The crystal structure of the second Z-DNA binding domain of human DAI (ZBP1) in complex with Z-DNA reveals an unusual binding mode to Z-DNA

Abstract

Mammalian DAI (DNA-dependent activator of IFN-regulatory factors), an activator of the innate immune response, senses cytosolic DNA by using 2 N-terminal Z-DNA binding domains (ZBDs) and a third putative DNA binding domain located next to the second ZBD. Compared with other previously known ZBDs, the second ZBD of human DAI (hZbeta(DAI)) shows significant variation in the sequence of the residues that are essential for DNA binding. In this article, the crystal structure of the hZbeta(DAI)/Z-DNA complex reveals that hZbeta(DAI) has a similar fold to that of other ZBDs, but adopts an unusual binding mode for recognition of Z-DNA. A residue in the first beta-strand rather than residues in the beta-loop contributes to DNA binding, and part of the (alpha3) recognition helix adopts a 3(10) helix conformation. The role of each residue that makes contact with DNA was confirmed by mutational analysis. The 2 ZBDs of DAI can together bind to DNA and both are necessary for full B-to-Z conversion. It is possible that binding 2 DAIs to 1 dsDNA brings about dimerization of DAI that might facilitate DNA-mediated innate immune activation.

Fig. 1.

Multiple sequence alignments of the first ZBDs (Zα) and the second ZBDs (Zβ). Zαs include human ADAR1 (hZα_ADAR1), mouse ADAR1 (mZα_ADAR1), human DAI (hZα_DAI), mouse DAI (mZα_DAI), goldfish PKZ (caZα_PKZ), zebrafish PKZ (drZα_PKZ), and Yaba-like disease poxvirus E3L (yabZα_E3L). Zβs include human DAI (hZβ_DAI), mouse DAI (mZβ_DAI), goldfish PKZ (caZβ_PKZ), and zebrafish PKZ (drZβ_PKZ). The secondary structures of hZβ_DAI and hZα_ADAR1 are shown by a rectangle indicating a helix or an arrow for a β-strand. The 3₁₀ helix is shown in green. Residues of hZβ_DAI and hZα_ADAR1 involved in DNA interactions are marked by blue and red circles, respectively. Highly conserved residues are highlighted in yellow. The residue numbers of both N and C termini are shown.

Fig. 2.

Structural comparison of hZβ_DAI and hZα_ADAR1 and their interactions with Z-DNA. (A) Overall structure of the hZβ_DAI/Z-DNA complex. The protein and DNA are drawn as a ribbon diagram and stick model, respectively. The N and C termini, the secondary structure elements of hZβ_DAI, and 5′ and 3′ of DNA are labeled. (B) The structural overlap of hZβ_DAI and hZα_ADAR1 near the Z-DNA binding site. The ribbon diagrams of hZβ_DAI (blue) and hZα_ADAR1 (green) are overlapped, and the stick model of a double-strand Z-DNA is drawn in brown and gray. Structural deviation near α3 and the β-wing are labeled and marked by red circles. (C) Stereoviews of the protein–DNA interfaces of hZβ_DAI (chain A)/Z-DNA (Left) and hZβ_DAI(Chain B)/Z-DNA (Right). The Cα chains of the ZBDs are colored sky blue, and the residues involved in DNA contact are depicted in sky blue stick models. The Z-DNA backbone is drawn as a red stick model, and bases are drawn as gray stick models. Water molecules are shown in green. Hydrogen bonds are drawn as dashed lines. (D) Schematic diagrams of the protein–Z-DNA interactions in hZβ_DAI (Left) and hZα_ADAR1 (Right). Hydrogen bonds and van der Waals contacts are represented by dashed black lines and pink lines, respectively. The CH–π interaction between the conserved Tyr and the C8 of a syn-guanine is indicated by black circles, and waters are shown by green circles. The protein–DNA interactions in hZα_ADAR1 are identical on both sides of the Z-DNA, thus only 1 side is shown.

Fig. 3.

The Z-DNA binding mode of hZβ_DAI. (A) Z-DNA recognition by R124 of hZβ_DAI. A magnified view near the wing region is drawn for the interfaces between hZβ_DAI and Z-DNA [chains A and C (Left) and chains B and D (Right)]. A σ-weighted 2 F_o − F_c omit map contoured at 1.2 σ was generated by omitting R124, R160, and the waters involved in the R124-mediated interaction. Possible hydrogen bonds among R124, water and the phosphate group of G2 are shown by dashed lines, and their distances are indicated in Å. (B) Surface charge distributions of the chain A (Left) and chain B (Right) viewed along the DNA binding cleft. The red and blue areas represent negatively and positively charged surfaces, respectively. Z-DNA chains are shown as stick models, and R124, K160 and each nucleotide are labeled.

Fig. 4.

B-to-Z-DNA conversion activity of hZβ_DAI and hZαβ_DAI. (A) The Z-converting activities were estimated by monitoring the CD signal at 255 nm for 40 min by using 60 μg/ml of double-stranded (dCdG)₆. The molar ratio of hZβ_DAI or its alanine mutants to ds(dCdG)₆ was 4:1. D139A was randomly chosen as a negative control. (B) WT hZαβ_DAI and 2 tyrosine mutants, hZαβ_DAI Y50A and hZαβ_DAI Y145A, were mixed with 15 μM double-stranded (dCdG)_6, at 2:1 and 4:1 molar ratios, and their CD signals were monitored at 255 nm for 60 min. As a control, the Z-DNA converting activity of a single ZBD (hZα_ADAR1) at a 4:1 molar ratio to DNA was also measured under the same condition.