Zα Domain of ADAR1 Binds to an A-Form-like Nucleic Acid Duplex with Low Micromolar Affinity

Abstract

The left-handed Z-conformation of nucleic acids can be adopted by both DNA and RNA when bound by Zα domains found within a variety of viral and innate immune response proteins. While Z-form adoption is preferred by certain sequences, such as the commonly studied (CpG)_n repeats, Zα has been reported to bind to a wide range of sequence contexts. Studying how Zα interacts with B-/A-form helices prior to their conversion to the Z-conformation is challenging as binding coincides with Z-form adoption. Here, we studied the binding of Zα fromHomo sapiens ADAR1 to a locked "A-type" version of the (CpG)₃ construct (LNA (CpG)₃) where the sugar pucker is locked into the C3'-endo/C2'-exo conformation, which prevents the duplex from adopting the alternating C2'/C3'-endo sugar puckers found in the Z-conformation. Using NMR and other biophysical techniques, we find that Zα^ADAR1 binds to the LNA (CpG)₃ using a similar interface as for Z-form binding, with a dissociation constant (K_D) of ∼4 μM. In contrast to Z-DNA/Z-RNA, where two Zα^ADAR1 bind to every 6 bp stretch, our data suggests that Zα^ADAR1 binds to multiple LNA molecules, indicating a completely different binding mode. Because Zα^ADAR1 binds relatively tightly to a non-Z-form model, its binding to B/A-form helices may need to be considered when experiments are carried out which attempt to identify the Z-form targets of Zα domains. The use of LNA constructs may be beneficial in experiments where negative controls for Z-form adoption are needed.

Figure 1.

A locked version of the (CpG)₃ duplex does not adopt the Z-form conformation by high-salt or in the presence of Zα. (a) Structural depictions of a B-form helix and its C2′-endo sugar pucker, an A-form helix and corresponding C3′-endo sugar pucker, and a Z-form helix and its alternating C2′-endo/anti C3′-endo/syn configuration (from 5′ to 3’). 3D depictions were made using PDB 6CQ3 (B-form helix), 2JXQ (A-form helix), and 4OCB (Z-form helix). (b) 2D cartoon schematic of the locked (CpG)₃ construct (left) and 3D structure of a fully locked duplex (right, using PDB: 2X2Q). The methylene bridge which locks the sugar pucker into C3’-endo/C2’-exo and thus the duplex into an A-type conformation is highlighted. (c) CD spectra from wavelengths of 220–320 nm of the d(CpG)₃, r(CpG)₃, and LNA (CpG)₃ duplexes in low salt (free, black solid lines), high-salt (6M NaClO₄, slashed black lines), and in complex with Zα (1:4 molar ratio of Nucleic Acid:Zα, red lines). The B-form, A-form, and Z-form spectral profiles are indicated. (d) Plots of the ellipticity at 285 nm measured by circular dichroism as a function of time after the addition of saturating amounts of Zα for the r(CpG)₃ and LNA (CpG)₃ duplexes. The temperature that the process was measured at is indicated at the top of the graphs.

Figure 2.

Zα^ADAR1 binds to the LNA (CpG)₃ using a similar interface as for Z-form nucleic acids and with low micromolar affinity. (a) Zoomed in insets of residues Tyr 177 and Ser 178 from the 2D ¹⁵N-HSQC titration of LNA (CpG)₃ into ¹⁵N-labeled Zα^ADAR1 (full spectra shown in Figure S2). The molar ratio of Zα^ADAR1:LNA (CpG)₃ is color-coded. Below the spectra are graphs plotting the ¹H and ¹⁵N chemical shifts (ppm) of the same residues throughout the titration. (b) 1D ¹H imino spectra following titration of Zα^ADAR1 into the LNA (CpG)₃ construct. The ratio of LNA (CpG)₃:Zα^ADAR1 is indicated on the right-hand side of the spectra. The imino assignment is indicated. (c) Chemical shift perturbations (CSPs) are plotted vs the residue number, and color coded according to the ratio of Zα^ADAR1:LNA (CpG)₃. (d) CSPs from (c) plotted onto the structure of Zα^ADAR1 bound to the r(CpG)₃ duplex in the Z-conformation (PDB: 2GXB). (e) The two interfaces revealed by clustering of CSPs (red is the Z-RNA binding interface, blue a second interface not known to be involved in nucleic acid binding). (f) Global fit of the dissociation constant (K_D) from the CSPs as a function of the LNA (CpG)₃ concentration. (g) Isothermal calorimetry thermogram of the LNA (CpG)₃ Zα^ADAR1 interaction. Extracted thermodynamic parameters are found in Table 1.

Figure 3.

The complex stoichiometry of LNA:Zα^ADAR1 binding is similar to that of Z-RNA. (a) Longitudinal relaxation rates R₁, which are sensitive to motions on the ps-ns timescale are plotted on the y-axis vs Zα^ADAR1’s residue number on the x-axis as a function of increasing LNA (CpG)₃ concentration (color-coded, as shown to the right-hand side of (c)). (b) Rotating frame relaxation rates R_1ρ, which are sensitive to potential slow motions on the μs-ms timescale in addition to the fast motion. (c) Residue-specific tumbling times τ_corr calculated from the ratio of R₂/R₁ (see methods for details). (d) Averages of the corresponding residue-specific R₁, R_1ρ, and τ_corr values from (a, b, and c) are plotted versus the molar ratio of Zα^ADAR1:LNA in the experiment. Measured values are from one set of relaxation rate experiments. Sedimentation coefficient distributions for the LNA (CpG)₃ (e), LNA (CpG)₆ (f), and r(CpG)₆ (g) at decreasing ratios of nucleic acid (NA) to Zα^ADAR1 as obtained from analytical ultracentrifugation (AUC). Predicted sedimentation coefficients for different theoretical nucleic acid Zα^ADAR1 complex stoichiometries are shown overlayed on top of the plots. Some of the predicted sedimentation coefficients are identical and are thus overlapped (e.g., for the LNA (CpG)₆, 2:2 and 1:4 had the sample predicted sedimentation coefficient).

Figure 4.

Model incorporating binding of Zα to an A-form helix prior to Z-RNA adoption and stabilization. Zα initially binds to multiple A-form helices with low micromolar affinity and has a fast exchange rate between the free and bound states. This is likely accompanied shuffling between the different duplexes brought into proximity. The bound RNA then transitions to the Z-conformation which is recognized by the Zα domains leading to a significantly lower dissociation constant and a slow off-rate, thus stabilizing the Z-RNA conformation.