Backbone Dynamics of the C-Terminal Domain of Escherichia coli Topoisomerase I in the Absence and Presence of Single-Stranded DNA

Abstract

The backbone dynamics of the C-terminal DNA-binding domain of Escherichia coli topoisomerase I has been characterized in the absence and presence of single-stranded DNA by NMR spectroscopy. ¹⁵N spin−lattice relaxation times (T₁), spin−spin relaxation times (T₂), and heteronuclear NOEs were determined for the uniformly ¹⁵N-labeled protein. These data were analyzed by using the model-free formalism to derive the model-free parameters (S², τ_e, and R_ex) for each backbone N−H bond vector and the overall molecular rotational correlation time (τ_m). The molecular rotational correlation time τ_m was determined to be 7.49 ± 0.36 ns for the free and 12.7 ± 1.07 ns for the complexed protein. Several residues were found to be much more mobile than the average, including 11 residues at the N-terminus, 2 residues at the C-terminus, and residues 25 and 31−35 which are located in a region of the protein that binds to DNA. The binding of ssDNA to the free protein causes a slight increase in the order parameters (S²) for a small number of residues and a slight decrease in the order parameters (S²) for the majority of the residues. In particular, upon binding to ssDNA, the mobility of the first α-helix and the two β-sheets was slightly increased, and the mobility of a few specific residues in the loops/turns was restricted. These results differ from the previous studies on the backbone dynamics of molecular complexes in which reduced mobilities were typically observed upon ligand binding.