Unique Structural Features of Mammalian NEIL2 DNA Glycosylase Prime Its Activity for Diverse DNA Substrates and Environments

Abstract

Oxidative damage on DNA arising from both endogenous and exogenous sources can result in base modifications that promote errors in replication as well as generating sites of base loss (abasic sites) that present unique challenges to maintaining genomic integrity. These lesions are excised by DNA glycosylases in the first step of the base excision repair pathway. Here we present the first crystal structure of a NEIL2 glycosylase, an enzyme active on cytosine oxidation products and abasic sites. The structure reveals an unusual "open" conformation not seen in NEIL1 or NEIL3 orthologs. NEIL2 is predicted to adopt a "closed" conformation when bound to its substrate. Combined crystallographic and solution-scattering studies show the enzyme to be conformationally dynamic in a manner distinct among the NEIL glycosylases and provide insight into the unique substrate preference of this enzyme. In addition, we characterized three cancer variants of human NEIL2, namely S140N, G230W, and G303R.

Keywords: DNA damage; DNA repair; NEIL2 glycosylase; SAXS; X-ray crystallography.

Figure 1.. Diagram of Fpg/Nei Glycosylase Domain Composition Highlighting Their Structural Features

NEIL enzymes are substantially larger than their bacterial counterparts. The eukaryotic glycosylases harbor disordered regions. NEIL2 is unique in that the flexible region is internal. Figure adapted from Liu et al. (2010).

Figure 2.. Structure of MdoNEIL2 and Comparison to Other Glycosylases

(A) Cartoon representation of the NEIL2 structure from N terminus (blue) to C terminus (red) with wild-type methionines highlighted with gray spheres and methionines engineered for additional phase verification shown in black. (B) Cartoon representation of NEIL2 (gray) and overlaid anomalous difference Fourier maps contoured at 3σ for the wild-type SeMet (orange), L-M SeMet (purple), sodium iodide (green), KAu(CN)₂ (blue), and K₂PtCl₄ (cyan). (C) Comparison of MdoNEIL2 to human NEIL1 (HsaNEIL1; PDB: 1TDH) (Doublié et al., 2004) and mouse NEIL3 (MmuNEIL3; PDB: 3W0F) (Liu et al., 2013b) displayed with the C-terminal domain in equivalent orientations and demonstrating the unique interdomain orientation of NEIL2. The N-terminal active-site residue is shown in pink, in space-fill mode. The N-terminal domain large insert (insert 1) and small insert (insert 2) unique to NEIL2 are disordered in the structure and shown as dotted lines.

Figure 3.. Activity of MdoNEIL2 toward DNA Lesions

(A) Glycosylase activity of MdoNEIL2 for ³²P-labeled single-stranded DNA (ssDNA) substrates run on urea-PAGE. Assays were performed using 25 nM DNA substrate and enzyme concentrations of 0, 25, 100, 400, and 800 nM. (B) Shown is the concentration-based activity titration for single-stranded abasic site substrate with data fit to a single exponential. (C) Shown is the summary of the maximum activity under the assay conditions expressed as fraction of strand cleavage for all single-stranded substrates andenzyme variants of MdoNEIL2 and HsaNEIL2. (D) Shown is the summary of the maximum activity for all double-stranded (dsDNA) substrates. Titration curves for all assays are provided in Figure S3.

Figure 4.. Comparison of the Open and Closed Forms of E. coli Endonuclease VIII (EcoNei) with MdoNEIL2

(A) Comparison of the unliganded forms of EcoNei (blue) and MdoNEIL2 (pink) oriented with the C-terminal domain on the left and N-terminal domain on the right. The zinc ions from the zinc finger are shown as spheres; the structures were superimposed on the C-terminal domain. (B and C) (B) The unliganded EcoNei (PDB: 1Q3B) (Golan et al., 2005) is shown in comparison to (C) DNA-bound EcoNei (PDB: 2EA0) (Golan et al., 2007). Both forms of EcoNei were overlaid based on their C-terminal domains and the DNA duplex for the DNA-bound form shown in both (B) and (C). A significant rotation (reported to be ~50°) of the N-terminal domain for the unliganded structure would be required for catalytic competency (Golan et al., 2005). (D and E) (D) The MdoNEIL2 structure is overlaid with the C-terminal expected conformation of (E), a predicted catalytically competent complex. A n angle of ~80° for rotation of the NEIL2 N-terminal domain between the two conformations was determined using Superpose within CCP4 (Winn et al., 2011). For (B)–(E), the C-terminal domain is oriented on the left and in black while the N-terminal domain is oriented to the right and shown in rainbow coloring indicating the N-terminal end containing the catalytic residues P2-E3 as blue and the C-terminal end of the domain as red. The structures in (B), (D), and (E) do not contain DNA, so the duplexes are shown in transparent mode for reference, as the DNA model is from the structure in (C) after C-terminal domain superpositions. The disordered large insert (insert 1) and small insert (insert 2) of the MdoNEIL2 N-terminal domain are shown as dotted lines.

Figure 5.. Comparison Using Least-Squares Superposition of the N-terminal Domain of Proteins in the Fpg/Nei Superfamily

The crystal structure of the mimivirus NEIL1 ortholog (MvNei1) (Imamura et al., 2009) is shown in complex with the abasic-site-containing duplex, colored gray, with key loops and the respective residues involved in catalytic function shown: P2-E3 and void-filling residues (L84, R114, and F116). The black spheres represent the abasic-site lesion. The MdoNEIL2 structure is shown in magenta upon independent superposition of the N-terminal and C-terminal domains onto the corresponding domains in MvNei1. Residues corresponding to HsaNEIL2 cancer variants S140 (green), G230 (orange), and G303 (cyan) are highlighted. The structure-based sequence alignment for the Fpg/Nei family was produced using PROMALS3D (Pei et al., 2008). Whereas the loop between β strands 4 and 5 containing the void-filling residue is consistent in length within the Fpg/Nei family, the loop between β strands 7 and 8 is quite variable and is significantly shorter in the NEIL2/3 orthologs. The structures used in the alignment are MvNei1 (PDB: 3A46) (Imamura et al., 2009), HsaNEIL1 (1TDH) (Doublié et al., 2004), MmuNEIL3 (3W0F) (Liu et al., 2013b), MvNei2/3 (4MB7) (Prakash et al., 2013), LlaFpg (1L1T) (Fromme and Verdine, 2002), and EcoNei (2EA0) (deposited but unpublished).

Figure 6.. SAXS Analysis for PCNA, EcoNei, MdoNEIL2, and MdoNEIL2cut

(A) Shown is the scattering curve for PCNA (experimental control) and fits to three crystal structures (PDB: 4ZTD [Hoffmann et al., 2016], 2ZVW [Strzalka et al., 2009], 1VYM [Kontopidis et al., 2005]), after omission of any ligands, using FoXS (Schneidman-Duhovny et al., 2016). (B) Shown is the SAXS profile for EcoNei under batch conditions (black) or upon elution from in-line SEC (gray). The fits shown represent the apo conformation (red and pink) and DNA bound (dark and light blue). (C) Scattering curves for batch and SEC-SAXS for full-length MdoNEIL2 (red and pink) and SEC-SAXS for MdoNEIL2cut (orange). For (A), (B), and (C), the inset shows the Guinier region. (D) Distance distribution function for all samples. (E) FoXS fitting for the MdoNEIL2cut.