The zalpha domain of the editing enzyme dsRNA adenosine deaminase binds left-handed Z-RNA as well as Z-DNA

Abstract

The Zalpha domain of human double-stranded RNA adenosine deaminase 1 binds specifically to left-handed Z-DNA and stabilizes the Z-conformation. Here we report spectroscopic and analytical results that demonstrate that Zalpha can also stabilize the left-handed Z-conformation in double-stranded RNA. Zalpha induces a slow transition from the right-handed A-conformation to the Z-form in duplex r(CG)(6), with an activation energy of 38 kcal mol(-1). We conclude that Z-RNA as well as Z-DNA can be accommodated in the tailored binding site of Zalpha. The specific binding of Z-RNA by Zalpha may be involved in targeting double-stranded RNA adenosine deaminase 1 for a role in hypermutation of RNA viruses.

Figure 1

The Z-RNA conformation can be stabilized by Zα, as shown by CD and Raman spectroscopy. CD studies: (A) Spectra are shown for 5 μM duplex r(CG)₆ in the A-form (–⋅–⋅–). All samples contained 10 mM Na₂HPO₄ (pH 7), 20 mM NaCl, and 0.5 mM EDTA. In 6.5 M NaClO₄, the typical Z-RNA spectrum is seen (– – –). The A-RNA spectrum changes as Zα is added (Zα has no CD signal above 250 nm, but a strong negative ellipticity below 250 nm). Spectra are shown for the addition of 5 μM Zα (-⋅⋅-⋅⋅-), which is 1 Zα:12 bp; 10 μM Zα (⋅ ⋅ ⋅ ⋅), 1:6; 15 μM Zα (- - - -), 1:4; and 30 μM Zα (——), 1:2. Inversion of the CD bands around 285 nm and the decrease in signal at 266 nm are characteristic of the A → Z transition. Raman spectroscopy: (B) The A-form of r(CG)₆ (5 mM duplex) has doublet peaks at 784 and 814 cm⁻¹, which are characteristic of the A-conformation. (C) Z-conformation of r(CG)₆ (5 mM duplex) induced by 6.5 M NaBr. The bands at 781 and 638 cm⁻¹ and markedly reduced intensity at 810 cm⁻¹ distinguish the Z-RNA conformation from the A-form (B). (D) Zα⋅r(CG)₆ complex containing 15 mM Zα and 5 mM r(CG)₆ duplex (1 Zα:4 bp RNA). This spectrum has several features identical to those of the salt-induced Z-RNA spectrum (C), notably intensities at 638, 781, and 810 cm⁻¹. (E) Difference spectrum created by digitally subtracting the Zα spectrum (F) from that of the Zα⋅RNA complex (D). (F) Zα peptide spectrum (15 mM Zα). Raman bands for the aromatic amino acids tryptophan and tyrosine are labeled.

Figure 2

Perturbation of Zα⋅r(CG)₆ complex results in reversion of Z-RNA to the A-conformation, as seen in CD spectra. (A) NaCl titration of Zα⋅r(CG)₆ complex (30 μM Zα, 5 μM r(CG)₆ duplex; 1 Zα:2 bp RNA). As NaCl is added, the broad Z-RNA peak around 285 nm gradually decreases, and then inverts, signifying reversion to the A-conformation. Representative points shown are 0.04 M NaCl (——), 0.5 M (- - - -), 0.6 M (⋅ ⋅ ⋅ ⋅), 0.8 M (–⋅⋅–⋅⋅–), and 1 M NaCl (–⋅–⋅–). The midpoint of the titration is ca. 0.7 M NaCl. (B) Urea denaturation of Zα⋅r(CG)₆ complex. The urea titration was monitored at 285 (■), 266 (○), and 230 nm (⋄), corresponding to major ellipticity signals of Z-RNA, A-RNA, and Zα protein, respectively. The relative amount of Z-RNA decreases coincident with an increase in the A-RNA signal, as Zα is denatured by increasing urea. (C) Thermal denaturation of the Zα⋅r(CG)₆ complex monitored at 285 (■), 266 (○), and 222 nm (⋄), corresponding to the major signal intensities for Z-RNA, A-RNA, and Zα, respectively. As the temperature is increased, the relative amount of Z-RNA decreases as Zα begins to denature (ca. 60°C). In addition, as the Z-RNA signal decreases, the A-RNA signal increases from 60°C up to approximately 80°C, above which the RNA denatures and the ellipticity drops at 266 nm, with a slight rise at 285 nm. Individual melting experiments yielded T_m values of 69, 86, and 77°C for Zα, r(CG)₆, and Zα⋅r(CG)₆, respectively.

Figure 3

Temperature dependence of the A → Z-RNA transition. (A) Kinetics of the A → Z transition as a function of temperature. CD traces at 285 nm of the Zα-induced conformational transitions are shown for 25, 32, 37, 45, and 55°C (the 50°C trace was omitted for clarity). The kinetics for the A → Z transition are first-order and were fit to single exponentials to obtain rate constants (Table 1). (B) The rates of the A → Z transition for the r(CG)₆ duplex at 45 and 50°C (285 nm) are comparable to the rate of B → Z transition of d(CG)₆ (264 nm) at 25°C, demonstrating the higher-energy requirements of the A → Z transition. (C) Temperature dependence for Z-conformational transition of r(CG)₆ (■) or d(CG)₆ (●). Arrhenius plots constructed from rate data as a function of temperature yielded activation energies of 38.1 ± 0.5 and 24.0 ± 0.8 kcal mol⁻¹ for r(CG)₆ and d(CG)₆, respectively. Correlation coefficients for the Arrhenius plots of r(CG)₆ and d(CG)₆ were 0.999 and 0.998, respectively.