Combining H/D exchange mass spectroscopy and computational docking reveals extended DNA-binding surface on uracil-DNA glycosylase

Abstract

X-ray crystallography provides excellent structural data on protein-DNA interfaces, but crystallographic complexes typically contain only small fragments of large DNA molecules. We present a new approach that can use longer DNA substrates and reveal new protein-DNA interactions even in extensively studied systems. Our approach combines rigid-body computational docking with hydrogen/deuterium exchange mass spectrometry (DXMS). DXMS identifies solvent-exposed protein surfaces; docking is used to create a 3-dimensional model of the protein-DNA interaction. We investigated the enzyme uracil-DNA glycosylase (UNG), which detects and cleaves uracil from DNA. UNG was incubated with a 30 bp DNA fragment containing a single uracil, giving the complex with the abasic DNA product. Compared with free UNG, the UNG-DNA complex showed increased solvent protection at the UNG active site and at two regions outside the active site: residues 210-220 and 251-264. Computational docking also identified these two DNA-binding surfaces, but neither shows DNA contact in UNG-DNA crystallographic structures. Our results can be explained by separation of the two DNA strands on one side of the active site. These non-sequence-specific DNA-binding surfaces may aid local uracil search, contribute to binding the abasic DNA product and help present the DNA product to APE-1, the next enzyme on the DNA-repair pathway.

Figure 1.

B-DNA docked to the DNA-bound structure of UNG (gray Cα backbone). (A) The 30 top-ranked B-DNA placements compared with bound DNA from 1SSP (blue phosphate backbone): 21 (yellow) at the active site; 6 (green) tightly clustered at a secondary site; 1 (magenta) between the active site and the secondary site; and 2 (orange) near the UNG N-terminus. (B) The larger active-site cluster (14 placements) replicates the UNG–DNA active-site contacts found in the 1SSP complex, including insertion of Leu 272 (black) into the DNA minor groove. These dockings also show direct contact of the complementary strand with residues 210–220 (magenta). In all, the active-site strand has the same 5′ to 3′ direction as the crystallographic DNA, as indicated by red coloring of the 3′-ends. The UNG backbone is colored by the DXMS results (see Figure 3). (C) The 2000 top-ranked B-DNA placements, represented by their geometric centers (spheres), are concentrated over the active site (indicated by the crystallographic DNA, blue, right), at the secondary site (indicated by docked B-DNA, green, left), and between the two sites.

Figure 2.

Percent deuterium incorporation for peptides after 30 (black), 300 (green) or 10000 s (magenta) are shown for UNG (top) and the UNG–DNA complex (middle). The change in deuteration (bottom) is shown for peptides common to both data sets, where a negative percentage indicates less deuteration in the UNG–DNA complex. Regions are defined as in Supplementary Figure S3 and Supplementary Tables S1–S3.

Figure 3.

Distribution of UNG regions showing significant solvent protection in the presence of DNA. (A) Peptides on the active-site face of UNG. Residues 142–157 (purple), 160–170 (blue green) and 210–220 (magenta) are highlighted on the UNG Cα backbone (right) and correspond to the deuteration profiles (left). Residues 210–220 show the greatest change in solvent protection, but have no contact with the bound DNA product (light blue phosphate backbone) in the 1SSP crystallographic structure. (B) Peptides that span the region between the active site and the secondary DNA-binding site predicted by computational docking. Residues 245–248 (green) and residues 251–264 (red) are highlighted on the UNG structure (right) and correspond to deuteration profiles. Residues 265–274 (orange) are shown on the UNG structure (left), but the deuteration profile is for residues 258–274, which partially overlap residues 251–264 (red). Residues 251–264 show the greatest change in solvent protection and make up part of the predicted secondary DNA-binding site (indicated by the docked DNA, green phosphate backbone), but have no DNA contacts in 1SSP.

Figure 4.

Model of the 30-bp product dsDNA bound to UNG. Both DNA strands in the model align with the crystallographic DNA (gray phosphate backbone) on the 5′ side of the active site. On the 3′ side of the active site, the active-site strand (light blue) contacts the groove created by residues 251–274, including the continuous surface formed by main-chain atoms of residues 251–264 (red) and 265–274 (orange). The complementary strand (blue) contacts the groove created by residues 210–220, including the surface created by the main-chain atoms (magenta), and may also contact the surface created by main-chain atoms of residues 251–258 (red).