Crystal structure of the EndoG/EndoGI complex: mechanism of EndoG inhibition

Abstract

EndoG is a ubiquitous nuclease that is translocated into the nucleus during apoptosis to participate in DNA degradation. The enzyme cleaves double- and single-stranded DNA and RNA. Related nucleases are found in eukaryotes and prokaryotes, which have evolved sophisticated mechanisms for genome protection against self-antagonizing nuclease activity. Common mechanisms of inhibition are secretion, sequestration into a separate cellular compartment or by binding to protein inhibitors. Although EndoG is silenced by compartmentalization into the mitochondrial intermembrane space, a nucleus-localized protein inhibitor protects cellular polynucleotides from degradation by stray EndoG under non-apoptotic conditions in Drosophila. Here, we report the first three-dimensional structure of EndoG in complex with its inhibitor EndoGI. Although the mechanism of inhibition is reminiscent of bacterial protein inhibitors, EndoGI has evolved independently from a generic protein-protein interaction module. EndoGI is a two-domain protein that binds the active sites of two monomers of EndoG, with EndoG being sandwiched between EndoGI. Since the amino acid sequences of eukaryotic EndoG homologues are highly conserved, this model is valid for eukaryotic dimeric EndoG in general. The structure indicates that the two active sites of EndoG occupy the most remote spatial position possible at the molecular surface and a concerted substrate processing is unlikely.

Figure 1.

Overall structure of the dEndoG/dEndoGI complex. Helices of dEndoG are illustrated as purple ribbons and strands of the central β-sheet in cyan. The small two-stranded β-sheet (β-strands D and E) involved in metal ion binding is highlighted in yellow, and the bound metal ion is shown as an orange sphere. The two wings forming an intermolecular β-sheet are shown in red (β-strands G and H). Dom1 and Dom2 of Endo GI, which sandwich the dEndoG dimer, are shown as brown ribbons. The missing linker between the two domains would start at Arg174 in Dom1 and connect the polypeptide chain with Leu219 in Dom2.

Figure 2.

Sequence conservation of EndoG. (A) Sequence alignment of EndoG homologues for D. melanogaster (NP_610737), Homo sapiens (NP_004426), Caenorhabditis elegans cps–6 (NP_491371) and S. cerevisiae Nuc1p (NP_012327). Identical residues are colored dark green and according to the decreasing similarity from light green through orange to yellow. Secondary structure elements are colored according to Figure 1. Helices are drawn as cylinders and strands as arrows. Residues which are involved in formation of the dEndoG homodimer interface are labeled with gray circles and residues directly involved in catalysis are marked with black triangles. (B) Conserved patches at the molecular surface of dEndoG. Left: A surface representation of one dEndoG monomer is colored according the amino acid sequence alignment in (A), and the second molecule is drawn as a ribbon model similarly to Figure 1. Right: the dEndoG homodimer turned by 180°, showing the conserved patch around the active site of dEndoG. (C) Active site of dEndoG. Residues involved in metal ion coordination and/or catalytic function are shown as stick models, the central Mg²⁺ ion as an orange sphere and water molecules from the metal ion hydration shell and solvent water molecules are shown as red spheres. An electron density map contoured at 3.0 σ for the omitted metal ion and water molecules is shown as a green mesh.

Figure 3.

Bookview of the EndoG/EndoGI complex. For simplicity, only one dEndoG monomer (left) and the corresponding Dom1 of dEndoGI (right) are shown as surface representations. Coloring is according to the electrostatic surface potential over the range of ±9 kT/e (blue/red).

Figure 4.

Nuclease activity assays of dEndoG. (A) Limited digestion of plasmid DNA. (B) Same sample as in (A) after melting. (C) pH-profile of dEndoG nuclease activity. The reaction was stopped after 1 min of incubation.

Figure 5.

Inhibition of the active site of dEndoG by dEndoGI. The interface between Dom1 of dEndoGI (brown) and the active site of one dEndoG monomer (violet) is shown in (A) and the equivalent interface between Dom2 and the second dEndoG molecule in (B). Residues involved in binding are shown as sticks and for simplicity only salt bridges of important arginine residues (dotted black lines) and hydrogen bonds to water molecules (dashed black lines) are presented.