Solution structure of the Zbeta domain of human DNA-dependent activator of IFN-regulatory factors and its binding modes to B- and Z-DNAs

Abstract

The DNA-dependent activator of IFN-regulatory factors (DAI), also known as DLM-1/ZBP1, initiates an innate immune response by binding to foreign DNAs in the cytosol. For full activation of the immune response, three DNA binding domains at the N terminus are required: two Z-DNA binding domains (ZBDs), Zα and Zβ, and an adjacent putative B-DNA binding domain. The crystal structure of the Zβ domain of human DAI (hZβ(DAI)) in complex with Z-DNA revealed structural features distinct from other known Z-DNA binding proteins, and it was classified as a group II ZBD. To gain structural insights into the DNA binding mechanism of hZβ(DAI), the solution structure of the free hZβ(DAI) was solved, and its bindings to B- and Z-DNAs were analyzed by NMR spectroscopy. Compared to the Z-DNA-bound structure, the conformation of free hZβ(DAI) has notable alterations in the α3 recognition helix, the "wing," and Y145, which are critical in Z-DNA recognition. Unlike some other Zα domains, hZβ(DAI) appears to have conformational flexibility, and structural adaptation is required for Z-DNA binding. Chemical-shift perturbation experiments revealed that hZβ(DAI) also binds weakly to B-DNA via a different binding mode. The C-terminal domain of DAI is reported to undergo a conformational change on B-DNA binding; thus, it is possible that these changes are correlated. During the innate immune response, hZβ(DAI) is likely to play an active role in binding to DNAs in both B and Z conformations in the recognition of foreign DNAs.

Fig. 1.

The solution structure of free hZβ_DAI and structural comparison with homologous proteins. (A) The solution structure of free hZβ_DAI is represented in the ribbon diagram. It contains an α + β helix-turn-helix fold typical of B-DNA binding proteins. (B) Superimposition of the solution structures defined by NMR spectroscopy, free hZβ_DAI, free hZα_ADAR1, and free vvZα_E3L (blue, red, and yellow, respectively). Dashed black circles indicate the N-terminal region of α3 and β turns.

Fig. 2.

Comparison of the free and DNA-bound hZβ_DAI structures. (A) Superimposition of the free (blue) and DNA-bound (magenta) hZβ_DAI structures (15). Z-DNA is depicted by a stick diagram. The secondary structures are labeled. (B) Comparison of the tyrosine conformation on the α3-helix in Z-DNA binding proteins; free hZβ_DAI and hZα_ADAR1 (blue and red, respectively) and DNA-bound hZβ_DAI and hZα_ADAR1 (magenta and green, respectively), and free vvZα_E3L (yellow). The tyrosine residue in Z-DNA bound structures (magenta and green) shows CH-π interactions (17) with the tryptophan residue in the β3-strand. A similar conformation is found with the free hZα_ADAR1 structure (red). (C) Superimposition of six residues contacting Z-DNA in the hZβ_DAI/Z-DNA complex (magenta) (15) with those in the ensemble of 20 lowest energy structure of free hZβ_DAI (blue). The Z-DNA backbone is in dark red; bases are in gray.

Fig. 3.

DNA binding experiments with CG6 and AT6. (A) Ribbon diagrams indicating the residues that show chemical-shift perturbations (Δδ) of the backbone; hZβ_DAI with CG6 (Left) and hZβ_DAI with AT6 (Right) (α, dark red, disappeared; β, red, ≥0.14 ppm; γ, orange, ≥0.10 ppm; δ, yellow, ≥0.06 ppm). (B) Surface mapping of the residues that show chemical-shift perturbations of the aliphatic side chains; with CG6 (Left) and hZβ_DAI with AT6 (Right) (α, purple, disappeared; β, blue, ≥0.10 ppm; γ, light blue, ≥0.075 ppm; δ, cyan, ≥0.05 ppm).