Metal ion and inter-domain interactions as functional networks in E. coli topoisomerase I

Abstract

Escherichia coli topoisomerase I (EcTopoI) is a type IA bacterial topoisomerase which is receiving large attention due to its potential application as novel target for antibacterial therapeutics. Nevertheless, a detailed knowledge of its mechanism of action at molecular level is to some extent lacking. This is partly due to the requirement of several factors (metal ions, nucleic acid) to the proper progress of the enzyme catalytic cycle. Additionally, each of them can differently affect the protein structure. Here we assess the role of the different components (DNA, metal ions, protein domains) in a dynamic environment as in solution by monitoring the catalytic as well as the structural properties of EcTopoI. Our results clearly indicated the interaction among these components as functionally relevant and underlined their mutual involvement. Some similarities with other enzymes of the same family emerged (for example DNA prevents divalent metal ions coordination at non selective binding sites). Interestingly, same interactions (C- and N-terminal domain interaction) appear to be peculiar of this bacterial topoisomerase which suggest they could be favorably exploited to the design of selective inhibitors for this class of enzyme.

Fig. 1

pBR 322 (0.15 μg) relaxation promoted by EcTopoI (0.7 nM) in the presence of variable metal ion concentration. In PANEL A, effect of increasing Mg²⁺ concentrations, according to two different protocols (see text). In PANEL B the percentage of relaxed DNA obtained after incubation of the nucleic acid with EcTopoI in the presence of increasing concentrations of metal ions according to two different protocols.

Fig. 2

pBR 322 (0.15 μg) relaxation promoted by EcTopoI (0.7 nM) in the presence/absence of 10 mM Mg²⁺ after increasing incubation time, according to two different protocols (see text). In Panel A, resolution of reaction products by agarose gel is reported, in Panel B the percentage of relaxed DNA obtained in the presence of Mg²⁺ subtracted for the relaxation induced in the absence of metal ion are plotted as a function of incubation time.

Fig. 3

Effect of divalent ions on the level of nicked plasmid DNA resulting from cleavage of supercoiled plasmid DNA (0.3 μg) by EcTopoI (200 ng). The nicked DNA from the covalent cleavage intermediate was separated from the closed DNA by agarose gel electrophoresis in the presence of ethidium bromide.

Fig. 4

Effect of divalent ions on cleavage of single-stranded DNA by topoisomerase I. 5’-³²P labelled single-stranded DNA 216 base in length (S) was incubated with 200 ng of EcTopoI or Top67 in the presence of 1 mM EDTA (E) or different divalent ions at 4 mM concentration.

Fig. 5

Main cleavage sites identified along a 32-residue sequence. The cleavage products produced on the same sequence after 30 min incubation at room temperature with EcTopoI and Top67 (1 μM), in the presence of increasing divalent metal ions concentrations (0-50 mM) in 20 mM Tris, pH 8.0, 100 μg/ml BSA, 12 % glycerol were resolved by DNA sequencing PAGE. Panel B, C and D refer to addition of Mg²⁺, Ca²⁺ and Mn²⁺, respectively. Lanes marked M and C refer to purine marker and DNA treated in the absence of the protein, respectively.

Fig. 6

CD spectrum of EcTopoI (0.35 μM) recorded in 50 mM Tris, 20 mM KCl, pH 7.7 (PANEL A) and melting profiles acquired by reading the protein CD signal at 220 nm (PANEL B) in the reported buffers.

Fig. 7

Dichroic spectra (PANEL A) and melting profiles (PANEL B) of EcTopoI and its N- and C-terminal domains (Top67 and ZD, respectively) recorded in 50 mM Tris, 150 mM KCl, pH 7.7. Protein concentration: 0.3 μM. Math refers to the algebraic combination of the Top67 and ZD domain.

Fig. 8

Melting profiles of 0.3 μM EcTopoI or Top67 (PANEL A and B, respectively) acquired by reading the protein CD signal at 220 nm in 50 mM Tris-HCl, 150 mM KCl, pH 7.7, in the presence/absence of 4 mM divalent metal ions. Protein concentration was 0.3 μM.

Fig. 9

Melting profiles of EcTopoI (Panel A) and its N- and C-terminal domains (Panel B and C, respectively) acquired by reading the protein CD signal at 220 nm in the presence (dotted line) or absence (solid line) of oligo32 in 50 mM Tris, 150 mM KCl, pH 7.7. The relative Molar Ellipticity variation (Δ Molar Ellipticity /(Δ Molar Ellipticity)_max) is reported. Protein concentration: 0.3 μM, DNA concentration: 0.3 μM.