Crystal structural analysis and metal-dependent stability and activity studies of the ColE7 endonuclease domain in complex with DNA/Zn2+ or inhibitor/Ni2+

Abstract

The nuclease domain of ColE7 (N-ColE7) contains an H-N-H motif that folds in a beta beta alpha-metal topology. Here we report the crystal structures of a Zn2+-bound N-ColE7 (H545E mutant) in complex with a 12-bp duplex DNA and a Ni2+-bound N-ColE7 in complex with the inhibitor Im7 at a resolution of 2.5 A and 2.0 A, respectively. Metal-dependent cleavage assays showed that N-ColE7 cleaves double-stranded DNA with a single metal ion cofactor, Ni2+, Mg2+, Mn2+, and Zn2+. ColE7 purified from Escherichia coli contains an endogenous zinc ion that was not replaced by Mg2+ at concentrations of <25 mM, indicating that zinc is the physiologically relevant metal ion in N-ColE7 in host E. coli. In the crystal structure of N-ColE7/DNA complex, the zinc ion is directly coordinated to three histidines and the DNA scissile phosphate in a tetrahedral geometry. In contrast, Ni2+ is bound in N-ColE7 in two different modes, to four ligands (three histidines and one phosphate ion), or to five ligands with an additional water molecule. These data suggest that the divalent metal ion in the His-metal finger motif can be coordinated to six ligands, such as Mg2+ in I-PpoI, Serratia nuclease and Vvn, five ligands or four ligands, such as Ni2+ or Zn2+ in ColE7. Universally, the metal ion in the His-metal finger motif is bound to the DNA scissile phosphate and serves three roles during hydrolysis: polarization of the P-O bond for nucleophilic attack, stabilization of the phosphoanion transition state and stabilization of the cleaved product.

Figure 1.

Measurements of the melting points for apo- and metal-bound N-ColE7 by circular dichroism. (A) The melting points of the apo- and each metal-bound N-ColE7 were measured by circular dichroism at a wavelength of 222 nm. The protein concentration used for all measurements was 0.1 mg/mL in 10 mM Tris-HCl (pH 8.0). The melting points were 74.3 ± 0.7°C for apo-N-ColE7, 75.5 ± 0.1°C for Mg-bound, 73.1 ± 0.8°C for Mn-bound, 80.8 ± 0.8°C for Zn-bound, and 80.8 ± 1.4°C for Ni-bound N-ColE7. (B) The zinc-bound N-ColE7 was mixed with buffers containing 10–100 mM MgCl₂. The melting points of N-ColE7 in 10 and 25 mM Mg²⁺ buffers were ~80°C, close to that of the Zn-bound enzyme, indicating that zinc was still bound in N-ColE7. However, the melting points of N-ColE7 in 50, 75, and 100 mM Mg²⁺ buffers were shifted to ~75°C, indicating that Zn²⁺ was not bound to N-ColE7 when Mg²⁺ concentrations were >50 mM.

Figure 2.

The metal-dependent endonuclease activity of N-ColE7 (the nuclease domain of ColE7). (A) The endonuclease activity of N-ColE7 was analyzed by plasmid digestion assays using pQE-70 as the substrate (see Control lane). Various amounts of apo-N-ColE7 (0.05, 0.27, 1.33, and 6.67 μM), did not cleave plasmids (100 ng). However, the endonuclease activity of apo-N-ColE7 was reactivated by the presence of one of the metal ions, Ni²⁺, Mn²⁺, Mg²⁺, and Zn²⁺. Ni-N-ColE7 cleaved plasmids at 0.05 μM; Mn- and Mg-N-ColE7 cleaved plasmids at 0.27 μM; and Zn-N-ColE7 cleaved plasmids at ~1.33 μM. (B) Different concentrations of Zn²⁺ and Mg²⁺ (0, 0.1, 1.0 10.0, 100.0 mM, as indicated in the figure) were added to Zn-bound and Mg-bound N-ColE7. Both Mg²⁺ (0.1 to 10 mM) and Zn²⁺ (0.1 to 100 mM) enhanced ColE7 endonuclease activity, but 100 mM of Mg²⁺-inhibited enzyme activity. (O) Open, (L) linear, and (S) supercoiled DNA.

Figure 3.

The endonuclease activities of N-ColE7 mutants analyzed by plasmid digestion assays. (A) Various amounts (0.01, 0.05, and 0.27 μM) of zinc-bound wild-type and mutated N-ColE7 were incubated with pQE-70 plasmids (100 ng) in 10 mM Tris-HCl (pH 8.0) for 20 min at 37°C. The digested DNA was resolved on 1% agarose gels. The wild-type N-ColE7 started to digest plasmids at 0.01 μM. The H573E and H573A N-ColE7 mutants showed reduced endonuclease activities, only starting to cleave DNA at ~0.05 μM. (B) The H545A and H545E mutants of N-ColE7 had no endonuclease activity when the enzyme concentration was increased from 0.01 to 0.27 μM. But H545Q mutant had residual endonuclease activity, cleaving DNA at ~0.27 μM. (O) Open, (L) linear, and (S) supercoiled DNA.

Figure 4.

The omit electron density maps of Zn-bound and Ni-bound endonuclease active sites in H545E/DNA/Zn²⁺ and N- ColE7/Im7/Ni²⁺ complexes, respectively. (A) Stereo view of the omit difference maps (F_o − F_c) contoured at 2.5 σ (blue) and 12.0 σ (red) shows that the DNA scissile phosphate (P5) is bound directly to the zinc ion. The tetrahedral geometry and bond distances around the Zn atom are schematically shown in the right panel. (B) Stereo views of the omit difference maps contoured at 2.5 σ (blue) and 18.0 σ (red) around the Ni-binding site in the two noncrystallographic-symmetry related molecules in N-ColE7/Im7/Ni²⁺ complex structure. In molecule A, Ni²⁺ is bound to three histidines and a phosphate in a tetrahedral geometry. In molecule B, Ni²⁺ is bound to three histidines, a phosphate, and a water molecule in a distorted trigonal bi-pyramidal geometry.

Figure 5.

Stereo view of the H545E/DNA/Zn²⁺ crystal structure. (A) The H-N-H motif (red) of N-ColE7 (blue and red ribbon) is bound at the minor groove of DNA 12-mer (yellow). DNA is bent ~20° (calculated by Curve [Lavery and Sklenar 1988]) away from N-ColE7. The zinc ion (displayed as a green ball) is located closely to one of the phosphates in the DNA backbone. (B) The zinc-bound active site in the N-ColE7/12-mer DNA complex (red) was superimposed with the metal-free active site (green) in the N-ColE7/8-mer DNA complex (PDB entry 1PT3). The two structures fitted well with an average RMS difference of 0.34 Å for 18 C_α atoms located in β-strand or α-helix regions used for superimposition. In the zinc-bound active site, the zinc ion is coordinated to the side chains of H544, H569, and H573 and the scissile phosphate in a tetrahedral geometry. In the zinc-free active site, only H573 shifted away slightly. The loop between residues 548 to 554 was disordered and not modeled in the H545E/DNA/Zn complex.

Figure 6.

Schematic presentations of the interactions between N-ColE7 and DNA. (A) The solid blue lines indicate hydrogen bonds or salt bridges (<3.50 Å) and the red arrows show van der Waals contacts (<3.35 Å) between N-ColE7 and DNA. Most of the interactions are between proteins side chains and DNA phosphate backbones. (B) DNA groove widths were plotted for each base step in H545E/12-mer DNA complex (this study), N-ColE7/8-mer DNA complex (PDB entry 1PT3), Vvn/DNA (PDB entry 1OUP), and I-PpoI/DNA (PDB entry 1A74). The DNA cleavage sites are aligned and marked by a solid arrow, shown at the bottom of the figure. The minor groove widths are widened to ~9 Å at the region bound to ββα-metal motif in all complexes. DNA is cleaved right at the 3′-side of the widened minor groove.