Volume 11, Issue 6, Pages 1647-1659 (June 2003) Structure and Specificity of the Vertebrate Anti-Mutator Uracil-DNA Glycosylase SMUG1  Jane E.A. Wibley, Timothy R. Waters, Karl Haushalter, Gregory L. Verdine, Laurence H. Pearl  Molecular Cell  Volume 11, Issue 6, Pages 1647-1659 (June 2003) DOI: 10.1016/S1097-2765(03)00235-1

Figure 1 SMUG1 Structure (A) Secondary structure cartoon of ΔN-xSMUG1, rainbow colored blue→red from N- to C terminus. This and all other molecular graphics were generated using PyMol (DeLano Scientific, San Carlos, CA; http://www.pymol.org). (B) As (A) but with α helices red, β strands green, and coil yellow. (C) Stereo-pairs of SMUG1 (N→C rainbow colored) viewed toward the cleft between the large and small lobes. (D) Molecular surface (N→C rainbow colored) showing the cleft and the recessed pyrimidine binding pocket. Molecular Cell 2003 11, 1647-1659DOI: (10.1016/S1097-2765(03)00235-1)

Figure 2 Structural Relationships in the UDG Superfamily (A) Secondary structure cartoons of examples of Family-1 (Herpes simplex type 1 UNG), Family-2 (E. coli MUG), and Family-3 (X. laevis SMUG1) uracil-DNA glycosylases. The common core is (N→C rainbow colored) with family specific insertions/extension shown in white. (B) Amino acid sequences of Xenopus and human SMUG1s, E. coli MUG, and the HSV-1 UNG aligned on the basis of their three-dimensional structures, using SSAP (Orengo et al., 1992). The N-terminal and C-terminal motifs that contribute catalytic residues and the walls of the pyrimidine binding pocket are highlighted in blue and red, respectively. The common phenylalanine residue that stacks against bound pyrimidines is highlighted in green, while the residue that forms the floor of the pocket (and in UNGs and SMUGs provides the major discrimination against cytosine) is highlighted in orange. Red cylinders and green arrows represent α helices and β strands, respectively. Molecular Cell 2003 11, 1647-1659DOI: (10.1016/S1097-2765(03)00235-1)

Figure 3 UDG Superfamily Active Sites Active sites of (A) xSMUG1; (B) HSV-1 UDG; (C) E. coli MUG (overview left, detail right), with the N- and C-catalytic motifs, floor, and side residues colored as in the sequence alignment in Figure 2. The catalytic residues are underlined in each case. Across the three UDG families, only the phenylalanine that provides the side wall of the pocket (green), and the glycine and proline in the N-terminal catalytic motif (blue) are completely conserved. Molecular Cell 2003 11, 1647-1659DOI: (10.1016/S1097-2765(03)00235-1)

Figure 4 SMUG1 – DNA Complexes (A) Stereo-pair of SMUG1 bound to a DNA duplex in a jilted complex. The terminal G:C base pair has been split with the estranged guanine directed away from the enzyme, while the cytosine is bound nonspecifically in a narrow channel between the large and small lobes of the enzyme over the mouth of the pyrimidine binding pocket. The surfaces of residues forming the active site and binding pocket are colored as in Figure 2, with the addition of residues 255–260 which are shown in brown. Arg254 penetrates the duplex from the minor groove, occupying the space left by the “flipped” pyrimidine, while Pro256 occupies the space left by the estranged guanine on the other strand. The abasic site generated by base excision of the central G:U mispair by SMUG1 during crystallization is indicated (ab). (B) As (A), but rotated 90° around the horizontal. (C) Close-up of the jilted complex with the flipped cytosine bound over the pyrimidine pocket. (D) In the poised complex, the uracil base of the βFU penetrates the pyrimidine pocket but does not make a productive interaction. Molecular Cell 2003 11, 1647-1659DOI: (10.1016/S1097-2765(03)00235-1)

Figure 5 DNA Interactions in the UDG Superfamily (A) In Family-1 UNGs, the scissile pyrimidine on the proximal strand is flipped into the active site, and the gap is occupied by a leucine residue from the enzyme (hUDG – PDB code 1SSP). The estranged base base on the distal strand remains fully stacked with its neighbors in the duplex. The asymmetric disruption of the base stacking generates an ≅45° kink in the path of the DNA. (B) In the Family-2 MUG enzyme, the scissile pyrimidine is again replaced by a leucine, but the stacking on the distal strand is also disrupted by intercalation of an arginine side chain between the estranged guanine and its 5′ neighbor, as part of a wedge penetrating the base stack of the DNA. (C) The Family-3 SMUG inserts a substantially larger “wedge” into the DNA, with both the scissile pyrimidine and its estranged partner being displaced into extrahelical conformations. The size of the wedge segment in SMUG requires that at least one base pair on the 3′ side of the scissile pyrimidine is also disrupted. Molecular Cell 2003 11, 1647-1659DOI: (10.1016/S1097-2765(03)00235-1)

Figure 6 Pyrimidine Specificity (A) Uracil binds in the pyrimidine pocket, making specific and selective hydrogen bonds with Asn174, and without displacing the water bound between the peptide nitrogens of Gly98 and Met102. Electron density is from an Fo-Fc with the base omitted from the calculation, and contoured at 2σ. (B) Schematic of (A). (C) Hydroxymethyluracil binds with the same specific hydrogen bonds as uracil. The bound water is displaced but the hydroxyl group satisfies the hydrogen bonds to NH-Gly98 and NH-Met102. Electron density is from an Fo-Fc with the base omitted from the calculation, and contoured at 2σ. (D) Schematic of (C). (E) The C5-methyl of thymine is too large to be accommodated without displacing the bound water but, unlike hydroxymethyluracil, it cannot replace the lost hydrogen bonds, and its binding is consequently energetically unfavorable. Molecular Cell 2003 11, 1647-1659DOI: (10.1016/S1097-2765(03)00235-1)