Structure determination and biochemical characterization of a putative HNH endonuclease from Geobacter metallireducens GS-15

Abstract

The crystal structure of a putative HNH endonuclease, Gmet_0936 protein from Geobacter metallireducens GS-15, has been determined at 2.6 Å resolution using single-wavelength anomalous dispersion method. The structure contains a two-stranded anti-parallel β-sheet that are surrounded by two helices on each face, and reveals a Zn ion bound in each monomer, coordinated by residues Cys38, Cys41, Cys73, and Cys76, which likely plays an important structural role in stabilizing the overall conformation. Structural homologs of Gmet_0936 include Hpy99I endonuclease, phage T4 endonuclease VII, and other HNH endonucleases, with these enzymes sharing 15-20% amino acid sequence identity. An overlay of Gmet_0936 and Hpy99I structures shows that most of the secondary structure elements, catalytic residues as well as the zinc binding site (zinc ribbon) are conserved. However, Gmet_0936 lacks the N-terminal domain of Hpy99I, which mediates DNA binding as well as dimerization. Purified Gmet_0936 forms dimers in solution and a dimer of the protein is observed in the crystal, but with a different mode of dimerization as compared to Hpy99I. Gmet_0936 and its N77H variant show a weak DNA binding activity in a DNA mobility shift assay and a weak Mn²⁺-dependent nicking activity on supercoiled plasmids in low pH buffers. The preferred substrate appears to be acid and heat-treated DNA with AP sites, suggesting Gmet_0936 may be a DNA repair enzyme.

Figure 1. PROMALS3D multiple sequence alignment of putative HNH endonucleases.

Consensus predicted secondary structure symbols: alpha-helix: h; beta-strand: e. Consensus amino acid symbols are: conserved amino acids are in bold and uppercase letters; aliphatic (I, V, L): l; aromatic (Y, H, W, F): @; hydrophobic (W, F, Y, M, L, I, V, A, C, T, H): h; alcohol (S, T): o; polar residues (D, E, H, K, N, Q, R, S, T): p; tiny (A, G, C, S): t; small (A, G, C, S, V, N, D, T, P): s; bulky residues (E, F, I, K, L, M, Q, R, W, Y): b; positively charged (K, R, H): +; negatively charged (D, E): −; charged (D, E, K, R, H): c. 4 h9d_chainA, Gme HNHE from G. metallireducens GS-15 (15 C-terminal residues EPSDGEGLEH₆ are disordered and not resolved in the structure. Other HNH endonuclease homologs in the alignment are: Gbe, G. bemidjiensis Bem; GspM21, Geobacter sp. M21; Gda, G. daltonii FRC-32; Gur, G. uraniireducens Rf4; GspM18, Geobacter sp. M18; Gsu, G. sulfurreducens PCA; Glo, G. lovleyi SZ; Cni, Calditerrivibrio nitroreducens DSM 19672; Dde, Deferribacter desulfuricans SSM1; Sfu, Syntrophobacter fumaroxidans MPOB.

Figure 2. Crystal structure of the Gmet_0936 protein, a putative HNH endonuclease from Geobacter metallireducens GS-15.

A. Schematic drawing of the structure of Gmet_0936 protein. The secondary structure elements are labeled. A bound zinc ion and its four Cys ligands are shown as a sphere (pink) and in stick models (in gray), respectively. Residues 53, 54, 68, 77 are shown as stick models (in green). B. Overlay of the structure of HNH endonuclease (in color) with the restriction endonuclease Hpy99I (in gray) . Residues in the zinc binding site and the active site are shown as stick models. The N-terminal residues of Hpy99I (1–105) are not shown. C. Molecular surface of the active site region of the HNH endonuclease. Residues His54, Asp53 and Asn77 are given different colors and labeled.

Figure 3. Structure of the Gmet_0936 protein dimer.

A. Schematic drawing of the Gmet_0936 protein dimer. The two molecules are colored in cyan and green. Residues Asp53-His54 in the putative active site are shown as stick models. The two-fold axis of the dimer is indicated by the red oval. B. Structure of Hpy99I dimer in complex with duplex DNA . The catalytic domains are colored in cyan and green, and the N-terminal segment in yellow. The DNA duplex is in orange.

Figure 4. Structural comparison of the conserved ββα-metal fold and catalytic sites of Gme HNHE, Hpy99I, phage T4 endonuclease VII, periplasmic nuclease Vvn from Vibrio vulnificus, and endonuclease I from bacterium Vibrio salmonicida.

Some additional secondary structures (β sheets) were inserted in the ββα-Me fold in Vvn nuclease and endonuclease I.

Figure 5. DNA mobility shift assay (DNA binding assay) in the absence of divalent cations and DNA nicking activity assays for 6xHis-tagged N77H in buffers with different metal ions.

A. Fluorescein-labeled duplex oligos were incubated with varying amount of 6xHis-tagged N77H or Gme HNHE at room temperature for 20 min and the DNA-protein complexes were resolved in a 10% PAG gel. ½ to 8× indicates the relative protein concentration (1x = 0.5 µg protein). Arrows indicate the substrates and the bound complexes. N.Bsu, Bacillus subtilis HNH nicking endonuclease. The binding buffer in this assay lacks divalent cations and therefore does not support DNA nicking. B. DNA nicking assay for WT and N77H (H₆). Lanes 1–3, φX174 RFI DNA (dsDNA); lanes 4–6, φX174 RFII DNA (dsDNA, nicked circular); lanes 7–9, φX174 ssDNA; lanes 10–12, M13 RFI (dsDNA); lanes 13–15, M13 ssDNA; lane 16, nicked M13 RFI DNA by Nb.BsmI; lane 17, 2-log DNA ladder.