure 2d, panel (viii)). Similarly, the Gly190 amide 1H resonance showed a slight downfield shift upon Z-RNA binding, whereas the ZDNA binding caused an upfield shift (Figure 2d, panel (vii)). In addition, the Trp195 side-chain showed little chemical shift perturbation when binding to Z-RNA but a large perturbation when binding to Z-DNA (Figure 2d, panel (ix)). In the α3 helix, Z-RNA binding caused distinct chemical shift changes in the Lys170, Glu171, Leu172, Arg174, and Val175 amide resonances compared to Z-DNA binding (Figure 2d, panels (i), (iii), and (iv)). Finally, the His159 amide resonance in the β1α2 region had a significantly different chemical shift perturbation between the hZαADAR1–d(CG)3 and hZαADAR1– r(CG)3 complexes (Figure 2d, panel (i)).

Figure 2. Chemical shift changes in hZαADAR1 upon binding to d(CG)3 and r(CG)3. (a) Histograms of the Δδavg values of 15N-hZαADAR1 upon binding to d(CG)3 (upper) and r(CG)3 (lower). Residues whose cross-peaks disappeared or became very weak during titration are indicated with asterisk. (b) Mapping the location of the residues having large Δδavg onto the crystal structure of hZαADAR1 complexed with d(CG)3 (left, PDB ID: 1QBJ)13 or r(CG)3 (right, PDB ID: 2GXB).21 The colors used to illustrate the Δδavg are: blue or red, >0.2 ppm; cyan or orange, 0.12–0.2 ppm; and pale green or yellow, 0.06–0.12 ppm (the same color coding is used in panel (a). (c) The difference in Δδavg between the hZαADAR1–d(CG)3 and hZαADAR1–r(CG)3 complexes. (d) Comparison of the 1H/15N-HSQC peaks of (i) His159, Lys170, and Asn173, (ii) Thr167 and Ser178, (iii) Glu171, (iv) Leu172, Arg174, and Val175, (v) Asp160 and Lys181, (vi) Lys187 and Ala189, (vii) Gly163 and Gly190, and (viii) Ser162 and Thr191 amide protons and (ix) Trp195 side-chain of hZαADAR1 in the free state (blue) and in complexes with d(CG)3 (green) and r(CG)3 (red) at 35 °C.

Chemical shift changes in hZαADAR1 upon binding to ZDNA. To further clarify the chemical shift perturbation results with d(CG)3, the 1H/15N-HSQC spectra of hZαADAR1 were

acquired at 35 °C as a function of the [N]t/[P]t ratio (Figure S9). Interestingly, most amide signals (for example, Ala180 in Figure 3a) for hZαADAR1 were strong at each titration point



(Figure S9), indicating that binding to d(CG)3 is a fast exchange process. The completion of the HSQC titration experiment was confirmed by the chemical shift changes (Figure 3b). However, five amide signals (Glu171, Asn173, Arg174, Leu176, and Y177), which had Δδavg > 0.3 ppm, exhibited slow exchange (Figure S9). We analyzed the HSQC titration curves exhibiting fast exchange based on the active B–Z transition model (Figure 1c). Kd,NP and Kd,ZP2 are the dissociation constants of the NP and ZP2 complexes, respectively, and KNZ,1 (= [ZP]/[NP]) is the equilibrium constant between the ZP and NP complexes. Because the independent fitting for each titration curve did not give reliable Kd values, during global fitting we have fitted simultaneously both the 1H (ΔδH,obs) and 15N (ΔδN,obs) chemical shift trajectories for 41 amide signals with eq 7 (Figures 4a) and the fZ data with eq 8 (Figure 4b), as described previously.21 The global fitting gave Kd,NP and Kd,ZP2 of 700), of the (c) hZαADAR1–d(CG)3 and (d) hZαADAR1–r(CG)3 complexes determined at 800 MHz. Solid lines indicate global best-fit for the CPMG data using a pseudo-three-state exchange model for the Z-DNA/Z-RNA binding in the hZαADAR1– d(CG)3 (using eq 1) and hZαADAR1–r(CG)3 complexes (using eq 2). (e,f) Mapping the location of the residues having large (e) ΔδFB and (f) ΔδNZ onto the crystal structures of hZαADAR1 complexed with d(CG)3 (left, PDB ID: 1QBJ)16 or r(CG)3 (right, PDB ID: 2GXB).31 The colors used to illustrate the Δδ are: purple, >1.4 ppm; red, 0.8–1.4 ppm; orange, 0.4–0.8 ppm; and yellow, 0.2–0.4 ppm.

Conformational Differences of hZαADAR1 Between Free and Bound States and Between A/B-form and Z-form Bound States. In addition to the rate constants for conformational exchange, the global fitting provides 15N chemical shift differences between the free and bound states as well as the A/B-form-bound and Z-form-bound states, using the relation, Δδ = Δω/(2πν0,N), where ν0,N is the Larmor frequency for 15N nucleus (Tables S2 and S3). Both DNA and RNA binding of hZαADAR1 caused significant chemical shift differences between the free and bound states (ΔδFB) in the α3, β1-α2, and β-hairpin regions (Figure 5e), similar to the chemical shift perturbation data (Figure 2a,b). Interestingly, for the DNA binding, the ΔδFB values for residues Glu171, Ile172, Asn173, Val175, and Tyr177 in the α3 helix are significantly smaller than the maximum chemical shift differences (Δδmax) in the 15N titration data (Table S2). Instead, these residues showed large

chemical shift differences between the B-form and Z-form binding states (ΔδNZ) (Figure 5f). These results show that the α3 helix in the B-form binding state exhibited distinct structural features compared to the Z-form binding state. For the RNA binding, similar results were observed for residues Glu171, Ile172, Asn173, Val175, and Tyr177 in the α3 helix (Figure 5e,f). Interestingly, residue Thr191 in the βhairpin had a much larger ΔδNZ value (= 2.34 ppm) for Z-RNA binding than for Z-DNA binding (ΔδNZ = 0.62 ppm) (Figure 5f). These results indicate that the β-hairpin region (specifically Thr191) plays an important role in the A–Z transition of RNA. 15N -Exchange Experiment on hZα z ADAR1 Bound to r(CG)3. To confirm the kFB and kBF values for RNA binding of hZαADAR1, we performed 2D TROSY-based 15Nz-exchange experiments on the hZαADAR1–r(CG)3 complex. In these spectra, apparent


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exchange cross-peaks (denoted F-to-B and B-to-F) indicate exchange of 15Nz-magnetization between the free and RNAbound states of hZαADAR1 during the mixing time following the 15N chemical shift evolution period (insets in Figure 6). When residues showed small chemical shift differences between the A-form and Z-form binding states of hZαADAR1 (ΔδNZ < 0.25 ppm), the kFB and kBF values were obtained by simultaneously best-fitting the intensities of the auto and exchange cross-peaks in the 15Nz-exchange experiment as a function of the mixing time using eq 12. The 15Nz-exchange dataset of the Ser162, Ser178, Ala180, and Lys196 amide protons was globally fitted to obtain a kFB of 14.7±0.5 s–1 and kBF of 41.8±1.6 s–1 (Figure 6a). This concentration-independent kBF value (= koff,ZP2) is similar to the value determined by the 15N CPMG relaxation dispersion experiment (34.9 s–1). The kFB values determined by the two experiments are different from each other, because the kFB depends on concentration (i.e., kFB = kon,ZP[ZP]). In the case of Glu171, the dataset was fitted to obtain the different kFB (9.2±0.4 s–1) and kBF values (25.9±1.2 s–1) (Figure 6b), because this amide proton exhibited significantly large chemical shift changes not only upon binding to RNA (ΔδFB = 1.28 ppm) but also upon A–Z transition of the RNA in complex (ΔδNZ = 2.47 ppm).

Figure 6. 15Nz-exchange profile of hZαADAR1 bound to r(CG)3. Relative intensities of the auto and exchange cross-peaks of (a) Ser162, Ser178, Ala180 and Lys196 and (b) Glu171 amide protons in the 15Nz-exchange spectra as a function of mixing time (Tmix). Solid lines indicated the best-fit curves using a simple model for a phenomenological pseudo-first-order exchange reaction.45 The inset in each panel shows the auto and exchange cross-peaks at Tmix = 20 ms.

DISCUSSION Human ADAR1 is one of the most widely studied Z-DNA binding proteins, and its Zα domain binds preferentially to ZDNA rather than B-DNA with high affinity [Kd = 30 nM for 6bp d(CG)3].32 It was reported that hZαADAR1 could also bind to a 12-bp Z-RNA [r(CG)6] with the same order of magnitude as the

affinity for a 12-bp Z-DNA.22 Our imino proton titration study found that hZαADAR1 binds to both 6-bp Z-DNA and Z-RNA substrates with similar binding affinity (Figures 1d and S6). Global analysis of the chemical shift perturbations of the caZαPKZ–dT(CG)3 complex based on multiple binding steps was able to show the structural differences between the B-DNA and Z-DNA binding conformations of caZαPKZ.21,33 Similarly, we also compared these structural differences in hZαADAR1 (Figure 3). However, data for some residues in the α3-helix (for example, Asn173 and Tyr177 of hZαADAR1 and Asn38 and Tyr42 of caZαPKZ) could not be analyzed in both complexes, because these amide signals disappeared or were severely broadened during titration. This approach also could not be applied to slow exchange system such as the hZαADAR1–r(CG)3 complex. Instead, we have performed global analysis on the CPMG data to determine the structural differences between the BDNA/A-RNA and Z-form binding conformations for the residues displaying slow exchange (Figure 5). This study suggested a pseudo-three-state model for both Z-DNA and ZRNA binding processes of hZαADAR1 in which two independent, two-state exchange process are at work: i) the fast exchange between free and DNA- or RNA-bound states of hZαADAR1; and ii) the slow process involving the B–Z transition of DNA or the A–Z transition of RNA in the bound state of hZαADAR1 (Figure 5b). We found that hZαADAR1 has a 30-fold smaller kBF (~ koff,ZP2) for RNA binding than DNA binding (Figure 5). These results were consistent with the 1H/15N-HSQC titration spectra of hZαADAR1, upon binding to d(CG)3 or r(CG)3 (Figures S9 and S10). In both the DNA and RNA binding process, hZαADAR1 exhibited significant chemical shift differences (ΔδFB) in the α3 helix and β-hairpin (Figure 5e). The slower dissociation rates of hZαADAR1 with Z-RNA than Z-DNA might be related to the distinct intermolecular H-bond interactions in the α3 helix and β-hairpin (Figure 1b). First, the crystal structural study revealed that, in the Z-RNA binding conformation, the hydroxyl group of Tyr177 showed H-bonding interaction with the rG2 phosphate and O2’ hydroxyl group, whereas this side-chain Hbonded with only the dG2 phosphate in the Z-DNA binding structure (Figure S15).16,31 Second, in the crystal structure of the hZαADAR1–Z-DNA complex, the Arg174 side-chain showed direct and water-mediated H-bonding interactions with the dC5 phosphate and the O4’ of dG6, respectively (Figure S15).16 However, when binding to Z-RNA, this side-chain formed a water-mediated intermolecular H-bond with the rC5 phosphate as well as an intramolecular H-bond with the Glu171 side-chain (Figure S15).31 These distinct H-bonding interactions were consistent with the chemical shift changes of the Lys170, Glu171, Leu172, Arg174, and Val175 amide resonances (Figure 2c). Third, the crystal structure of the hZαADAR1–Z-RNA complex showed that the side-chain of His159 exhibited a distinct orientation due to a water-mediated H-bonding interaction with the Lys169 side-chain (Figure S15),31 which plays an important role in the intermolecular H-bond interactions (Figure 1b). These structural features might be correlated with the significant chemical shift difference of the His159 amide resonance between the hZαADAR1–d(CG)3 and hZαADAR1–r(CG)3 complexes (Figure 2d). Fourth, the crystal structural study also showed that, in the Z-RNA binding conformation, the γ-OH of Thr191 formed H-bonding interaction with only the rC3 phosphate, whereas this hydroxyl group H-bonded with both the phosphate and the O4’ of dC3 in the Z-DNA binding structure (Figure S15).16,31 This was



supported by our NMR data that residues in the β-hairpin region such as Gly190 and Thr191 displayed distinct chemical shift changes from those of the Z-DNA binding structure (Figure 2c). When we assume hZαADAR1 has a Kd,ZP2 with the same order of magnitude in both cases, the kon,ZP value for RNA binding is also about 30-fold smaller than that of DNA binding. These results indicate that hZαADAR1 not only dissociates from but also associates with a Z-RNA helix much more slowly compared to a Z-DNA helix. Our previous studies on the caZαPKZ–dT(CG)3 complex reported that the increase of [NaCl] from 10 to 100 mM caused a 13-fold larger kon,ZP.21 We suggested that the distinct conformational features of free caZαPKZ with varying salt concentration might be related to the association rates of caZαPKZ with Z-DNA.21 In this study, it was found that hZαADAR1 in the complex with Z-RNA exhibited significant structural differences compare to that of Z-DNA (Figure 2). Thus, we concluded that, because of this unusual structural feature, hZαADAR1 required more time to achieve the complete association with Z-RNA. Using this approach, we could determine the rate constants (kZN) for the Z-to-B conversion of DNA as well as the Z-to-A transition of RNA in complex with hZαADAR1. When hZαADAR1 binds to Z-DNA, the kZN value (12.7±0.6 s–1) can explain why the imino proton spectra exhibit a slow exchange of NMR resonances, in contrast to the results from the 1H/15N HSQC spectra related to the kBF value (1,050±64 s–1). In the case of ZRNA binding by hZαADAR1, the values of kZN (2.15±0.08 s–1) and kBF (34.9±0.8 s–1) are consistent with the slow exchange behavior of both spectra. Similar to the kBF values, the kZN value for the RNA duplex is 6-fold smaller than that for the corresponding DNA (Figure 4). This is consistent with a previous report that the A–Z transition of RNA required more activation energy (~1.2 kcal∙mol–1∙bp–1) than the corresponding DNA.22 We found that the Glu171, Asn173, Val175, and Tyr177 amide groups showed significantly large conformational changes (ΔδNZ > 1.0 ppm) upon the B–Z transition of DNA as well as the A–Z conversion of RNA (Figure 5f), indicating that the α3 helix is located close to the functional core that plays an important role in conversion to the Z-form helix. Surprisingly, the Thr191 amide group underwent substantial structural change during the conversion of dsRNA to Z-form (ΔδNZ = 2.34 ppm), whereas only a slight change was observed in the case of DNA (ΔδNZ =0.62 ppm). These results are likely related to the unique intermolecular H-bonding features of the Thr191 side-chain in the Z-RNA binding conformation as described above (Figure S15),16,31 which also contributes to A-to-Z conformational change of dsRNA. We suggested that the maximum 15N chemical shift change (Δδmax) was contributed by not only the exchange between the free and bound states (ΔδFB) but also the transition between the BN and BZ states (ΔδNZ). However, when ΔδNZ is > 0.2 ppm, the ΔδFB and ΔδNZ values did not satisfy this suggestion (Tables S2 and S3). These results were caused by no limitation on the ΔδFB and ΔδNZ during this analysis. To determine more accurately the ΔδFB and ΔδNZ values, the analysis should be performed with the CPMG data collected under various [N]t/[P]t ratio (that is, various pF), which will be performed in future. ADAR1 is the interferon-induced isoform present in the cytoplasm but absent from the nucleus.34 It is reported that some RNA viruses transmit infection through A-to-I genomic hypermutation.35 ADAR1 has higher editing activity on dsRNA bearing CG repeats than on RNAs without these Z-forming sequences.22 It was also reported that full-length ADAR1 had much higher binding affinity to RNA than an ADAR1 mutant

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lacking the Zα domain.36 Thus, the interferon-induced ADAR1 might play an antiviral role by targeting dsRNA regions with virally encoded Z-RNA forming sequences. Our relaxation dispersion study revealed that hZαADAR1 in complex with dsRNA had significantly longer lifetimes for the RNA-binding state (smaller kBF) as well as the Z-form binding state (smaller kZN) compared to dsDNA (Figure 5). We suggest that these longer lifetimes provide more time for the binding of ADAR1 to dsRNA with CG-repeat sequences, after which the adenosine at specific sites can be deaminated to inosine. SUMMARY We have performed structural and dynamics analyses of the interactions of hZαADAR1 with DNA during its B–Z transition as well as RNA during its A–Z transition. Global analysis of relaxation dispersion showed that hZαADAR1 associated with and dissociated from Z-RNA more slowly (30-fold) than Z-DNA, even though it had similar binding affinity. We also found that the α3 helix is located close to the functional core that plays an important role in conversion to a Z-form helix. Our results suggest that hZαADAR1 displays unique structural and dynamic features in the Z-RNA binding conformation that may be involved in targeting ADAR1 for a role in recognition of RNA substrates. MATERIALS AND METHODS Sample Preparation. The DNA and RNA oligomers were purchased from M-biotech Inc. (Korea branch of IDT Inc.), purified by reverse-phase HPLC, and desalted using a Sephadex G-25 gel filtration column. The amount of DNA/RNA was represented as the concentration of the double-stranded sample. The coding sequence for residues Glu140 – Gln202 of hZαADAR1 was cloned into E. coli expression plasmid pET28a (Novagen, WI, USA). To produce 15N-labeled hZαADAR1, BL21(DE3) bacteria were grown in M9 medium that contained 1 g/L 15NH Cl. The 15N-labeled hZα 4 ADAR1 proteins were purified with a Ni-NTA affinity column and a Sephacryl S-100 gel filtration column, as described elsewhere.19,37 The DNA, RNA, and protein samples were dissolved in a 90% H2O/10% D2O NMR buffer containing 10 mM sodium phosphate (pH 6.0) and 100mM NaCl. The duplex formation of a 6-bp DNA/RNA dissolved in NMR buffer was confirmed by its melting temperature [0.2 mM d(CG)3: 51.4 °C; 0.2 mM r(CG)3: 48.6 °C] calculated using the program PairFold.38 Biolayer Interferometry (BLI) Experiments. The BLI experiments were performed using the BLITZ system (Fortebio, CA, USA). DNA oligonucleotides of 5’-biotindATTATAT(CG)6-3’ and 5’-d(CG)6-3’ and RNA oligonucleotides of 5’-biotin-rAUUAUAU(CG)6-3’ and 5’r(CG)6-3’ were purchased (IDT, CA, USA) and annealed in buffer containing 5 mM HEPES (pH 7.5) and 10 mM NaCl. The annealed DNA and RNA samples were immobilized into the streptavidin-coated biosensor (Fortebio, CA, USA) and reacted with various concentrations of hZαADAR1 (0.5 μM to 10 μM). The equilibrium binding constant (Kd) was determined from the BLI data at various concentrations using the global fitting method provided in data analysis software version 7.0 (Fortebio, CA, USA).


Page 9 of 13 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


NMR Experiments. All of the 1H and 15N NMR experiments were performed on an Agilent DD2 700-MHz NMR spectrometer (GNU) or a Bruker Avance-III 800-MHz NMR spectrometer (KBSI, Ochang) equipped with a cold probe. The imino proton and 1H/15N-HSQC spectra were obtained for complexes prepared by addition of 15N-labeled hZαADAR1 to 0.2 mM DNA/RNA or addition of DNA/RNA to 0.5 mM 15Nlabeled hZαADAR1 at 35 °C. One-dimensional (1D) NMR data were processed with FELIX2004 (FELIX NMR, USA) or Mnova 12.0.0 (Mestrelab, Spain) software, while the 2D data were processed with NMRPIPE39 and analyzed with Sparky.40 External 2-2-dimethyl-2-silapentane-5-sulfonate was used for the 1H, 13C, and 15N references. The relative populations of Zform (fZ) in DNA/RNA duplexes were determined by eq 3: 𝑓Z = 𝐼

G2z

2 × 𝐼G2z

(3)

+ (𝐼G4z + 𝐼G2n + 𝐼G4n)

where IG2z and IG4z are the peak intensities of the G2 and G4 imino resonances of Z-form helix, respectively and IG2n and IG4n are the peak intensities of the G2 and G4 imino resonances of B-form/A-form helix, respectively. The average chemical shift differences (Δδavg) of the amide proton and nitrogen resonances between the two states were calculated by eq 4: ∆δavg = (∆δH) + (∆δN 5.88) 2

2

(4)

where H and N are the chemical shift differences of the amide proton and nitrogen resonances, respectively. Binding Models. The HSQC titration curves were analyzed by assuming an active model of B–Z or A–Z transition (Figure 1c), where P is the free form of hZαADAR1; NP and ZP are the singly bound forms to B-form/A-form and Z-form nucleic acid, respectively; ZP2 is the doubly bound hZαADAR1 to Z-form DNA or RNA; and N is the B-form of free d(CG)3 or A-form of free r(CG)3. The concentrations of N, NP, ZP, and ZP2 forms, [N], [NP], [ZP] and [ZP2], respectively, were expressed using [N]t, [P]t, Kd,NP, Kd,ZP2, and KNZ,1, [N] = [N]t

𝐾d,NP𝐾d,ZP2 𝐾d,NP𝐾d,ZP2 + (1 + 𝐾BZ,1)𝐾d,ZP2[P] + 𝐾BZ,1[P]2

[BP] = [N]t [ZP] = [N]t

𝐾d,ZP2[P] 𝐾d,NP𝐾d,ZP2 + (1 + 𝐾BZ,1)𝐾d,ZP2[P] + 𝐾BZ,1[P]2 𝐾BZ,1𝐾d,ZP2[P]

𝐾d,NP𝐾d,ZP2 + (1 + 𝐾BZ,1)𝐾d,ZP2[P] + 𝐾BZ,1[P]2

[ZP2] = [N]t

𝐾BZ,1[P]2 𝐾d,NP𝐾d,ZP2 + (1 + 𝐾BZ,1)𝐾d,ZP2[P] + 𝐾BZ,1[P]2

(5)

where [N]t is the total concentration of nucleic acid duplex; Kd,NP and Kd,ZP2 are the dissociation constants for the NP and ZP2 complexes, respectively; KNZ,1 (= [ZP]/[NP]) is the equilibrium constant between NP and ZP forms; and [P] is the concentration of the free hZαADAR1, which is a solution of the following cubic equation, as described previously:21 3

2

(6)

[P] + 𝑎[P] + 𝑏[P] + 𝑐 = 0 𝑎 = 2[N]t ― [P]t + (1 + 1 𝐾BZ,1)

∆δobs =

[BP] [P]t

∆δB +

[ZP] + 2[ZP2] [P]t

(7)

∆δZ

where ΔδB and ΔδZ are the 1H and 15N chemical shift differences of the B-DNA- and Z-DNA-bound forms relative to the free form, respectively. The relative Z-DNA population (fZ) was determined from the integration of new resonances in the imino proton spectra. The observed fZ value determined from imino proton resonances is described as: 𝑓Z =

[ZP] + [ZP2]

(8)

[N]t

15N

CPMG Relaxation Dispersion Study. The 15N amide CPMG relaxation dispersion experiments were performed using free 15N-labeled hZαADAR1 and 15N-labeled hZαADAR1 complexed with d(CG)3 and r(CG)3 at 35 °C.41 Experiments employed a constant relaxation delay (Trelax) of 60 ms and 12 values of νCPMG = 1/(2τCP) ranging from 33 to 1,000 Hz, where τCP is the delay between consecutive pluses. Transverse relaxation rates R2,eff were calculated for each cross-peak signal at each value by: 𝑅2,eff(νCPMG) = ― 𝑇

1 relax

ln

{

𝐼(νCPMG) 𝐼0

}

(9)

where I(νCPMG) and I0 are the peak intensity at a given value of νCPMG with delay time of 60 and 0 ms, respectively. hZαADAR1 in complex with Z-DNA/Z-RNA shows two kinds of independent exchange processes, the conformational exchange of free protein and the association/dissociation of ZDNA/Z-RNA. In this case, the relaxation dispersion data of hZαADAR1 complexed with Z-DNA/Z-RNA (R2,effcomp) in fast exchange on the NMR chemical shift timescale were fitted to: 𝑅comp 2,eff (νCPMG)

=

𝑅02

+

𝑅free 2,eff(νCPMG)

+

pFpB(∆ω)2 𝑘𝑒𝑥

{

1―

(

(10) R2,efffree

where is the relaxation dispersion data of free hZαADAR1; pF and pB (= 1 – pF) are the relative populations of the free and bound states, respectively; R20 is the intrinsic transverse relaxation rate; kex is the exchange rate between the free and bound states; and Δω is chemical shift difference (in Hz) between the free and bound states.42 The Δω value was used to calculate the 15N chemical shift differences (N) by the equation, N = Δω/(2πν0,N), where ν0,N is the Larmor frequency for 15N nucleus at specific magnetic field.42 In the limit of slow exchange and when kFB, kBF

NMR Dynamics Study Reveals the ZÎ± Domain of Human ADAR1

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