Simulations of DNA topoisomerase 1B bound to supercoiled DNA reveal changes in the flexibility pattern of the enzyme and a secondary protein-DNA binding site

Abstract

Human topoisomerase 1B has been simulated covalently bound to a negatively supercoiled DNA minicircle, and its behavior compared to the enzyme bound to a simple linear DNA duplex. The presence of the more realistic supercoiled substrate facilitates the formation of larger number of protein-DNA interactions when compared to a simple linear duplex fragment. The number of protein-DNA hydrogen bonds doubles in proximity to the active site, affecting all of the residues in the catalytic pentad. The clamp over the DNA, characterized by the salt bridge between Lys369 and Glu497, undergoes reduced fluctuations when bound to the supercoiled minicircle. The linker domain of the enzyme, which is implicated in the controlled relaxation of superhelical stress, also displays an increased number of contacts with the minicircle compared to linear DNA. Finally, the more complex topology of the supercoiled DNA minicircle gives rise to a secondary DNA binding site involving four residues located on subdomain III. The simulation trajectories reveal significant changes in the interactions between the enzyme and the DNA for the more complex DNA topology, which are consistent with the experimental observation that the protein has a preference for binding to supercoiled DNA.

Figure 1.

Molecular structure of the hTop1B in covalent complex with a 240 bp negatively supercoiled DNA after 20 ns of atomistic molecular dynamics, showing the formation of the secondary protein–DNA binding site.

Figure 2.

Per-residue RMSF of the protein in hTop1Blin (black line) and hTop1Bsc (red line) complexes. The different protein domains are defined by vertical lines.

Figure 3.

Clustering of the structures of the linker domain, comprising residues Ala625-Lys712, in the simulation of hTop1Blin (A) and hTop1Bsc complexes (B). The centroids of the four families representing 96% of total structures in hTop1Blinear and of the only one representing 98% of total structures in hTop1Bsc are reported in red.

Figure 4.

Protein–DNA hydrogen bonding network within the secondary binding site.

Figure 5.

(A) Time average and S.D. error of Δtw for each base-pair step. Catalytic site (A and A′), linker binding site (B) and secondary (distal) binding site (C) are highlighted in the plot. ΔTw of the disrupted DNA bases are shown in green. (B) Cleavage site of the linear DNA (left panel) and of the supercoiled DNA plasmid (right panel). The presence of the hydrogen bond between the −1/+1 bases in the linear DNA is displayed in green.