Crystal structure of NucB, a biofilm-degrading endonuclease

Abstract

Bacterial biofilms are a complex architecture of cells that grow on moist interfaces, and are held together by a molecular glue of extracellular proteins, sugars and nucleic acids. Biofilms are particularly problematic in human healthcare as they can coat medical implants and are thus a potential source of disease. The enzymatic dispersal of biofilms is increasingly being developed as a new strategy to treat this problem. Here, we have characterized NucB, a biofilm-dispersing nuclease from a marine strain of Bacillus licheniformis, and present its crystal structure together with the biochemistry and a mutational analysis required to confirm its active site. Taken together, these data support the categorization of NucB into a unique subfamily of the ββα metal-dependent non-specific endonucleases. Understanding the structure and function of NucB will facilitate its future development into an anti-biofilm therapeutic agent.

Figure 1.

The nuclease activity of BlNucB against high molecular weight DNA. (A) BlNucB was incubated with (+) and without (–) calf thymus DNA for 15, 30 and 60 min at 37°C for 1 h. Samples of the digestion products were separated by agarose (0.8% w/v) gel electrophoresis and the DNA made visible by staining with ethidium bromide. HindIII-digested λ DNA (λ) is included as a marker. (B) BlNucB was incubated with a mixture of nicked (N) and supercoiled (S) pBR322 DNA at room temperature for 15 s, 30 s, 1 min, 2 min, 5 min, 10 min, 20 min and 30 min (labeled ¼ to 30 min). 10 mM EDTA (E) was added to the reaction mix prior to the addition of BlNucB for 30 min. Undigested (–) and BamHI-linearized (L) pBR322, along with HindIII-digested λ DNA (λ) are included as markers. The digestion products were separated by agarose (0.8% w/v) gel electrophoresis and the DNA made visible by staining with GelRed.

Figure 2.

The nuclease activity of BlNucB against oligodeoxynucleotide substrates. (A) Sequence of the oligodeoxynucleotides used, with the position of the phosphorothioate linkages indicated with the blue asterisks. (B) BlNucB was incubated with fluorescent double stranded oligodeoxynucleotide DNA at room temperature for 1 and 15 min, before separation by 17% (polyacrylamide) denaturing gel electrophoresis and visualization on a Typhoon scanner (GE Healthcare). The non-fluorescent complementary oligodeoxynucleotide used in all reactions is sequence 7. Undigested TAMRA-labelled 30mer and FITC-labelled 18mer oligodeoxynucleotides are used as size controls, and the schematic above the gel indicates which oligodeoxynucleotide was used; phosphorothioates are indicated by the blue circles.

Figure 3.

The crystal structure of BlNucB. (A) Overview of the structure of BlNucB as a cartoon, colour-ramped from blue to red, from N- to C-terminus. The secondary structure elements are labelled. (B) Topology diagram of BlNucB; α-helices are shown as red cylinders and β-strands as pink arrows. The ββα motif that defines the nuclease superfamily to which BlNucB belongs is highlighted by the grey box. (C) The surface of BlNucB coloured by sequence conservation (68), drawn in the same view as panel A. Note the concave depression in approximately the middle of the structure the mouth of which is mostly coloured deep purple, which indicates a high degree of sequence conservation. Non-conserved regions are coloured blue, with a purple-blue gradient between the two extremes. (D) Electrostatic potential mapped to the surface of BlNucB shown in the same view as above; positively-charged regions are coloured blue, negatively-charged red and uncharged regions are white. Note how the mouth of the depression is positively-charged whereas the bottom of the cavity is negatively-charged.

Figure 4.

BlNucB belongs to the ββα family of non-specific endonucleases. (A) The structure of BlNucB (cyan) indicates that it belongs to a novel subgroup of the ββα (or His-Me finger) family of non-specific endonucleases, as exemplified by Smendo (magenta; PDBid: 1SMN; 45). The ββα motif that defines the active site is labelled in each structure by the corresponding secondary structure element (top row). Other than this motif, the structural homology between BlNucB and Smendo is not extensive, but the surface representation in the same view (bottom row) indicates that they share the same surface depression that houses the active site. (B) A superposition of the active sites of Smendo (magenta) vs BlNucB (cyan), showing the conservation of the metal binding site in the latter. Asn119 in Smendo forms the only direct proteinaceous contact to the bound Mg²⁺ ion (pale pink sphere), and the water shell around the metal ion is stabilized by interactions to Gln114 and the general base, His89. The structural equivalents in BlNucB are Asn117, Asp93 and Glu94, respectively. Residues are coloured by structure, cyan for BlNucB and magenta for Smendo.

Figure 5.

NucB nuclease activity ex vivo. (A) BsNucB mutants are defective in nuclease activity. Cells were grown in Schaeffers media to induce sporulation at 37°C. Supernatant were then mixed with chromosomal DNA in the ratio 1:3 for 3 and 6 h at 37°C before DNA was visualized using 1% agarose gel stained with SYBR Gold. Wild-type (HM715), ΔnucB (AK361), nucB^H47A (AK453), nucB^D87A (AK447), nucB^D87N (AK449), nucB^D88A (HM1766) nucB^D102A (AK451), nucB^N111A (HM1767). (B) The active site of BlNucB is shown after rotation of the view in Figure 4A around a horizontal axis of ∼90°. The equivalent residues in BlNucB that were mutated in BsNucB (Figure 5A) are labelled in cyan; their counterparts in BsNucB are additionally labelled in black. Though BsNucB His47 is removed from the active site, mutation of it and immediate neighbours presumably affects nuclease activity because of the loss of a stabilizing network of hydrogen bonds involving Asp87 and Asp102; the equivalents in BlNucB are His53, Asp93 and Ser108.

Figure 6.

A model of the interaction of BlNucB with DNA. A model for the interaction of BlNucB (cyan) with post-cleavage DNA (orange worm) based on the shared structural feature of the ββα motif in Vvn endonuclease (silver; PDBid: 1OUP; 52). Key amino acids for Vvn DNA recognition are shown as sticks and are labelled in silver K28, E79, R99 and N127, with cyan labelled BlNucB structural equivalents R92, D93, R77 and N117. Vvn K100 does not seem to have a structural equivalent in BlNucB and the interaction of Vvn residues W85 and W94 with the DNA backbone remote from the active site is likely performed by BlNucB R70. The scissile phosphate is labelled with a red ‘0’ and the –1 phosphate is also indicated.