Figure 4.

Metal cofactor interactions with Tdp2. (A) Intrinsic tryptophan fluorescence of mTdp2^cat was used to monitor a conformational response to divalent metal ion binding. Either Mg²⁺ or Ca²⁺ were titrated in the presence or absence of 5′-P DNA, and the tryptophan fluorescence was monitored with an excitation wavelength of 280 nm and emission wavelength of 350 nm using 10 nm band pass filters. Both Mg²⁺ and Ca²⁺ induce a conformational change which elicits an increase in tryptophan fluorescence of mTdp2^cat in the presence and absence of DNA, while D358N active site mutant of mTdp2^cat is unresponsive to Mg²⁺. (B) mTdp2^cat activity assayed on a T5PNP substrate as a function of Mg²⁺ and Ca²⁺ concentration. PNP release (monitored by absorbance at 415 nm) as a function of Mg²⁺ concentration and in the absence or presence of 1 or 10 mM Ca²⁺ is shown; error bars, s.d. n = 4. (C) σ-A weighted 2Fo-Fc electron density map (blue) and model-phased anomalous difference Fourier (magenta) maps for the mTdp2^cat–DNA–Mn²⁺ complex (PDB entry 5INP) show a single Mn²⁺ (cyan) is bound with expected octahedral coordination geometry. A 53σ peak in the anomalous difference Fourier map (data collected at λ = 1.5418 Å) supports Mn²⁺ as the identity of this atom. (D) Comparison of Ca²⁺ (green Ca²⁺ ion, orange DNA) (PDB entry 5INQ), and Mg²⁺ (magenta Mg²⁺ ion, yellow DNA) (PDB entry 4GZ1) mTdp2^cat–DNA structures shows that Ca²⁺ distorts the 5′-phosphate binding mode.