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 Å resolution structure of free wild-type Nei and two structures of its inactive mutants, Nei-E2A (2.3 Å) and Nei-R252A (2.05 Å). 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 ∼50° 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.

Structural Characterization of a Viral NEIL1 Ortholog Unliganded and Bound to Abasic Site-containing DNA

“…As mentioned earlier, a significant conformational change was reported in EcoNei between the free and liganded forms of the enzyme, which adopted an open and closed conformation (30). In contrast, superposition of the MvNei1⅐THF complex structure onto unliganded MvNei1 structures (Form II crystal) showed that the protein structures are identical except the zincless finger tip region and a few amino acid side chains (root mean square deviation of C␣ atoms are 0.7 and 0.8 Å, see Table 1).…”

“…In the unliganded EcoNei structure, the conformational change occurs via the flexible hinge region (Leu-122 to Pro-127), which connects the two Nei domains. The open conformation is stabilized via hydrogen bonds between the hinge region residues Leu-122 and Gln-123 and residues Arg-50 and Arg-147 (30). These interactions are lost in the EcoNei⅐DNA complex (23).…”

“…In trapped Fpg⅐DNA complexes (22,51) and Fpg complexes with DNA containing an AP site (51-53) the lesion binding loop was observed to be disordered. In contrast, this region is ordered in unliganded Thermus thermophilus Fpg (TthFpg) (24), EcoNei (30), and hNEIL1 (29). It is also ordered in complexes with DNA containing a lesion (48).…”

“…MvNei1 shares significant structural similarity with human NEIL1 (29). In contrast to E. coli Nei where a dramatic conformational change was observed upon binding DNA (30), the structure of MvNei1 bound to furan containing DNA does not reveal any substantial conformational change compared with the unliganded protein. The MvNei1 loop corresponding to the ␣F-␤9/10 lesion recognition loop of Fpg is ordered in both the unliganded and furan-bound structures, unlike what was reported for other Fpg/Nei enzymes where the loop is generally ordered when the enzyme is unliganded or bound to a lesion, and disordered otherwise.…”

The Fpg/Nei Family of DNA Glycosylases

“…The Fpg/DNA complexes include Schiff base intermediates, non-covalent complexes with AP-site analogs, and recognition or end-product complexes. Although the structure of EcoNei as a Schiff base intermediate in a complex with DNA was solved (56), it wasn’t until recently that the unliganded structure of EcoNei was determined which revealed a unique and interesting interdomain global conformational change upon DNA binding (62). Furthermore, the structures of unliganded human NEIL1 (61), unliganded MvNei1 and MvNei1 in a complex with THF were subsequently obtained (67).…”