Solution structure of the Z-DNA binding domain of PKR-like protein kinase from Carassius auratus and quantitative analyses of the intermediate complex during B-Z transition

Abstract

Z-DNA binding proteins (ZBPs) play important roles in RNA editing, innate immune response and viral infection. Structural and biophysical studies show that ZBPs initially form an intermediate complex with B-DNA for B-Z conversion. However, a comprehensive understanding of the mechanism of Z-DNA binding and B-Z transition is still lacking, due to the absence of structural information on the intermediate complex. Here, we report the solution structure of the Zα domain of the ZBP-containing protein kinase from Carassius auratus(caZαPKZ). We quantitatively determined the binding affinity of caZαPKZ for both B-DNA and Z-DNA and characterized its B-Z transition activity, which is modulated by varying the salt concentration. Our results suggest that the intermediate complex formed by caZαPKZ and B-DNA can be used as molecular ruler, to measure the degree to which DNA transitions to the Z isoform.

Figure 1.

Interaction of caZα_PKZ with DNA and its dependence of NaCl concentrations. (A) Multiple sequence alignment of Z-DNA binding proteins. Numbering and secondary structural elements for caZα_PKZ are shown above the sequence. Yellow and gray bars indicate residues important for Z-DNA recognition and protein folding, respectively. The key aromatic residue, tyrosine, is indicated by an orange bar. The asterisks indicate four highly conserved residues which play important roles in Zα function. (B) Residues of caZα_PKZ involved in intermolecular interaction with dT(CG)₃ reported in a previous study (10). Intermolecular H-bonds and van der Waals contacts indicated by solid lines and dashed lines, respectively. Three water molecules in key positions within the protein–DNA interface are indicated by orange ovals. (C) Mechanism for the B–Z conformational transition of a 6-bp DNA by two ZBPs. Black arrows indicate the primary transition mechanism. (D) 1D imino proton spectra of dT(CG)₃ at 35°C upon titration with caZα_PKZ in NMR buffer (pH = 8.0) containing 10 (left), 100 (middle) or 250 mM NaCl (right). The resonances from B-form are labeled as G2b and G4b and those from Z-form are labeled as G2z and G4z. (E) Relative Z-DNA populations (f_Z) of dT(CG)₃ induced by caZα_PKZ at 10 (red circle), 100 (blue square) or 250 mM NaCl (green triangle) as a function of [P]_tot/[N]_tot ratio. Solid lines are the best fit of the emerging G2z resonance to Equation (8).

Figure 2.

Solution structure of free caZα_PKZ and comparison with other structures. (A) Superimposed backbone traces of the 10 lowest energy caZα_PKZ structures. (B) Superimposition of the 3D structures of free caZα_PKZ (violet) and caZα_PKZ–dT(CG)₃ complex (PDB ID = 4KMF, pale green).

Figure 3.

Quantitative assessments of DNA binding by caZα_PKZ using HSQC data. (A) A representative region of the ¹H/¹⁵N-HSQC of caZα_PKZ upon titration with dT(CG)₃ at 10 (left) or 100 mM NaCl (right). The cross-peak color changes gradually from blue (free) to red (bound) according to the [N]_tot/[P]_tot ratio. (B) Global fitting of the f_Z data and (C and D) the ¹H/¹⁵N-HSQC titration curves for caZα_PKZ with dT(CG)₃ as a function of [N]_tot/[P]_tot ratio. Data for the global fitting were derived from ¹H (upper) and ¹⁵N (lower) chemical shift changes of HSQC cross-peaks of caZα_PKZ at (C) 10 or (D) 100 mM NaCl. Solid lines are the best fits to Equation (8) (in B) or Equation (7) (in C and D).

Figure 4.

DNA binding patterns differ with salt concentration and DNA conformation. (A) Histograms of the Δδ_avg values of ¹⁵N-caZα_PKZ for the B-DNA and Z-DNA binding at 10 (left) or 100 mM NaCl (right). Residues whose cross-peaks disappear or become very weak during titration are represented with green square symbols. The asterisks indicate residues whose cross-peaks overlap with other resonances during titration. (B and C) Mapping the location of the residues having large Δδ_avg onto the NMR structure of free caZα_PKZ for the (B) Z-DNA and (C) B-DNA binding at 10 (left) or 100 mM NaCl (right). The colors used to illustrate the Δδ_avg are: red or blue, >0.18 ppm; orange or cyan, 0.12–0.18 ppm; and yellow or pale green, 0.08–0.12 ppm (the same color coding is used in panel A). In both panels, the purple spheres indicate residues whose cross-peaks disappear or become very weak during titration. (D) The average chemical shift differences (Δδ_avg) between [NaCl] of 10 and 100 mM for free caZα_PKZ (upper) and caZα_PKZ complexed with B-DNA (middle) and Z-DNA (lower). (E) The calculated ¹H/¹⁵N-HSQC cross-peaks of caZα_PKZ complexed with B-DNA and Z-DNA in buffer containing 10 or 100 mM NaCl.

Figure 5.

Quantitative assessments of DNA binding by caZα_PKZ using CPMG data. (A) Two-state models of association/dissociation of a protein–ligand complex (upper) and the caZα_PKZ–Z-DNA complex (lower). (B) The ¹⁵N CPMG NMR relaxation dispersion data for free caZα_PKZ with [NaCl] of 10 (left) or 100 mM (right). (C) The simulated ¹H/¹⁵N-HSQC cross-peaks of H40 of the caZα_PKZ–dT(CG)₃ complex at 10 (upper) or 100 mM NaCl (lower). The ¹⁵N chemical shifts and calculated [P]/[P]_tot ratio are shown left and right of the spectra, respectively. (D) The ¹⁵N CPMG NMR relaxation dispersion data for the caZα_PKZ–dT(CG)₃ complex at 10 (left) or 100 mM NaCl (right). The microscopic rate constant (k_off,ZP2) was extracted from k_ex and the [P]/[P]_tot ratio.

Figure 6.

Contribution of H-bonding interaction of K56 with Z-DNA phosphate to B–Z transition. (A) 1D imino proton spectra of d(CG)₃ at 35°C upon titration with caZα_PKZ in NMR buffer (pH = 8.0) containing 10 (left), 100 (middle) or 250 mM NaCl (right). The resonances from B-form are labeled as G2b and G4b and those from Z-form are labeled as G2z and G4z. (B) Relative Z-DNA populations (f_Z) of d(CG)₃ induced by caZα_PKZ in NMR buffer containing 10 (red circle), 100 (blue square) or 250 mM NaCl (green triangle) as a function of [P]_tot/[N]_tot ratio. Solid lines are the best fit to Equation (8). (C) The exchange rate constants (k_ex) of the imino protons of the free dT(CG)₃ (left) and caZα_PKZ–dT(CG)₃ (right, [P]_tot/[N]_tot = 3.4) at 35°C. (D) The k_ex of the imino protons of the free d(CG)₃ (left), caZα_PKZ–d(CG)₃, (middle, [P]_tot/[N]_tot = 6.0) and hZα_ADAR1–d(CG)₃, (right, previous data (11)) at 35°C.

Figure 7.

Quantitative description of the energy landscape of the first (lef) and second DNA binding (right) of caZα_PKZ at 10 (red) or 100 mM NaCl (blue). Gibbs free energies for the DNA binding and B–Z transition steps were calculated using the equation, ΔG^o = RTlnK_d, where R is the gas constant and K_d is the dissociation constant for DNA binding (K_d,BP or K_d,ZP2) or ΔG^o = –RTlnK_BZ,1, where K_BZ,1 is the equilibrium constant for B–Z transition. The activation energy difference (ΔΔG^‡_ZP2) for the Z-DNA binding of caZα_PKZ between 10 and 100 mM NaCl condition was calculated using the equation, ΔΔG^‡_ZP2 = ΔG^‡_ZP2^100mM – ΔG^‡_ZP2^10mM = –RTln(k_on,ZP^100mM/k_on,ZP^10mM), where k_on,ZP^10mM and k_on,ZP^100mM are the association rate constants for Z-DNA binding of caZα_PKZ at 10 and 100 mM NaCl, respectively.