The solution structure of the N-terminal domain of E3L shows a tyrosine conformation that may explain its reduced affinity to Z-DNA in vitro

Abstract

The N-terminal domain of the vaccinia virus protein E3L (Z alpha(E3L)) is essential for full viral pathogenicity in mice. It has sequence similarity to the high-affinity human Z-DNA-binding domains Z alpha(ADAR1) and Z alpha(DLM1). Here, we report the solution structure of Z alpha(E3L) and the chemical shift map of its interaction surface with Z-DNA. The global structure and the Z-DNA interaction surface of Z alpha(E3L) are very similar to the high-affinity Z-DNA-binding domains Z alpha(ADAR1) and Z alpha(DLM1). However, the key Z-DNA contacting residue Y48 of Z alpha(E3L) adopts a different side chain conformation in unbound Z alpha(E3L), which requires rearrangement for binding to Z-DNA. This difference suggests a molecular basis for the significantly lower in vitro affinity of Z alpha(E3L) to Z-DNA compared with its homologues.

Fig. 1.

3D solution structure of Zα_E3L.(A) Stereoview of the ensemble of the 20 lowest energy structures of E3L. The first and last residue of the 3 α-helices (red) (α1, α2, α3) and 3 β-strands (cyan) (β1, β2, β3) are numbered. (B) Stereoview of the backbone ribbon of the mean structure illustrating the (α plus β) helix–turn–helix fold of Zα_E3L. The N- and C-termini are labeled with N and C, respectively. (C) The secondary structure of Zα_E3L is shown with the amino acid sequence directly under it.

Fig. 2.

(A) The conformation of substrate DNA in the presence of Zα_E3L. The CD spectrum of d(CG)₆T₄(CG)₆ substrate DNA in the presence of Zα_E3L (blue curve) shows a conventional B-DNA conformation. In contrast, the CD spectrum of d(CG)₆T₄(CG)₆ in the presence of Zα_E3L and [Co(NH₃)₆]³⁺, which is known to promote the conversion from B- to Z-DNA (17), shows an inversion of ellipticity at 295 and 255 nm characteristic for duplex DNA in the Z-conformation (red curve). For control, Zα_E3L alone (green curve) and Zα_E3L in the presence of [Co(NH₃)₆]³⁺ (gray curve) show zero ellipticity between 320 and 250 nm. (B) Chemical shift map of the Zα_E3L/substrate DNA interaction. The ¹⁵N-HSQC NMR spectrum of Zα_E3L in the presence of d(CG)₆T₄(CG)₆ and [Co(NH₃)₆]³⁺ (red peaks) shows several large chemical shift changes compared with Zα_E3L alone (green peaks). In addition, there are three vanishing resonances (residues underlined). This finding indicates a selective interaction between Zα_E3L and d(CG)₆T₄(CG)₆ in the vicinity of the affected residues. The ¹⁵N-HSQC NMR spectrum of Zα_E3L in the presence of d(CG)₆T₄(CG)₆ substrate DNA shows no chemical shift alterations as compared with Zα_E3L alone (Fig. 4), indicating no discernible interaction under these conditions. Further, the spectrum of Zα_E3L in the presence of [Co(NH₃)₆]³⁺ is identical to Zα_E3L alone (not shown). (C) The Z-DNA-binding site of Zα_E3L. In the presence of d(CG)₆T₄(CG)₆ and [Co(NH₃)₆]³⁺, the H atoms of Zα_E3L that show large averaged chemical shift changes (≥0.082 ppm; bold labels in B) are shown as cyan spheres. H atoms with medium shifts (≥0.068 ppm; italic labels in B) are shown as dark red spheres. Atoms of Zα_E3L that show vanishing resonances (underlined labels in B) are shown as dark blue spheres in the 3D structure of Zα_E3L. Chemical shifts are listed in Table 2, which is published as supporting information on the PNAS web site. These data indicate a contiguous Z-DNA-binding site made up of helices α3 and α2 and the area around W66.

Fig. 3.

Superposition of the 3D structures of Zα_E3L (blue), Zα_ADAR1 (light green), and Zα_DLM1 (gray) in stereo. (A) The secondary structure elements of Zα_E3L, Zα_ADAR1, and Zα_DLM1 superimpose well except for the loop containing P63 (side chain labeled). Also, the side chains of K40, R41, N44, K45, and W66, which contact the bound Z-DNA in the co-crystal structures of Zα_ADAR1 and Zα_DLM1, show very similar positions. Only the side chain of Y48 adopts a distinct position in Zα_E3L (red) as compared with Zα_ADAR1 and Zα_DLM1.(B) Distinct Y48-W66 distance between Zα_E3L and Zα_ADAR1. Superposition of Y48 and W66 between the 20 lowest energy structures of E3L (blue), unbound Zα_ADAR1 (red), and bound Zα_ADAR1 (light green). Whereas Y48 residues are within van-der-Waals distance to W66 in both Zα_ADAR1 structures, it adopts two solvent exposed rotamer positions in Zα_E3L, which are 7.2 and 10.8 Å apart from W66.