Structure of the uncomplexed DNA repair enzyme endonuclease VIII indicates significant interdomain flexibility

Abstract

Escherichia coli endonuclease VIII (Nei) excises oxidized pyrimidines from DNA. It shares significant sequence homology and similar mechanism with Fpg, a bacterial 8-oxoguanine glycosylase. The structure of a covalent Nei-DNA complex has been recently determined, revealing critical amino acid residues which are important for DNA binding and catalysis. Several Fpg structures have also been reported; however, analysis of structural dynamics of Fpg/Nei family proteins has been hindered by the lack of structures of uncomplexed and DNA-bound enzymes from the same source. We report a 2.8 A resolution structure of free wild-type Nei and two structures of its inactive mutants, Nei-E2A (2.3 A) and Nei-R252A (2.05 A). All three structures are virtually identical, demonstrating that the mutations did not affect the overall conformation of the protein in its free state. The structures show a significant conformational change compared with the Nei structure in its complex with DNA, reflecting a approximately 50 degrees rotation of the two main domains of the enzyme. Such interdomain flexibility has not been reported previously for any DNA glycosylase and may present the first evidence for a global DNA-induced conformational change in this class of enzymes. Several local but functionally relevant structural changes are also evident in other parts of the enzyme.

Figure 1

Overall structure of the DNA-free Nei. (a) Stereoview comparison of the three Nei structures described in the present paper (main-chain atoms only), demonstrating the high structural similarity between WT Nei (cyan), Nei-E2A (magenta) and Nei-R252A (yellow). (b) Schematic ribbon diagram, showing the overall structure of the molecule and its secondary structure elements. This scheme shows the DNA-binding elements of the enzyme, as determined from the Nei–DNA structure (17). These elements include the N-terminal loop (green), Lys-52 (pink), the DNA-inserted tripeptide including Gln69, Leu70 and Tyr71 (yellow), the H2TH motif (blue), and the zinc finger motif (red). The hinge polypeptide is shown in cyan and the ‘missing loop’ is shown in purple. The zinc atom is represented by a gray sphere (top). (c) A similar presentation of free Nei from a view perpendicular to (b). Here the polypeptide chain is ‘Rainbow’ color-coded, so that it starts (N-terminus) in dark blue and ends in red (C-terminus).

Figure 2

Solvent-accessible surface of free Nei, colored according to electrostatic potential (positive in blue, negative in red and neutral in gray), demonstrating the highly positive DNA-binding cleft of the enzyme (left center). Locations of three important residues involved in DNA binding are indicated by arrows.

Figure 3

Superposition of free Nei on the Nei–DNA complex, as resulted from a least-squares fit of the C-terminal domains of the free Nei structure (red) with the structure of the Nei–DNA covalent complex (1K3W). In the complex, the protein is shown in a ribbon diagram (green) and the DNA is shown in a ball-and-stick model (backbone, blue; bases, cyan). The zinc atom (bottom right) is shown as a magenta/yellow ball in the free/complex structures, respectively. (a) A view perpendicular to the long axis of the Nei molecule in the Nei–DNA complex, demonstrating the 50° rotation of the two domains of the free enzyme relative to each other. (b) The same superposition model viewed along the axis of the bound DNA [approximately orthogonal to (a)], demonstrating the induced fit of the free protein onto the DNA in the complex.

Figure 4

Conformational changes in the flexible hinge connecting the two Nei domains. (a) Cα trace of the free Nei structure. The areas involved in interactions with DNA are shown in yellow. The hinge region (residues 121–127) is shown in red (center). The movements of the protein elements from the open to the closed state are indicated by arrows (large arrows, protein domains; small arrows, hinge). (b) A close view on the hinge (residues 122–128) in the free Nei structure (carbons in green), with the corresponding section of the experimental electron density map (‘omit’ map at contour level of 4.0σ, blue), demonstrating the unequivocal model building of this part of the structure. Superimposed on it, is the same polypeptide chain in the Nei–DNA complex (carbons in yellow), showing the major conformational differences in this area between free and complexed Nei. (c) The main hydrogen bonds (dotted lines) stabilizing the open state of the free protein. Carbon atoms of the hinge residues are shown in dark green, carbon atoms of the interacting residues, in light green. The main chain of the hinge is colored beige, except for the closed turn (residues 122–125), which is colored purple.

Figure 5

The zinc finger motif of Nei. An electron density map is shown around the bound zinc atom [‘omit’ map at contour levels of 5σ (blue), and 17σ (green)]. The four Cys residues (with typical Zn–S bond lengths of 2.28–2.38 Å) are shown in a ball-and-stick representation and the two anti-parallel β-strands are shown in a ribbon diagram (beige). Superimposed on this region is the corresponding region of the Nei–DNA complex (red), demonstrating a movement of the tip of the zinc finger between the free and the DNA-bound forms of the enzyme.

Figure 6

The ‘missing’ polypeptide loop in Nei and Fpg. A superposition of the polypeptide chain in free Nei (red), and the corresponding region of the DNA-complexed Nei (green) with modeled thymine glycol (Tg, orange; other parts of the DNA are shown in cyan) and the loop region as described previously (17). Also superimposed is the same region in the crystal structure of Bacillus stearothermophilus Fpg complexed with DHU-containing DNA (PDB 1R2Z) (22) (blue). Similar parts of the polypeptide chain are shown in khaki. Note the major conformational change of this flexible loop (residues 212–223) in the different structures and upon DNA binding (left), closing onto the modeled Tg binding pocket.

Figure 7

Stereoview superposition of free Tth-Fpg (blue) and free Nei (purple). Two crystallographically independent Tth-Fpg molecules (PDB 1EE8) are shown in dark and light blue, respectively. All three structures (shown here as Cα diagrams) have been superimposed based on their C-terminal domains. Note the relatively small interdomain flexibility of the two Tth-Fpg molecules, in comparison with the significant interdomain rotation in the free Nei structure.