The Monomeric dUTPase from Epstein-Barr Virus Mimics Trimeric dUTPases Nicolas Tarbouriech, Marlyse Buisson, Jean-Marie Seigneurin, Stephen Cusack, Wim P. Burmeister  Structure  Volume 13, Issue 9, Pages 1299-1310 (September 2005) DOI: 10.1016/j.str.2005.06.009 Copyright © 2005 Elsevier Ltd Terms and Conditions

Figure 1 Sequence and Structure of the EBV dUTPase (A) Cα representation of the EBV dUTPase structure with every tenth amino acid labeled. The disulfide bridge is shown in orange, dUMP is shown in a ball-and-stick representation, and conserved motifs involved in the active site formation are shown in color. Motif 1: green; motif 2: blue; motif 3: red, motif 4: yellow. The figure was made with MOLSCRIPT (Kraulis, 1991). (B) Stereoview of a superposition of the Cα chain of the complexes of EBV dUTPase with dUMP (blue) or α,β-imino-dUTP (yellow). The inhibitor α,β-imino-dUTP is shown together with its electron density from an Fo-Fc omit map contoured at the 1σ (green) and 3σ (red) level. The red sphere depicts a water molecule, and the cyan sphere depicts the magnesium ion. Aspartate residues important for the rearrangement of loop 115–132 are shown together with the position of Gln115, which delimits the flexible loop. (C) Structure-based alignment of known dUTPase sequences made with INDONESIA (Li et al., 2003). Domain I and domain II + III of the EBV dUTPase have been aligned independently. Residues are highlighted in gray, dark gray, or yellow depending on whether they are, respectively, 60%, 80%, or 100% conserved. The five motifs are shown with boxes of the same color as in (A), with motif 5 in black. Ecol: E. coli, Mtub: M. tuberculosis, EIAV: equine infectious anemia virus, Hsap: H. sapiens, N: EBV domain I, C: EBV domain II + III. Residues with side chains interacting with the substrate analog or with a role in catalysis in the EBV dUTPase structures are printed in magenta. Secondary structures of H. sapiens and EBV dUTPase are shown above and below the corresponding sequences. Secondary structure elements are named according to Mol et al. (1996). For EBV dUTPase, an “N” precedes the name for domain I and a “C” precedes the name for domains II and III. Structure 2005 13, 1299-1310DOI: (10.1016/j.str.2005.06.009) Copyright © 2005 Elsevier Ltd Terms and Conditions

Figure 2 Comparison of EBV and E. coli dUTPases Stereoview. Helices are colored in green, the β strands are in blue, and the loop structures are in yellow (EBV) or pink (E. coli [Barabas et al., 2004], pdb entry 1rn8). Products or substrate analogs are colored in orange. The figure was generated with PYMOL (DeLano, 2002). (A and B) View perpendicular to the 3-fold axis of the E. coli enzyme looking onto the active site. (C and D) Top view onto the 3-fold axis of the E. coli enzyme. Structure 2005 13, 1299-1310DOI: (10.1016/j.str.2005.06.009) Copyright © 2005 Elsevier Ltd Terms and Conditions

Figure 3 Superposition of the Two dUTPase-like Domains of EBV dUTPase Stereoview in which domain I is colored in green and domain II is in red. The superposition used the CCP4MG software. Secondary structure elements are labeled according to Figure 1C, with N or C specified if the element is specific for domains I or II. Structure 2005 13, 1299-1310DOI: (10.1016/j.str.2005.06.009) Copyright © 2005 Elsevier Ltd Terms and Conditions

Figure 4 The Active Site (A) Schematic organization of the trimeric dUTPases and localization of the conserved sequence motifs I–V around the active sites represented by red dots. (B) Schematic organization of the monomeric dUTPases. (C) Stereoview of the superposition between the EBV dUTPase complex with dUMP (green carbon atoms), with α,β-imino-dUTP (salmon carbon atoms), and with the E. coli dUTPase-α,β-imino-dUTP (blue carbon atoms, pdb entry 1rn8) complex. Alternate conformations are shown for Arg84, Arg171, and Ser172 of the complex with dUMP. Two important water molecules are shown: W1 at the top and W23 at the bottom. The two EBV structures were aligned based on their Cα trace, and the E. coli one was aligned with the dUMP complex based on the position of the deoxyuridine group. (D) Stereoview of the comparison of the active site from the human dUTPase-dUDP complex (white carbon atoms, pdb entry 1q5h) and the EBV dUTPase in complex with dUMP (yellow carbon atoms). The deoxyuridine groups have been superposed. For clarity, the diphosphate group of the human enzyme has been truncated. The structure at 1.5 Å resolution of the EBV enzyme allows the unambiguous assignment of the orientation of the imidazole ring of His71 based on an even distribution of the atomic temperature factors in case of the correct orientation. Structure 2005 13, 1299-1310DOI: (10.1016/j.str.2005.06.009) Copyright © 2005 Elsevier Ltd Terms and Conditions