Figure 6: Stabilization of the central channel hinders conformational coupling with the active sites in dUTPases.

(a) Sequence comparison of the allosteric loop region within the dUTPase superfamily. The alignment was created by clustalW with minimal manual editing. Allosteric loop, yellow highlight; the uracil binding cleft, underlined; conserved motifs, grey highlight; uracil ring coordination, bold. Amino acid conservation is distinguished by: identity (*), strong similarity (:) and weak similarity (.). (b) Superposition of the 3D structures of representative dUTPase superfamily enzymes from panel (a) (PDB: E. coli DCD – 1XS1, hDUT – 2HQU, EIAV DUT – 1DUC, E. coli DUT – 1RN8, MTB DUT – 3HZA). The superposition was performed by the alignment of the bound nucleotides using PyMol. Only the main chain atoms of the proteins and the nucleoside part of the ligands are shown for clarity. The color code refers to the proteins in panel (a). Note the structural variance in the allosteric loop. (c–e) Cross section of the central channel of hDUT (1Q5H), E. coli DUT and MTB DUT at the level of the uracil binding pocket. The side chains within the central channel and the bound ligands are shown as sticks with atomic coloring. The Mg²⁺ ion is represented as blue non-bound sphere. In panel (d) the longitudinal view of the threefold trimer interface (two subunits are shown) of the E. coli DUT is also shown using surface representation with atomic coloring to highlight the hydrophobic character of the central channel.