A poxvirus protein forms a complex with left-handed Z-DNA: crystal structure of a Yatapoxvirus Zalpha bound to DNA

Abstract

A conserved feature of poxviruses is a protein, well characterized as E3L in vaccinia virus, that confers IFN resistance on the virus. This protein comprises two domains, an N-terminal Z-DNA-binding protein domain (Zalpha) and a C-terminal double-stranded RNA-binding domain. Both are required for pathogenicity of vaccinia virus in mice infected by intracranial injection. Here, we describe the crystal structure of the Zalpha domain from the E3L-like protein of Yaba-like disease virus, a Yatapoxvirus, in a complex with Z-DNA, solved at a 2.0-A resolution. The DNA contacting surface of Yaba-like disease virus Zalpha(E3L) closely resembles that of other structurally defined members of the Zalpha family, although some variability exists in the beta-hairpin region. In contrast to the Z-DNA-contacting surface, the nonbinding surface of members of the Zalpha family are unrelated; this surface may effect protein-specific interactions. The presence of the conserved and tailored Z-DNA-binding surface, which interacts specifically with the zigzag backbone and syn base diagnostic of the Z-form, reinforces the importance to poxvirus infection of the ability of this protein to recognize the Z-conformation.

Fig. 1.

Sequence and secondary structural comparison of yabZα_E3L with the other Z-DNA-binding domains and related poxvirus E3L domains. (A) Multiple sequence alignment of the Zα proteins. Numbering and secondary structure elements for the Zα domains of E3L from YLDV (yabZα_E3L), YMTV (ymtvZα_E3L), vaccinia virus (vvZα_E3L), Orf virus (ovZα_E3L), and Swinepox virus (svZα_E3L), along with human ADAR1 (hZα_ADAR1) and mouse DLM1 (mZα_DLM1) (accession numbers as in ref. , except YMTV NC 005179). Secondary structure is drawn on top of the sequence. The α-helix and β-sheet are represented by tubes and arrows, respectively. The conserved sequences of all Zα family are colored yellow. Residues from yabZα_E3L that make contact with the DNA are marked with a blue triangle. (B) The overall structure of the yabZα_E3L:Z-DNA complex. The N and C terminus and secondary structure elements are labeled. (C) Structural superposition of yabZα_E3L:Z-DNA (sky blue), hZα_ADAR1-Z-DNA (red), mZα_DLM1-Z-DNA (green), and vvcZα_E3L (royal blue). All DNA atoms and structurally aligned Cα atoms were used for the superposition (64 Cα for hZα_ADAR1 and 57 Cα for Zα_DLM).

Fig. 2.

Interaction of yabZα_E3L with Z-DNA. (A) Z-DNA binding of yabZα_E3L as measured by CD. Unbound (dC-dG)₆ is in the B-form under the buffer conditions used here (green curve). In the presence of a 2.5:1 (base pair:protein) molar ratio of DNA to protein (yabZα_E3L in blue, hZα_ADAR1 in red and mZα_DLM-1 in green), the DNA adopts the Z-conformation, as shown by the CD spectra. All curves are identical. (B) Kinetic measurement of DNA changing from B-DNA to Z-DNA in the presence of yabZα_E3L, hZα_ADAR1, or mZα_DLM1. The change in DNA conformation with time is measured by the change in ellipticity at 255 nm. hZα_ADAR1 and yabZα_E3L show similar kinetics of conversion, whereas mZα_DLM1 acts slightly more slowly.

Fig. 3.

Comparison of contacts between yabZα_E3L or hZα_ADAR1 with Z-DNA_. (A) Stereo view of the protein-DNA interface of the yabZα_E3L:Z-DNA between chain A and chain C. (B) Stereo view of the protein-DNA interface of Zα_ADAR1:Z-DNA. Both diagrams are viewed from the C-terminal end of the recognition helix (α3). Water molecules are shown in green. Hydrogen bonds are drawn as dashed lines. The tyrosine on the recognition helix is oriented in an edge-on view. (C and D) Schematic diagrams of the protein-DNA interactions in the yabZα_E3L:Z-DNA (C) and hZα_ADAR1:Z-DNA (D) complexes. Hydrogen bonds are represented by dashed lines and van der Waals contacts by open circles. The CH-π interaction between the conserved Tyr and a syn-guanosine is indicated by filled circles. Waters are shown by green ovals.

Fig. 4.

Surface charge distributions of yabZα_E3L, hZα_ADAR1, mZα_DLM1, and vvZα_E3L (from left to right) viewed along the DNA-binding cleft (Upper) and rotated 180° on the back side (Lower). The red and blue areas represent the negatively and positively charged surfaces, respectively. Skeletal representations showing the bound Z-DNA strand are found in the first three diagrams.