Structural characterization of a viral NEIL1 ortholog unliganded and bound to abasic site-containing DNA

Abstract

Endonuclease VIII (Nei) is a DNA glycosylase of the base excision repair pathway that recognizes and excises oxidized pyrimidines. We determined the crystal structures of a NEIL1 ortholog from the giant Mimivirus (MvNei1) unliganded and bound to DNA containing tetrahydrofuran (THF), which is the first structure of any Nei with an abasic site analog. The MvNei1 structures exhibit the same overall architecture as other enzymes of the Fpg/Nei family, which consists of two globular domains joined by a linker region. MvNei1 harbors a zincless finger, first described in human NEIL1, rather than the signature zinc finger generally found in the Fpg/Nei family. In contrast to Escherichia coli Nei, where a dramatic conformational change was observed upon binding DNA, the structure of MvNei1 bound to DNA does not reveal any substantial movement compared with the unliganded enzyme. A protein segment encompassing residues 217-245 in MvNei1 corresponds to the "missing loop" in E. coli Nei and the "alphaF-beta10 loop" in E. coli Fpg, which has been reported to be involved in lesion recognition. Interestingly, the corresponding loop in MvNei1 is ordered in both the unliganded and furan-bound structures, unlike other Fpg/Nei enzymes where the loop is generally ordered in the unliganded enzyme or in complexes with a lesion, and disordered otherwise. In the MvNei1.tetrahydrofuran complex a tyrosine located at the tip of the putative lesion recognition loop stacks against the furan ring; the tyrosine is predicted to adopt a different conformation to accommodate a modified base.

FIGURE 1.

A, ribbon diagram of unliganded MvNei1. The model from form II crystals comprises residues 2–289. The secondary structure elements were defined by DSSP (59) and are as follows: αA (4–18), β1 (22–27), αB (41–45), β2 (50–58), β3 (61–67), β4 (75–81), β5 (87–89), β6 (96–102), β7 (107–111), β8 (118–122), αC (125–133), αD (157–162), αE (173–183), αF (196–215), αG (226–229), β9 (265–267), and β10 (279–281). Helices are shown in light green and β-strands in magenta. The putative lesion binding loop is highlighted in a darker shade of green. B, comparison of MvNei1 with E. coli Nei (EcoNei). Superposition of MvNei1 (pink) with unliganded EcoNei (gray; PDB code 1Q3B (30) and the EcoNei trapped DNA complex (black; PDB code 1K3W (23)). C, close-up of the zinc-finger motif. Shown are residues 230–262 for EcoNei, 263–290 for hNEIL1, and 253–289 for MvNei1. The asterisks indicate the position of the Cα of the conserved arginine (Arg-252 in EcoNei and Arg-277 in hNEIL1 and MvNei1).

FIGURE 2.

A, MvNei1·THF complex structure. The zincless finger, H2TH, catalytic proline and glutamic acid, and conserved arginine are highlighted in pink. The DNA is shown in gray. The putative lesion binding loop is shown in dark green. B, schematics of MvNei1·DNA interactions. Nucleotides are numbered beginning from THF0, with positive numbers toward the 5′-end. Hydrogen bonds are represented with black arrows pointing toward the acceptors. Tyr-221 stacks with THF.

FIGURE 3.

A, close-up view of THF and interacting residues with overlaid simulated annealing omit map contoured at 5 σ (lime green). MvNei1 is shown in green and DNA in gray. Hydrogen bonds are represented by black dashed lines. B, superposition of the MvNei1·THF complex onto LlaFpg·AP site complexes (THF and Pr). The MvNei1·THF complex is shown in green, LlaFpg/Pr complex (PDB code 1PJI (52)) in beige, and the LlaFpg·THF complex (PDB code 1PM5 (52)) in orange. The hydrogen bond is shown as a black dashed line. The blue dashed line indicates the distance between the amino group of Pro-2 and C1′ of THF.

FIGURE 4.

Determination of the apparent dissociation constant of MvNei1 bound to DNA containing THF. Plot of percent MvNei1 bound to a 23-mer 5′-³²P-labeled duplex containing THF as a function of enzyme concentration. Error bars represent the standard error on the average of three separate experiments.

FIGURE 5.

A, superposition of the loop region of the three Nei enzymes with known structures: MvNei1 (pink), EcoNei (gray; PDB code 1Q3B (30)), and hNEIL1 (cyan; PDB code 1TDH (29)). Two asterisks indicate the start and end of the putative lesion binding loop in MvNei1. B, superposition of the MvNei1 loop (pink) with the αF–β10 loop of the BstFpg·8-oxoG complex (blue; PDB code 1R2Y (48)). C, superposition of the MvNei1·THF complex with the BstFpg·DHU complex (PDB code 1R2Z (48)). The MvNei1 complex is shown in green and the BstFpg complex in blue.