Figure 5: Mg²⁺ binding to the central channel reduces the flexibility of the dUTPase trimer.

(a) Predicted Mg²⁺ binding sites (pink) within the central channel of human dUTPase (PDB: 1Q5H, colored by subunits). The residues constituting the channel wall are shown as surface while the rest of the molecule is shown as cartoon representation. The Tyr residues possibly responsible for the change in the near UV spectra upon Mg²⁺ binding are shown in blue. Active sites are highlighted by the bound dUDP (shown as sticks with atomic coloring). (b) Near UV and Far UV (inset) spectra of hDUT^F158W in the presence (dash-dot-dot) and in the absence (solid line) of 5 mM MgCl₂. The spectrum of the buffer is marked by dash-dot line. (c) Thermal unfolding of hDUT^F158W in the presence and in the absence of 5 mM Mg²⁺. Smooth lines through the data are Boltzmann fits (Equation (4)). The melting temperatures (transition midpoints) are Tm = 59.8 ± 0.2 °C in the presence of MgCl₂ and Tm = 58.4 ± 0.2 °C in the absence of MgCl₂ (n = 3). Errors represent SD. (d) Limited trypsinolysis of hDUT^F158W and mtDUT^H145W performed in the presence and in the absence of 5 mM MgCl₂. The open gray arrow head, the black arrow head and the open black arrow head indicate the intact, the N-terminal cleaved and the N- and C-terminal cleaved enzymes, respectively. The densitometric analysis of the relative amount of the core enzyme (intact enzyme + N-terminal cleaved enzyme + N- and C-terminal cleaved enzyme) is shown in Supplementary Fig. 5d.