The RNA-dependent protein kinase (PKR) plays an important role in the innate immune response against viral infection. 1 PKR recognizes double-stranded RNA (dsRNA) in cytosol via two dsRNA binding domains (dsRBDs). 1 In several fish species, PKZ contains two left-handed Z-DNA binding domains (ZBDs) instead of dsRBDs to recognize heterogeneous nucleic acids. 2-5 Similar to PKR, the phosphorylation of eIF2α by PKZ is activated by binding to Z-DNA instead of dsRNA. 5 Therefore, PKZ is a functional orthologue of PKR, even though it binds to Z-DNA. 5 Z-DNA forms under high salt condition, negative supercoiling and complex formation with Z-DNA binding proteins (ZBPs). 6-8 The crystal structures of the ZBDs of human ADAR1 (hZα ADAR1 ), 9 mouse DAI (mZα DAI ), 10 yatapoxvirus E3L (yabZα E3L ), 11 and goldfish PKZ (caZα PKZ ) 12 in complex with 6-base-paired (6-bp) dT(CG) 3 have been reported. These structural studies revealed that the intermolecular interaction with Z-DNA is mediated by residues in the α3 helix and in the β-hairpin (β2-L4-β3) (Figure 1 (a)). 9-12 Interestingly, the K56 side-chain in the β-hairpin of caZα PKZ is involved in H-bonding with the phosphate of Z-DNA in the complex unlike other ZBPs (Figure 1(b)). 12 Although the overall structure of free caZα PKZ is very similar to the caZα PKZ -Z-DNA complex, the β-hairpin shows the different orientation from the crystal structure (Figure 1 (c)). 13 This study also revealed that these interaction of K56 plays an important role in its B-Z transition activity. 13 To investigate the structural and dynamic properties of caZα PKZ when it induces the B-Z transition in a DNA duplex, we have performed NMR backbone dynamics experiments on the free caZα PKZ and caZα PKZ -dT(CG) 3 complex. The results revealed that caZα PKZ exhibits a unique dynamics feature of the β-hairpin for efficient Z-DNA binding. This study provides valuable insights into the molecular mechanism of the Z-DNA recognition of caZα PKZ .
ExperimentalThe DNA duplex dT(CG) 3 was purchased from M-Biotech Inc. (Seoul, Korea), purified by reverse-phase HPLC and Sephadex G-25 gel filtration column. To produce uniformly 15 N-labeled caZα PKZ , BL21(DE3) bacteria cells were grown in M9 medium containing 1 g/L 15 NH 4 Cl. The expression and purification of 15 N-labeled caZα PKZ were described in a previous report. 13 All NMR experiments were performed on an Agilent DD2 700-MHz spectrophotometer (GNU, Jinju, Korea) equipped with cold probe. All NMR data were processed with NMRPIPE 14 and analyzed with Sparky. 15 Backbone dynamics parameters, longitudinal R 1 relaxation and transverse R 2 relaxation rates were measured using 1.0 mM 15 N-labeled free caZα PKZ or caZα PKZ -dT(CG) 3 complex at a protein/DNA molar ratio (P/N ratio) of 2.0. The R 1 relaxation rates were measured in a series of 1 H/ 15 N-HSQC spectra with 10 delays of 50, 100, 150, 200, 250, 300, 350, 400, 500, and 600 ms. R 2 measurements were taken from a series of spectra with 10 relaxation delays of 10, 30, 50, 70, 90, 110, 130, 150, 190, and 23...
